Doctor of Philosophy, The Ohio State University, 2023, Pharmacy

Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases, yet it disproportionately contributes to 35% of breast cancer deaths. In addition, chemotherapy resistance is a significant challenge in TNBC treatment. In this study, we aimed to identify potential synthetic lethal targets for cisplatin/doxorubicin treatment in TNBC and investigate the potential of a cell-death double knock-out library to discover synergistic lethal gene pairs for TNBC therapy. Initially, we identified the MDA-MB-231 cell line as the most representative model for TNBC chemotherapy-poor responders by comparing genomic profiles from TNBC cell lines and patient samples. We then conducted a genome-wide CRISPR-Cas9 screen and RNAseq analysis in MDA-MB-231 cells. Our analysis confirmed the involvement of known essential genes in DNA damage repair and regulation of DNA replication pathways, such as BCL2L1, ATM, CDC25B, and NBN, in sensitizing cells to cisplatin/doxorubicin. Moreover, we identified hundreds of previously unrecognized genes and pathways related to DNA repair, G2/M DNA damage checkpoint, AMPK signaling, and mTOR signaling. The observed differences between transcriptomic responses and essential pathways from the CRISPR screen suggest a complex regulatory system in the cellular response to DNA-damaging drugs. By integrating various data analysis methods and biological experimental approaches, we pinpointed several promising genes, such as MCM9 and NEPPS, which could serve as potential drug targets to overcome chemoresistance. We also utilized our lab's custom CRISPR double knock-out library, which leverages the XDeathDB database for candidate gene selection. This comprehensive platform provides insights into 12 cell death modes and 149 cell death hallmark genes. We aim to create a cell-death double knock-out library using these genes and perform double knock-out screening on MDA-MB-231 cells. The identified synergistic lethal gene pairs may serve as potentia (open full item for complete abstract)

Committee: Lang Li (Advisor) Subjects: Bioinformatics; Pharmacology; Pharmacy Sciences

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

Osteosarcoma (OS) is the most common malignant primary bone tumor in pediatric patients. Therapeutic target identification in osteosarcoma has been extremely challenging due to the high degree of heterogeneity in osteosarcoma genetic profile and the lack of specific oncogenic driver genes. Despite the great research efforts, the treatment and survival outcomes for osteosarcoma patients have stagnated over the past 30 years, and osteosarcoma remains a lethal cancer due to metastasis and development of resistance to current chemotherapies. Study of chemo-resistance and identification of novel targets are therefore critical to better understand osteosarcoma chemo-resistance mechanisms and ultimately lead to new therapies. In the hope of providing an innovative angle for osteosarcoma study, we first performed a comprehensive multiplatform analysis on osteosarcoma tumors, including somatic copy number alteration, gene expression and methylation, and identified three molecularly distinct and clinically relevant subtypes for osteosarcoma. Previously unappreciated osteosarcoma-type-specific features were revealed based on the subgrouping, providing insights into refining osteosarcoma therapy and relationships to other types of cancer. Additionally, a CRISPR functional knockout screen is an ideal new strategy to address the genetic complexity of osteosarcoma. The CRISPR/Cas9 gene editing system with customized libraries of guide RNAs (gRNAs) can be used to test the contribution of each individual gene to osteosarcoma therapy resistance on a genome-wide scale. By comparing the differences in the abundance of gRNAs between the control and phenotyped samples, the genes responsible for the observed phenotype can be identified. Genome-wide CRISPR screen with low-dose drug treatment was conducted on the selected osteosarcoma cell line SaOS2 to identify the genes that are essential for osteosarcoma cell proliferation under different drug treatments. The CRISPR screen, together wi (open full item for complete abstract)

Committee: Moray Campbell (Committee Member); Ryan Roberts (Committee Member); Jeffrey Parvin (Committee Member); Li Lang (Advisor) Subjects: Bioinformatics; Biology; Pharmaceuticals; Pharmacy Sciences

Doctor of Philosophy in Biomedical Sciences (Ph.D.), University of Toledo, 2021, Biomedical Sciences (Cancer Biology)

Cancer remains a significant public health burden worldwide, and advances in treatment are aimed at delivering a deathblow to cancer cells, while sparing normal cells. Hence, the ongoing search for novel targets has led to the discovery of a cytoplasmic cis ATR (Ataxia Telangiectasia and Rad3 Related) protein whose function is normal in cells at homeostasis, but when predominant in the cytoplasm, it provides an oncogenic drive through evasion of apoptosis. ATR is a member of the PIKK (Phosphatidylinositol 3-kinase-related kinase) family of protein kinases and nuclear ATR plays a crucial role in DNA damage responses (DDR) by phosphorylating hundreds of downstream proteins. However, ATR is also present in the cytoplasm as either a cis or trans isomeric form, depending on Pin1 which converts cis ATR to trans ATR. Following DNA damage, Pin1's isomerization of cytoplasmic ATR is inhibited leading to an increase in cytoplasmic cis ATR levels. This DNA damage can be from an acute insult or from effects of accumulated damage from the aging process. Following DNA damage, cytoplasmic cis ATR, via its BH3 domain, binds to t-Bid at the outer mitochondrial membrane and sequesters t-Bid to prevent t-Bid binding of pro-apoptotic Bax/Bak protein thus, suppressing apoptosis. Therefore, the levels of cis ATR in the cytoplasm could determine cell fate: death or immortality. With a predominance of cis ATR in cell cytoplasm, there is an inability of the cells to be killed by apoptosis, leading to DNA damage accumulation, genomic instability, and thus oncogenesis. This phosphorylation-dependent peptidyl-prolyl isomerization of ATR by Pin1 serves as a regulatory tool that can be taken advantage of since PP2A can dephosphorylate the key phosphorylated residue in ATR that Pin1 recognizes. With PP2A regulation of ATR, it is possible to shift the balance between the cis and trans forms of cytoplasmic ATR. This is particularly important because it provides a potential strategy in enhancing can (open full item for complete abstract)

Committee: Yue Zou Ph.D. (Advisor); Kathryn Eisenmann Ph.D. (Committee Chair); Caitlin Emily Baum Ph.D. (Committee Member); Saori Furuta Ph.D. (Committee Member); Phillip R. Musich Ph.D. (Committee Member) Subjects: Biochemistry; Biomedical Research; Cellular Biology; Molecular Biology; Oncology

Master of Science (MS), University of Toledo, 2020, Pharmaceutical Sciences (Pharmacology/Toxicology)

According to the 2020 World Drug Report, amphetamine type stimulant (ATS) is the third most used illicit drug with 27 million users globally. Methamphetamine (MA) is a potent central nervous system (CNS) stimulant with a high abuse potential and addiction. MA use has become an increasing concern worldwide especially due to the adverse effects it possesses. MA is also known as the poor man's cocaine since it is more easily accessible “illegally” and inexpensive compared to cocaine. The euphoric feeling from MA also lasts longer than cocaine due to its long half-life (10-12h) and lipophilicity that allows MA to better penetrate the blood brain barrier (BBB) easily. The primary aim of this study was to study the acute toxic effects of MA that may contribute to acute lethality as well as to long-term adverse effects associated with MA use. The hypothesis of this study is that death in 5-dpf larval zebrafish is due to apoptosis in the cardiovascular system and/or in the brain secondary to acute MA exposure. MA works by causing an increase in the levels of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and preventing the re-uptake of these neurotransmitters (NT) by blocking monoamine transporters, which then increases the levels of the NT in the synaptic cleft. An increase in DA, NE, 5-HT causes users to experience intense euphoric feelings, increased heart rate (tachycardia), hyperthermia, increased energy, hypertension and decrease in appetite. Some of the chronic abuse of MA leads to neurotoxicity, agitation, aggression, anxiety, psychosis, renal and liver failure, seizure, and cardiac arrhythmias, heart attacks. In this study, we used 5-day old larval zebrafish and exposed them to three different concentrations (0mM, 5mM and 15mM) of MA for 5-hr to assess change in heart rate and other lethality parameters such as lack of heartbeat, opaque appearance and notochord bend (lordosis). Subjects were then processed for cleaved caspase-3 immunofluorescence assay an (open full item for complete abstract)

Committee: Frederick Williams (Advisor); Scott Hall (Committee Member); Zahoor Shah (Committee Member) Subjects: Pharmacology; Toxicology

Master of Science (MS), Wright State University, 2020, Pharmacology and Toxicology

Cancer is a life-threatening illness and innovative research is therefore required to fuel the development of new anti-cancer therapies. As an anti-proliferative drug, hydroxyurea (HU) has been used in the treatment of various neoplastic and non-neoplastic diseases such as sickle cell anemia, psoriasis, and viral infections. HU is a well-known inhibitor of ribonucleotide reductase (RNR), an enzyme that generates dNTPs for DNA replication and repair. In our genetic screen in fission yeast looking for mutants with defects in checkpoint response, we also found a set of mutants that are highly sensitive to HU but with a functional checkpoint response. It is likely that in addition to the RNR suppression, HU induces cell lethality by a previously less understood mechanism involving perturbations of various metabolic pathways. This study is to identify new metabolic genes whose mutations sensitize the cells to HU in order to better understand the cell-killing mechanisms of HU. We have identified new mutation in erg12 gene, which encodes the mevalonate kinase enzyme in the ergosterol biosynthesis pathways. While characterizing the erg12 mutant, we have unexpectedly found five multi-copy suppressors including mns1, uge1, chs1, mug109 and hba1. Results from this study may help to better understand the cell-killing mechanisms of HU, the side effects, and drug resistance associated with the HU-based chemotherapies. They may also promote therapeutic innovations for the benefits of patients with cancer or fungal infections.

Committee: Yong-jie Xu M.D., Ph.D. (Advisor); Michael Kemp Ph.D. (Committee Member); Jeffrey B. Travers M.D., Ph.D. (Committee Member) Subjects: Genetics; Pharmacology; Toxicology

Doctor of Philosophy, University of Toledo, 2020, Experimental Therapeutics

Novel synthetic psychostimulants (i.e., `bath salts') have begun to replace common drugs of abuse like methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) in recreational drug markets. The rate at which novel synthetic psychostimulants are produced has rapidly outpaced their pharmacological evaluation and has led to an alarming increase in reports of potentially life threating adverse events. The mechanisms underlying synthetic psychostimulant related toxicity, lethality, and their propensity for abuse are poorly understood. This is partly due to the sheer number, the potential for unpredictable targets, as well as the likelihood of adulterated preparations with licit and illicit substances. In order to determine which of the synthetic psychostimulants and/or combinations of these compounds hold the greatest threat for adverse events, rigorous controlled studies are required in an animal model capable of high-throughput evaluation. The zebrafish has become a widely used model in neurobehavioral research due to its high-throughput capabilities and diverse behavioral repertoire. The goal of this study was two-fold; first, to begin understanding the mechanisms related to the toxicity, lethality, and abuse potential of synthetic psychostimulants; second, to present the zebrafish as a viable high-throughput model capable of addressing these questions at a pace comparable to the street chemists' synthesis rate.

Committee: Frederick Williams (Committee Chair); Caren Steinmiller (Committee Member); F. Scott Hall (Committee Member); Isaac Schiefer (Committee Member) Subjects: Pharmacology

Master of Science (MS), Wright State University, 2019, Biological Sciences

Two closely related Caenorhabditis species, C. briggsae and C. nigoni are cross fertile and produce viable adult progeny. From C. nigoni mothers, F1 adult females are viable and fertile, F1 males are viable but sterile. In crosses that utilize C. nigoni males and C. briggsae hermaphrodites produce viable adult F1 females but F1 males arrest during embryogenesis. A mutation in the Cbr-him-8 gene is a recessive maternal-effect suppressor of male-specific lethality. Hybrid crosses with cbr-him-8 mutant mothers produce viable adult male progeny. The HIM-8 protein in C. elegans is required for the pairing of X-chromosomes during meiosis. This function is likely conserved in C. briggsae. Unpaired chromosomes are transcriptionally silenced in a wide variety of taxa. Based on this information it's been proposed that the meiotic silencing of unpaired chromosomes (MSUC) is suppressing an X-linked hybrid lethal gene responsible for male specific lethality. Multiple co-suppression assays identified two genes as candidate hybrid lethal genes, CBG30927 and CBG00239. These genes were knocked out with RNAi and CRISPR to evaluate if either of these genes were a hybrid lethal gene. sgRNA/Cas9 complexes and dsRNA of the candidate hybrid lethal genes was injected into C. briggsae hermaphrodites. Injections using CRISPR were able to disrupt expression of control targets but not the candidate hybrid lethal genes. Both RNAi and CRISPR injected nematodes were mated with C. nigoni males and the resulting progeny were scored for viable F1 males. From injections of dsRNA containing copies of CBG30927 or Cbr-hig-1, male progeny were derived. Cbr-hig-1 has a syntenic ortholog in C. nigoni that is not present in any other Caenorhabditis species. Several regions were identified in the C. briggsae and C. nigoni transcripts including an exon 5 extension that is responsible for a change in the predicted structure of the proteins that could be responsible for the dysgenic interactions.

Committee: Scott Everet Baird Ph.D (Advisor); Quan Zhong Ph.D. (Committee Member); Labib Rouhana Ph.D. (Committee Member) Subjects: Biology; Cellular Biology; Genetics; Organismal Biology

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

Ribosomal protein L13a plays an extra-ribosomal function in translational silencing of GAIT (IFN-gamma-activated inhibitor of translation) element bearing mRNAs encoding inflammatory proteins but the underlying molecular mechanism of translational silencing and ribosomal incorporation of L13a remains poorly understood. Also, our laboratory showed that L13a acts as a physiological defense against uncontrolled inflammation in macrophage-specific knockout (KO) mice. However, the consequence of a total knockout of L13a in mammals remains unexplored. Therefore, our current study is focused on (i) identifying the amino acid residue(s) of L13a essential for incorporation and translational silencing of target mRNAs and (ii) studying the consequences of systemic loss of L13a in a mouse model. To address the first question, we compared the prokaryotic L13 structure with human L13a, which depicted the presence of an α-helical extension of ~55 amino acids at the C-terminal end of human L13a. We observed that deletion of this helix impairs ribosomal incorporation and the translational silencing ability of L13a. We have identified the amino acids within this helix at position 159(K) and 161(K) that are essential for ribosomal incorporation. CryoEM studies of the human ribosome showed the interaction of the amino acids at position 185(V), 189(I) and 196(L) of L13a with RP L14. We found that mutating these residues abrogates the ribosomal incorporation of L13a. Importantly, we also showed that mutation of the amino acids at position 169(R), 170(K) and 171(K) to Ala abrogate translational silencing activity, but not ribosomal incorporation, showing mutually exclusive ribosome incorporation and translational silencing domain. To address the second question, we generated heterozygous L13a mice (L13a+/-). However, the homozygous KO (L13a-/-) mice are embryonically lethal at an early stage. We have identified the KO embryos in the pre-implantation (morula) stage, suggesting an essential (open full item for complete abstract)

Committee: Barsanjit Mazumder (Advisor); Anton A. Komar (Committee Member); Crystal M. Weyman (Committee Member); William M. Baldwin (Committee Member); Girish Shukla (Committee Member); William C. Merrick (Committee Member) Subjects: Developmental Biology; Molecular Biology

Master of Science (MS), Wright State University, 2018, Biological Sciences

In the cross of C. nigoni males to C. briggsae hermaphrodites, all F1 males arrest during embryogenesis. However in the reciprocal cross there are some viable F1 male progeny. This unidirectional male-specific lethality in the F1 hybrids has been attributed to a hybrid lethal gene in a 500 Kb region of the X chromosome of C. briggsae. Cbr-him-8 is a recessive maternal suppressor of the male-specific lethal phenotype, due to the requirement of the him-8 protein for proper X chromosome pairing. Without proper pairing of any one of the chromosomes in the Caenorhabditis genome, genes present on the unpaired chromosome will be silenced due to a process known as meiotic silencing of unpaired chromosomes (MSUC). It has been proposed that MSUC-based silencing of the X-linked hybrid lethal gene is the mechanism by which the male-specific lethality is suppressed. Based on this model, a co-suppression assay was used to identify the hybrid lethal gene. Transgenic strains of C. briggsae were constructed via microinjection of bacterial artificial chromosomes (BACs) of small portions of the X chromosome in which the hybrid lethal gene resides. The BACs were mixed with pCFJ909, a plasmid containing a functional cbr-unc-119 gene, this mixture was then microinjected directly into the gonad of cbr-unc-119 mutant hermaphrodites. A proportion of the resulting progeny incorporated the injected DNA into their nucleus and formed heritable extra-chromosomal arrays. These offspring were then selected based on the rescue of the unc-119 phenotype. Transgenic hermaphrodites were then mated to C. nigoni males and scored for viable F1 male progeny. Two BAC rescued the male specific hybrid lethal phenotype. Multiple other BACs failed to rescue the lethality phenotype. Focusing on a single BAC clone, using gene groupings and pCFJ909 the number of possible genes have been narrowed to two candidate hybrid lethal genes within the BAC 08G05. As well as 5 candidate hybrid lethal genes in the non-adjacen (open full item for complete abstract)

Committee: Scott Baird Ph.D. (Committee Chair); Labib Rouhana Ph.D. (Committee Member); Jeffrey Peters Ph.D. (Committee Member) Subjects: Biology; Genetics; Microbiology; Organismal Biology

PhD, University of Cincinnati, 2015, Medicine: Molecular Genetics, Biochemistry, and Microbiology

Polo-like kinase 3 (Plk3) is a member of a conserved family of serine/threonine kinases that primarily regulate cell cycle progression and mitotic events. In response to ionizing radiation, Plk3 facilitates the activation of G1/S checkpoint arrest. Further, published data from our lab suggested that Plk3 may be capable of activating G1/S checkpoint arrest independent of p53 signaling. The tumor suppressor protein p53 is the central hub of DNA damage response signaling and the most frequently deleted or mutated gene in cancers. A Plk3-dependent, p53-independent mechanism for G1/S checkpoint arrest could partially compensate for the loss of p53 function. Therefore, in our first project, we investigated the hypothesis that the combined loss of p53 and Plk3 would be synthetic lethal with cell death resulting from the abolishment of the G1/S checkpoint and the accumulation of unrepaired DNA damage. However, our experimental results did not support our hypothesis and we decided to shift to a new project. The Plk literature also suggests that Plk3 may have additional roles associated with normal cell function. Here, mouse embryonic fibroblasts generated from Plk3 knockout or wildtype mice were compared, to identify alternative functions for Plk3 in addition to its canonical role. Specifically, Plk3 has been reported to associate with key proteins involved in cytoskeletal organization; co-localizing with f-actin and directly phosphorylating ß-tubulin. These and other data suggested a role for Plk3 in regulation of the cytoskeleton and cell morphology. To this end, given the importance of dynamic cytoskeletal rearrangement to cell motility, we analyzed whether Plk3 is involved in cell migration, attachment and/or invasion using Plk3 knockout mouse embryonic fibroblasts.

Committee: Peter Stambrook Ph.D. (Committee Chair); El Mustapha Bahassi Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); Anil Menon Ph.D. (Committee Member); William Miller Ph.D. (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy, The Ohio State University, 2013, Pharmacy

Pancreatic cancer is among the most lethal of all human diseases. With a 5-year survival of less than 6%, there is a dire need for new therapeutic options. Molecular understanding of the disease is vital to overcome barriers to effective treatment. Here we evaluate the expression of noncoding RNAs in patient tissues, cell lines, in vitro model systems and in a genetically engineered mouse model that recapitulates the disease progression in humans. Two different classes of noncoding RNAs were studied, microRNA (miRNA) and transcribed ultraconserved elements (T-UCRs). Corroborating previously published data, we identify a miRNA cluster (miR-216a, miR-216b, and miR-217) that is consistently down-regulated in the different models of the disease. These miRNAs, are pancreas enriched, and appear to be acinar cell specific. A large percentage of the 482 known T-UCRs had increased expression in pancreatic cancer tissues, cell lines and in an in vitro system that mimics the pancreatic desmoplastic reaction. We hypothesized that loss of miR-216/-217 expression might be a crucial step in regulating acinar-to-ductal metaplasia (ADM). Although historically believed to originate in the ductal cells, compounding evidence now has shifted the paradigm to an acinar cell origin of the malignancy. miR-216/-217 expression decreases using an in vitro model that mimics the ADM process. Adenovirus overexpression of miR-217 during the in vitro ADM attenuates acinar to epithelial transdifferentiation. We present evidence of indirect regulation of the epithelial cell marker CDH1 by miR-217, suggesting that miR-217 could regulate ADM though suppression of ductal markers in acinar cells. We also considered that the miRNA cluster may target master regulatory transcription factors. We demonstrate that miR-217 targets REST, a major transcriptional suppressor. We show REST protein to be up-regulated in the patient protein analyzed and that REST target genes are up-regulated in gene profiling (open full item for complete abstract)

Committee: Thomas Schmittgen (Advisor); Mamuka Kvaratskhelia (Committee Member); Kalpana Goshal (Committee Member); Mark Bloomston (Committee Member) Subjects: Molecular Biology; Oncology; Pharmaceuticals

MS, University of Cincinnati, 2003, Medicine : Environmental Health Sciences

PBBs are known to cause negative health effects in humans and in animals. It is unknown if these effects are mediated by an underlying genetic component of the AHR phenotype. In this study, we examined the developmental effects of both coplanar and noncoplanar PBBs on mouse lines having both high and low affinity AHR phenotype with C57BL/6J background. We determined a coplanar PBB high-affinity AHR-dependent neonatal lethality and immunotoxicity, coupled with the induction of CYP1A1 in embryonic liver and brain at gestational day 18.

Committee: Dr. Daniel W. Nebert (Advisor) Subjects: Health Sciences, Toxicology

Doctor of Philosophy, The Ohio State University, 2009, Food Science and Nutrition

High pressure processing (HPP; 100-700 MPa at temperatures < 45°C) and pressure-assisted thermal processing (PATP) (500-700 MPa; 90-120°C) have been used to inactivate pathogenic and spoilage bacteria and produce high quality foods. The objectives of this dissertation were to evaluate the influence of various pressure-temperature combinations on quality, microbial lethality and thermophysical properties of selected foods. Experiments were conducted to investigate the influence of process temperature (95-121°C) at different pressures (0.1, 500-700 MPa) on carrot quality. Results indicated that under comparable process temperatures (up to 105°C), pressure-assisted thermal processing (PATP) retained the carrot quality attributes such as color and carotene content better than thermal processing (TP). However, process and preprocess thermal history greatly influenced carrot textural change. Pressure protective effects on product hardness at elevated temperatures (110-121°C) were less pronounced. Subsequently, experiments were conducted to evaluate the role of pressure during sequential (pressure pre-treatment at ambient temperature followed by TP) or simultaneous (PATP) treatment in preserving product quality attributes. To learn how different food matrices are influenced by various pressure-heat combinations, experiments were also carried out using carrot, jicama, red radish, zucchini, and apricot. Results showed that TP degraded product texture severely but HPP followed by TP improved texture retention. In comparison to TP alone, PATP better retained texture and color. The beneficial effects of PATP may come from the densification of the tissue due to pressurization or biochemical changes of the pectic substances. Texture retention was product dependent, with jicama being the least influenced among the foods tested. An instrumental based crunchiness index (CI) was developed and validated using sensory data. CI was able to describe textural transformation of various p (open full item for complete abstract)

Committee: V.M. Balasubramaniam PhD (Advisor); Sudhir Sastry PhD (Committee Member); Ahmed Yousef PhD (Committee Member); Luis Rodriguez-Saona PhD (Committee Member) Subjects: Food Science

Doctor of Philosophy (PhD), Ohio University, 2006, Chemistry (Arts and Sciences)

Lacking both alleles of the tumor suppressor gene p53 results in the lethality of a subset of 129 female mice (16%) in early embryogenesis due to exencephaly. In this study we show that novel genetic modifier genes of p53 can exacerbate embryonic abnormalities. Using a mouse model in which CE mice were crossed with the p53-null 129 mice, a subset of p53+/- and -/- male and female embryos died during gestation. Our hypothesis based on the skewed ratio from Mendellian inheritance in the F2 generation is two genetic modifiers of a p53 null allele lead to an increase in embryonic lethality. We have identified a recessive modifier of the p53 (mop1) locus from CE mice on chromosome 11 centromeric to p53, and a dominant modifier of p53 (mop2) locus from 129 mice on chromosome 11 telomeric to p53. Sequencing of candidate genes within these two regions revealed a transverse mutation (321P to 321S) in the Ovca1 gene within the mop2 locus of CE mice. The mutation increased both mRNA and protein levels of Ovca1 in various tissues in CE mice compared to 129 mice. CE primary cells cultured from different tissues proliferated more rapidly than 129 cells. In addition, CE cells were in G1, S, and G2/M phases while 129 cells have a higher percentage in the G1 phase. Transfection of wild-type Ovca1 into CE cells caused a higher G1 phase arrest. OVCA1 protein localized to punctate bodies clustered around the nucleus in CE mice, while in 129 mice OVCA1 was diffused throughout the cell. Immunohistochemical staining showed that p53+/- abnormal embryos have more proliferating cells than normal embryos, but no obvious difference was observed in differentiated neuronal cells and apoptotic cells. p53-/- small embryos have less differentiated neuronal cells and proliferating cells as well as more apoptotic cells than the normal embryos. Normal and abnormal p53+/- F2 embryos both have the Ovca1 mutation. Thus, Ovca1 combined with mop1, genetically modifies embryonic lethality in p53 deficient m (open full item for complete abstract)

Committee: Susan Evans (Advisor) Subjects:

Doctor of Philosophy, Case Western Reserve University, 2013, Biomedical Engineering

Uncontrolled hemorrhage comprises 60-70% of trauma-associated mortality in the absence of other lethal conditions (e.g. damage to central nervous or cardiac system). Immediate intervention is critical to improving chances of survival. While there are several products to control bleeding for external wounds including pressure dressings, tourniquets or topical hemostatic agents there are few, if any, effective treatments that can be administered in the field to help staunch internal bleeding. Intravenous hemostatic nanoparticles that augment blood clotting when administered after trauma have been previously shown to half bleeding times in a femoral artery injury model in rats. The aims of the present study were to: 1) Determine their efficacy in a lethal hemorrhagic liver injury model, 2) determine the impact of targeting ligand concentration on hemostasis, and 3) test them in a clinically relevant porcine model of hemorrhage. Nanoparticle administration (GRGDS-NP1, 40 mg/kg) after lethal liver resection in the rat increased 1-hour survival to 80% compared to 40-47% in controls. Targeting ligand conjugation was then increased 100-fold (GRGDS-NP100), and a dosing study performed. GRGDS-NP100 hemostatic nanoparticles (2.5 mg/kg) were efficacious at doses 8-fold lower than GRGDS-NP1, and increased 1-hour survival to 92%. In vitro analysis using rotational thromboelastometry (ROTEM) confirmed the increased dose-sensitivity of GRGDS-NP100 and laid the foundation for methods to determine optimal ligand concentration parameters. Hemostatic nanoparticles were then tested in a clinically relevant porcine liver injury model, which elucidated an unexpected adverse reaction, comprised of a massive hemorrhagic response. A naive (uninjured) porcine model was then employed. These experiments revealed an adverse reaction consistent with complement activation related pseudoallergy (CARPA), which could be mediated by tuning nanoparticles' zeta potential. Neutralizing the nanopa (open full item for complete abstract)

Committee: Erin Lavik Sc.D. (Committee Chair); Jeffrey Ustin M.D. (Committee Member); Horst von Recum Ph.D. (Committee Member); Robert Miller Ph.D. (Committee Member) Subjects: Biomedical Engineering; Biomedical Research; Medicine