Master of Science in Pharmaceutical Science (MSP), University of Toledo, 2016, Pharmaceutical Sciences (Industrial Pharmacy)

Abstract: Purpose: The current work intends to study the activity of tanshinone IIA on secretion of VEGF and expression of HIF-1a in human retinal pigment epithelial cells (ARPE-19 cells) under hypoxic condition. Methods: The cytotoxicity of Tanshinone IIA was tested in ARPE-19 and bovine corneal endothelial cells by MTT assay. ARPE-19 cells were incubated with different concentrations of cobalt chloride (100, 150, and 200 µM) for 12 hours and levels of expressed HIF-1a and secreted VEGF were quantified through Western blot and ELISA, respectively. Further, ARPE-19 cells were pretreated for one hour with different concentrations of Tanshinone IIA (5, 10, 15 and 18 µM). After one hour, the cells were subjected to hypoxic condition using 150 µM cobalt chloride for 12 hours in the presence and absence of Tanshinone IIA. The cells were then harvested and the secreted VEGF and expressed HIF-1a was studied. Results: Tanshinone IIA at concentrations 5, 10, 15 and 18 µM did not show cytotoxicity in both ARPE-19 and bovine corneal endothelial cells. Chemical hypoxia induced by cobalt chloride caused a significant increase in VEGF level in a dose dependent manner and HIF-1a expression peaked at 150 µM. Based on the data, cobalt chloride concentration was maintained at 150 µM for further studies. Tanshinone IIA decreased the level of HIF-1a and VEGF secretion in a dose dependent manner under hypoxic condition. Conclusion: This study is the first report on the effects of Tanshinone IIA on VEGF secretion and HIF-1a level in ARPE-19 cells. Tanshinone IIA could be a potential new treatment option for wet AMD.

Committee: Sai Boddu (Advisor); Zahoor Shah (Committee Member); Jerry Nesamony (Committee Member) Subjects: Alternative Medicine; Pharmacy Sciences

PhD, University of Cincinnati, 2005, Medicine : Cell and Molecular Biology

Lung development is analogous to a well orchestrated symphony, and depends on successful interactions between various cellular components within the lung. The pulmonary vasculature is essential for normal lung development and is a key member of the lung development ensemble. Impairment of pulmonary vascular development is the hallmark of neonatal diseases such as respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). Members of the VEGF family of growth factors and receptors are critical mediators of vascular development. Two members of the VEGF family, VEGF-A and VEGF-D are highly expressed in developing lung. VEGF-A is known to play a role in pulmonary vascular development, but its role in vessel specification in the developing lung is poorly understood. In addition, we hypothesize that VEGF-D plays a role in pulmonary lymphatic development based on its high expression in developing lung mesenchyme and its ability to bind the lymphatic receptor VEGFR-3. Through conditional regulation of VEGF-A, we have characterized differential temporal effects of the pulmonary vasculature on lung organogenesis, unique to early (embryonic day E10.5-E12.5), middle (E12.5-E16.5), and late (E16.5-E18.5) phases of development. Induction of VEGF-A in distal lung from E14.5 to E18.5 resulted in disorganization of endothelial ultrastructure, increased endothelial cell proliferation and rearrangement of the established vascular plexus, altering alignment with the epithelium. Remarkably, VEGF-A induction caused a 3.3-fold increase in small lymphatic vessels and a 1.3-fold increase in arteries. This preferential induction indicates that VEGF-A influences lymphatic specification in late stages of lung development. In addition, we have characterized differential time-sensitive effects of VEGF-A on other aspects of embryonic lung development. VEGF induction during the perinatal period disrupted terminal branching morphogenesis, inhibited formation of primitive alveolar sept (open full item for complete abstract)

Committee: Ann Akeson (Advisor) Subjects:

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

Age-related macular degeneration (AMD) is one of the most common ocular diseases, typically affecting people aged over sixty. Millions of people are diagnosed with AMD, which is projected to continuously increase with growing aging populations. As a retinal disorder, the pathogenesis of AMD is complicated because of the heavy involvement of genetic and environmental factors. However, it is widely believed that reactive oxygen species (ROS) generated during the biological aging act as a driving force of the development of AMD. The high level of oxidative species and associated functional loss of mitochondria in the retina are believed to result in cell death and para-inflammation. Consequently, vascular endothelial growth factors (VEGF) are secreted and induce choroidal neovascularization (CNV). The newly formed blood vessels near the central retina can further cause irreversible retinal damage and permanent vision loss once they bleed and scar. To halt the progression of AMD, the monthly intravitreal injection of anti-VEGF is the current gold standard treatment, which relies on deactivating free VEGF in the eye. However, frequent invasive injections are associated with potential complications and low patient compliance. Previous research has focused on developing micro-sized and nano-sized drug delivery devices composed of poly(lactic-co-glycolic acid) (PLGA) or polydimethylsiloxane (PDMS). Unfortunately, the aforementioned devices could not release protein therapeutics sustainably and were limited by the release duration of four months, which reduces potential treatment efficacy. To address these problems, we have developed three drug delivery systems in the forms of implant, microparticle, and nanoparticle, respectively. The three devices are polymer-based and biodegradable. They demonstrate several advantages over previously reported devices: i) extending release duration by improving the drug loading rate and reducing the initial burst release; ii) protecting (open full item for complete abstract)

Committee: Katelyn Swindle-Reilly Dr. (Advisor); Andre Palmer Dr. (Committee Member); Jessica Winter Dr. (Committee Member); Prabhakara Nagareddy Dr. (Committee Member) Subjects: Chemical Engineering

PhD, University of Cincinnati, 2020, Medicine: Molecular and Developmental Biology

The formation of a functional vascular system is required for proper embryonic development in all vertebrates. Being one of the first organ systems to form during embryogenesis, ¬the cardiovascular system, comprising the heart, arteries, veins and capillaries, transports nutrients, oxygen, metabolites, waste, immune cells and hormones throughout the body. Thus, the formation of organ systems during development and subsequent maintenance throughout life is reliant upon a normal functional vascular system. This dissertation focuses on the molecular mechanisms that govern vascular development in zebrafish embryos. We utilize zebrafish embryos due to their rapid development, optical transparency and high amenability to genetic manipulation.

Committee: Saulius Sumanas Ph.D. (Committee Chair); Elisa Boscolo Ph.D. (Committee Member); Brian Gebelein Ph.D. (Committee Member); Joshua Waxman Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member) Subjects: Cognitive Psychology; Developmental Biology

Doctor of Philosophy, University of Toledo, 0, Biomedical Engineering

Cellulose and chitosan (CS) are two of the most abundant biopolymers in nature. CS is highly explored in tissue engineering applications mainly because of its unique chemical structure of possessing cationic amine groups. This bestows it with the ability to have specific interaction with extracellular matrix components such as glycosaminoglycan and proteoglycans making it even more attractive in bone regeneration. This study focused on the application of these biopolymers towards the development of microparticles and hence robust injectable bone scaffolds. The current strategy of applying MPs for bone regeneration involves their direct injection to the defect site. This technique most of the time, however, leads to the improper localization of those MPs. This becomes more problematic when these MPs are loaded with therapeutic agents or growth factors as the intended localized delivery of those agents could not be achieved. Another strategy that has been implemented for the use of MPs in bone regeneration involves the development of preformed solid scaffolds created mostly through chemical agglomeration or sintering techniques. This technique, however, takes away the injectable property of MPs making them like the conventional solid scaffolds. To address this issue related to the applications of the MPs and to develop effective therapeutics releasing scaffolds, the concept of embedding microparticles into a thermosensitive gel was used. This MPs-gel composite was developed to stay as a liquid at a lower temperature enabling an easy injection, but it undergoes a temperature-induced transition to gel at physiological temperature (37 oC). This gel enables the proper localization of MPs at the target defect site enabling efficient therapeutic effects. The blend of methylcellulose (MC) and alginate (Alg) was used to develop thermosensitive gel. The gelation occurs primarily due to the physical interaction among the hydrophobic chains of MC and is further strengthened by (open full item for complete abstract)

Committee: A. Champa Jayasuriya (Committee Chair); Arunan Nadarajah (Committee Member); Sarit Bhaduri (Committee Member); Ahalapitiya H. Jayatissa (Committee Member); Jiayong Liu (Committee Member) Subjects: Biomedical Engineering

PhD, University of Cincinnati, 2018, Medicine: Molecular and Developmental Biology

            The cardiovascular system is one of the earliest organ systems to form in the developing embryo. Development of blood vessels is a strictly regulated process that is crucial for embryonic development as well as for organ functionality throughout the life of an organism. The circulatory system is necessary for the transportation of fluid, oxygen, metabolites, waste products, hormones, and immune cells and cytokines throughout the body. These functions cannot be carried out without proper formation of blood and lymphatic vessels. The focus of this dissertation is increasing the understanding of the mechanisms and pathways necessary for blood and lymphatic vessel development.             Previous studies have implicated mixed models of de novo vasculogenesis and angiogenesis in the development of blood vessels in organs. These studies, carried out in mouse and chick embryos, lack the ability to track vessel development in live embryos and therefore identify the source of cells forming these vessels. Here we identify a novel mechanism of organ vascularization in which endothelial cells in existing vessels migrate to form new vessels. We utilize zebrafish due to the rapid development, transparent embryos, and amenability to genetic manipulation to study vascularization of the embryonic gut tube. Vegfaa expression in the presumptive endoderm drives ventral migration of endothelial cells out of the posterior cardinal vein (PCV), which then coalesce to form the sub-intestinal vein (SIV) and supra-intestinal artery (SIA). These results represent a novel mechanism of organs vascularization that is likely to occur in other organs and in other vertebrate organisms.             We also investigate the role of ETS transcription factor, Etv2, in lymphatic development/lymphangiogenesis. Prior studies have demonstrated the requirement of Vegfc/Vegfr3 signaling in lymphatic development, however questions about the process remain, including how this signaling is regulate (open full item for complete abstract)

Committee: Saulius Sumanas Ph.D. (Committee Chair); Elisa Boscolo Ph.D. (Committee Member); Tony J. DeFalco Ph.D. (Committee Member); Joshua Waxman Ph.D. (Committee Member); Chunyue Yin Ph.D. (Committee Member) Subjects: Developmental Biology

BS, Kent State University, 2016, College of Arts and Sciences / Department of Biological Sciences

Vascular endothelial growth factor (VEGF) is necessary for angiogenesis and tumor growth, but VEGF splice isoforms have distinct characteristics. The smallest isoform, VEGF121 is diffusible, allowing for more distant angiogenic signaling. VEGF189 is found within the cell and the adjacent extracellular matrix, while VEGF165 is the most abundant and somewhat diffusible (although less diffusible than VEGF 121). In low level oxygen (hypoxia) induction of VEGF expression depends on transcription factors such as the hypoxia factor, HIF1a and Wilms' Tumor 1 (WT1). However, the mechanisms regulating alternative splicing and variation in VEGF isoform levels in hypoxia is not clear. Based on literature review, both hypoxia and WT1 have been shown to regulate serine arginine rich protein kinase 1 (SRPK1), a kinase that phosphorylates splicing factors. We asked whether this HIF1a /WT1¿ SRPK1 pathway would control alternative splicing of VEGF isoforms in leukemia cells. Another study has suggested that WT1 down-regulates the VEGF 121 isoform. We hypothesized that in leukemia cells, both WT1 and VEGF levels would increase under hypoxic conditions, but that levels of the VEGF 165 splice form would be enhanced greater than VEGF121 levels (given elevated levels of the repressor, WT1). Our findings showed that hypoxia increased expression of WT1, total VEGF and VEGF 165, but not VEGF 121 in myeloid and lymphoid derived leukemia cells. However, changes in VEGF isoform levels were cell specific. These results shed light on the hypoxia pathway and indicate that effective anti-angiogenic therapy will need to be tailored for each type of leukemia.

Committee: Gail Fraizer Ph.D (Advisor); Steven Kuerbitz MD (Committee Member); Gary Koski Ph.D (Committee Member); Alexander Seed Ph.D (Committee Member) Subjects: Biology; Biomedical Research

Master of Science (MS), Wright State University, 2016, Microbiology and Immunology

Chronic wounds have become a major clinical and economic burden in our society. New approaches that accelerate wound healing are desperately needed. Angiogenesis and vascularization play a critical role in healing. One of the essential angiogenic factors that promote the formation of vascular beds is vascular endothelial growth factor (VEGF). Moreover, adipose-derived stem cells (ASC), through their regeneration and differentiation properties, may promote healing when transplanted into a wound bed. I have proposed to develop a novel organotypic wound model to facilitate the study of wound healing process. I hypothesize that administration of genetically-modified ASC secreting VEGF via an adeno-associated viral vector serotype (AAV5) directly into the wound site will accelerate rejuvenation of ischemic tissue. To test this hypothesis, the VEGF-165 gene was synthesized (IDT) and cloned (Clontech) into two AAV5 plasmids, pAAV5-CMV-GFP and pAAV5-CAG-GFP and transfected in HEK293, Cos-7 and primary ASC cells for gene expression. Based on the higher transfection efficiency and VEGF secretion in pAAV5-CAG-VEGF-GFP, AAV5-CAG-VEGF-GFP virus was produced by the University of Iowa using the Baculovirus system. I used AAV5-CAG-VEGF-GFP and AAV5-CMV-GFP (control virus) to transduce the ASC. Wound healing assays in vitro measured by scratch assay were performed on monolayers of ASC and were observed for healing. In vivo wound healing assay was performed on 2mm or 3mm organotypic wound models by treating them with ASC-AAV5-CAG-VEGF-GFP, ASC-AAV5-CMV-GFP, non-transduced ASC or ASC media. In vitro wound healing assays showed complete closure of non-transduced ASC in three days whereas the ASC-AAV5-CAG-VEGF-GFP healed within two days indicating the efficiency of VEGF in accelerating closure. In vivo wound healing assays on organotypic wounds showed some closure in wounds treated with ASC compared to control wounds treated with media alone. However wounds treated with ASC-AAV5-CAG- (open full item for complete abstract)

Committee: Katherine J.D.A. Excoffon Ph.D. (Advisor); R. Michael Johnson M.D., FACS (Committee Member); Barbara Hull Ph.D. (Committee Member) Subjects: Biology

PHD, Kent State University, 2016, College of Arts and Sciences / Department of Biological Sciences

An important aspect of leukemia therapy is identification of genes that are abnormally expressed in patients. This helps determine prognostic groups so that treatment regimens and relapse probabilities in patients can be efficiently determined. Additionally, knowledge of gene expression patterns also contributes to our understanding of molecular mechanisms underlying leukemogeneis, which in turn, assists in the development of effective therapies. Thus, here we evaluated using quantitative real time PCR (qPCR) analysis, the expression of Wilms tumor 1 (WT1), Vascular Endothelial Growth Factor (VEGF) and Cyclin A1 (CCNA1) genes in bone marrow samples from pediatric Acute Lymphocytic Leukemia (ALL) and Acute Promyelocytic Leukemia (AML-M3) patients and asked whether WT1 regulated VEGF and CCNA1 expression. We found that compared to the expression in non-leukemia control bone marrow samples (NBM), all of the AML-M3 samples expressed high levels of WT1 and CCNA1 mRNA while VEGF levels varied, although were moderately high in majority. In ALL, majority of the samples expressed WT1 and CCNA1 levels comparable to NBM while VEGF levels were significantly lower. WT1 mutations in pediatric acute leukemias were not identified but a single nucleotide polymorphism (SNP rs16754) was observed in ~15% of samples. A coordinate expression pattern for WT1 and VEGF was observed when leukemia cell lines were grown under hypoxia conditions. A direct relationship was also seen when WT1 expressing plasmids were transiently transfected into K562 cells and CCNA1 expression analyzed. The effect of WT1 expression on the VEGF and CCNA1 mRNA levels could be mediated through transcriptional regulation as WT1 binding sites were identified in the promoter region of these genes. Binding of WT1 to the VEGF gene promoter in the chromatin of LNCaP cells has been previously demonstrated. Similarly, here we show WT1 binding to the CCNA1 promoter in chromatin of K562 cells. Luciferase reporter assays als (open full item for complete abstract)

Committee: Gail Fraizer (Committee Chair); Jennifer McDonough (Committee Member); Gary Koski (Committee Member); Steven Kuerbitz (Committee Member); Hamza Balci (Committee Member) Subjects: Cellular Biology

Master of Science, The Ohio State University, 2016, Vision Science

Purpose: Age-related macular degeneration is the leading cause of permanent blindness in developed countries.1-3 Higher levels of depression and disability and lower quality of life has been measured in this population.4-7 The purpose of this study was to measure perceived stress and it relationship to visual function in patients with AMD using the Perceived Stress Scale. Methods: Patients with AMD were enrolled during visits to a retinal specialist for evaluation of the need for treatment with intraocular anti-VEGF injection. Visual acuity was assessed using a backlit ETDRS chart and by-letter scoring. Four surveys were then administered to the patient. They included the Perceived Stress Scale (PSS), Impact of Vision Impairment survey (IVI), and the ENRICHD Social Support Inventory (ESSI), and the Center for Epidemiologic Studies Depression Scale (CES-D).Participants completed a large-print paper version of the surveys when possible, and the survey was read aloud to patients who were unable to complete it for visual reasons. Rasch analysis of PSS responses was performed using Winsteps with the Andrich rating scale model. Item fit to the Rasch model was assessed using infit iii mean square statistics. Response category functioning was assessed using category probability curves, and survey targeting for the study population was assessed by comparing Rasch item measures with person measures for all participants. Results: We had a total of 79 participants in this particular part of the study. The mean age was 81.19 with 48.1% bring female and 51.9% male. Rasch analysis showed that the PSS was valid to use in this population and measured the desired construct, stress. During our analysis we found that visual acuity in the better and worse eye were correlated with age. Age was also positively correlated with IVI results. Visual acuity in the better eye was correlated to visual acuity in the worse eye. PSS was not correlated with visual acuity in the worse or bette (open full item for complete abstract)

Committee: Bradley Dougherty O.D., PhD (Advisor) Subjects: Ophthalmology; Psychology

MS, Kent State University, 2015, College of Arts and Sciences / School of Biomedical Sciences

Vascular Endothelial Growth Factor-a (VEGF-a) is a key factor in promoting leukemia growth and progression. VEGF-a can be alternatively spliced generating several isoforms with distinct biological characteristics. One such isoform, VEGF121, was reported to be significantly high in bone marrow of patients with acute myeloid leukemia and was correlated with poor clinical outcome. However, little is known about the regulation of VEGF-a isoforms especially in leukemia. Recently, WT1 was found to regulate the process of blood cell early development (hematopoiesis) by controlling the ratio of VEGF121 to VEGF165. This suggests a potentially significant contribution of WT1 to hematologic malignancies (leukemia) by dysregulating VEGF-a isoform expression. The goal of our research is to determine the effect of potential regulators on the expression of VEGF-a isoforms in leukemia. Using human leukemia cells, we studied the effect of WT1, as an inhibitor of SRPK transcription and, a specific inhibitor of the kinase function of SRPK that is required for multiple splicing factors. We also examined the effect of Epidermal growth factor, as a known activator of SRPK on VEGF-a major isoforms. Our hypothesis is that in leukemia cells, WT1 over-expression, splicing kinase inhibition, and EGF induction would change expression of VEGF isoforms. Using isoform specific quantitative real time PCR (qRTPCR) assays we measured VEGF-a isoforms (VEGF121 and VEGF165) in leukemia cells. We have observed that WT1 reduced the VEGF121 but did not change VEGF165. However, our results showed that WT1-suppression of VEGF121 was not mediated through SRPK as described previously in other systems. On the other hand, SRPIN340 treatment (a specific kinase inhibitor of SRPKs) reduced expression of both isoforms. Consistent with it`s effect in solid tumors, EGF induction resulted in a significant up-regulation of both VEGF 121 and 165 isoforms in leukemia cells. Interestingly, SRPK inhibition was able to bl (open full item for complete abstract)

Committee: Gail Fraizer (Advisor); Gary Koski (Committee Member); Steven Kuerbitz (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology

BS, Kent State University, 2015, College of Arts and Sciences / Department of Biological Sciences

Hypoxia is known to be a characteristic of the inner tumor environment. In order to restore oxygen to the tumor, cancer cells will increase production of vascular endothelial growth factor (VEGF) which is necessary for angiogenesis and, therefore, tumor growth. VEGF is present in different splice forms, with the predominant in cancer cells being VEGF121 and VEGF165. These splice forms have distinct characteristics which allow for different patterns of blood vessel formation within the tumor. VEGF121 lacks the heparin binding domain that is present in VEGF165, and thus is capable of diffusing away from the site of origin through the extracellular matrix and recruiting larger, pre-existing blood vessels. VEGF165, however, can bind to the heparin in the extracellular matrix and is sequestered locally, allowing it to create more internal microvessels than VEGF121. VEGF121 has also been implicated in more aggressive, metastatic cancer types. We hypothesized that VEGF expression in prostate cancer cells would increase in response to hypoxic conditions, favoring the VEGF121 isoform. We examined VEGF splice form mRNA production in both LNCaP and PC3 cells, which are prostate cancer cell lines with different metastatic potentials. Our findings suggest that hypoxia does cause an increase in both splice forms relative to expression under normoxic conditions, with a greater increase in fold change observed for VEGF121. These results of elevated VEGF121 indicate an increase in the potential for angiogenic and metastatic prostate cancer growth. Overall this work highlights the role of the hypoxic tumor microenvironment in regulating the functionally distinct VEGF isoforms in cancer cells.

Committee: Gail Fraizer PhD (Advisor); Paul Sampson PhD (Committee Member); Helen Piontkivska PhD (Committee Member); Mary Ann Raghanti PhD (Committee Member) Subjects: Biology; Biomedical Research

PhD, University of Cincinnati, 2015, Medicine: Molecular and Developmental Biology

Proper blood vessel development is crucial not only in embryonic development, but also in maintaining proper organ functionality throughout adulthood. The heart and blood vessels transport oxygen, metabolites, waste products, and immune response signals to all tissues of the body. Without proper development of arteries and veins, these functions would not be possible. This dissertation focuses on understanding how these two major vessel types are developed. We investigate the roles of two proteins, Stabilin-2 (Stab2) and Ets Varient 2 (Etv2), and how they function in relation to Vascular endothelial growth factor (Vegf) signaling to regulate vascular endothelial cell differentiation and arterial venous specification.

Committee: Saulius Sumanas Ph.D. (Committee Chair); Richard Lang Ph.D. (Committee Member); Jerry Lingrel Ph.D. (Committee Member); Joshua Waxman Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member) Subjects: Developmental Biology

Doctor of Philosophy, Case Western Reserve University, 2015, Molecular Medicine

CD36 is a multi-ligand scavenger receptor present in many cells including microvascular endothelial cells (MVEC) in which it induces anti-angiogenic signaling via its cognate protein ligand thrombospondin-1 (TSP-1). Cell-derived microparticles (MP), less than or equal to 1µm vesicles shed from many cell types during activation or apoptosis bind CD36 via phosphatidylserine (PS) exposed on their surface. We hypothesized that CD36-MP interactions on MVEC inhibits angiogenesis. We found that MP generated in vitro from different cellular sources (THP1, human umbilical vein endothelial cells (HUVEC), MVEC and RBCs) and endogenous circulating human MP inhibit angiogenic activities of MVEC using in vitro and in vivo migration, and in vitro tube-formation assays. Experiments on different passages and batches of MVEC with different levels of CD36 expression, murine CD36 transfection in HUVEC that do not express CD36 and cd36-/- mice showed a CD36 dependent inhibition of MVEC migration by MP. Studies with annexin-V showed a PS dependent inhibitory effect. Mechanistically, MP-mediated inhibition was via induction of reactive oxygen species (ROS) in a NADPH oxidase and Fyn kinase dependent manner. CD36 forms a complex with vascular endothelial growth factor receptor-2 (VEGFR2) in MVEC and inhibits VEGF signaling. Src homology 2 domain containing protein tyrosine phosphatase (SHP)-2 is constitutively associated with the VEGFR2-CD36 signaling complex and TSP-1-CD36 interaction induces SHP-1 association with the complex. Mechanism(s) of TSP-1-mediated inhibition of VEGFR2 signaling was not known. We hypothesized that CD36-TSP-1 interaction recruits SHP-1 to VEGFR2-CD36 complex, to dephosphorylate VEGFR2 and attenuates VEGF signaling. Immunohistochemistry of MVEC exposed to a recombinant protein containing the CD36 binding domain of thrombospondin-1 (known as the TSR domain) induced association of SHP-1 with CD36. Silencing CD36 expression in MVEC by siRNA showed that TS (open full item for complete abstract)

Committee: Roy Silverstein MD (Advisor); Keith McCrae MD (Committee Chair); Tatiana Byzova PhD (Committee Member); Heather Gornik MD (Committee Member); Candece Gladson MD (Committee Member) Subjects: Biology; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2014, Chemistry

Cancer, a major health issue in the United States, is the second leading cause of death after disease of heart. The formation of new blood vessels, known as angiogenesis, which new capillaries sprout from the existing vessels, is believed to promote the tumor growth. Due to the dependence of tumor progression on angiogenesis, it has been an interesting field in the search of antiangiogenesis agents which stop the progression of tumor formation and also different types of receptors accounting for angiogenesis by the ever-evolving positron emission tomography (PET) technology. Phospholipids oxidation in biological systems contributes to diverse pathological processes and is also believed to introduce age-related diseases. Oxidative cleavage of phospholipids containing docosahexaenoic acid (DHA) yields the reactive electrophilic species such as 4-hydroxy-7-oxohept-enoates (HOHA) which would later on covalently bond to e-amino group of protein lysine residue and turn into the 2-(¿-carboxyethyl)pyrrole (CEP) protein adducts. The CEP adducts is found to be associated with age-related macular degeneration (AMD) by triggering the angiogenesis in the retina. Recent study also revealed the novel molecular pattern between CEP adducts and Toll-like receptor 2 (TLR2) which leads to the angiogenesis response. And it provides an alternative therapeutic target especially in cases cancers resistant to the conventional anti-VEGF therapy. Therefore it is necessary to synthesize the gamma radioactive isotope labeled CEP adducts and apply them for the targeting of TLR2 with PET imaging technique. A convenient synthesis of putative fluorine-18-labeled-CEP was achieved with satisfied yields and the fluorination condition was optimized as well because limited half-life of fluorine-18 (109.7min) required the reaction to be accomplished in a short time-frame. Meanwhile in order to overcome the limited stability of CEP adducts which prevents its long-term usage as standards in immunosorbent (open full item for complete abstract)

Committee: Robert Salomon (Advisor); Irene Lee (Committee Chair); Gregory Tochtrop (Committee Member); Rajesh Viswanathan (Committee Member); Yanming Wang (Committee Member) Subjects: Analytical Chemistry; Biochemistry; Organic Chemistry

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

Epidermal keratinocytes, the predominant cell type in the epidermis, play a crucial role in two processes in the skin: skin carcinogenesis and cutaneous wound healing. Non-melanoma skin cancer (NMSC) is the most prevalent type of cancer, with 3.5 million cases diagnosed each year in the US. These cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are primarily caused by exposure to ultraviolet (UV) light from the sun. Wound healing is a key process in many aspects of medicine. In addition to injury and trauma, millions of surgeries are performed each year. Chronic wounds, which do not heal properly, can lead to hospitalizations, amputations, death and affect 6.5 million patients every year at an estimated cost of $12 billion dollars. Therefore, it is critical to understand how keratinocytes function in both of these processes. Angiogenesis, the growth and expansion of new blood vessels, occurs during both NMSC and wound healing. Vascular endothelial growth factor (VEGF) promotes angiogenesis by causing the proliferation, migration and survival of vascular endothelial cells. VEGF is produced by the skin in response to UV and promotes NMSC indirectly through the induction of angiogenesis. Additionally, wounds contain high levels of VEGF. VEGF receptor 1 (VEGFR-1) has now been identified on epidermal keratinocytes, suggesting that VEGF can affect keratinocytes directly. Therefore, we hypothesize that VEGF may influence wound healing and skin carcinogenesis by directly affecting keratinocytes via VEGFR-1. To test this, a unique conditional knockout mouse with VEGFR-1-deficient keratinocytes (cKO) was developed and was utilized in acute and chronic UV-induced skin carcinogenesis studies as well as wound healing studies. Immunohistochemical analysis of human and murine NMSC samples revealed that VEGFR-1 is highly expressed in skin tumors. Furthermore, in vitro studies indicated that keratinocyte VEGF and VEGFR-1 expression is regulated (open full item for complete abstract)

Committee: Traci Wilgus PhD (Advisor); Tatiana Oberyszyn PhD (Committee Member); Gregory Lesinski PhD (Committee Member); Amanda Toland PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, The Ohio State University, 2011, Microbiology

Her-2/neu (ErbB2) is a member of the epidermal growth factor family of receptors and is overexpressed in about 30% of breast cancers. Targeting this receptor is a very attractive strategy for antitumor therapy using both peptides and monoclonal antibodies. Trastuzumab and Pertuzumab are humanized monoclonal antibodies that both target the extracellular domain of the Her-2 receptor. Great limitations still exists with these treatments due to their high cost and limited duration of action, thereby necessitating repeated administration of the drugs. The overexpression of Her-2 leads to overexpression of another protein known as vascular endothelial growth factor (VEGF) which turns on the angiogenic switch aimed at causing increased blood flow and oxygen to the tumors. Without an increase in blood flow, the tumors cannot increase in size, hence angiogenesis is required for tumor growth and metastasis. VEGF is known to interact with one of its receptors VEGFR-2 before it can stimulate angiogenesis. The disruption of this interaction is considered another attractive target for cancer therapy and monoclonal antibodies like Bevacizumab have been developed to prevent this interaction. This work is mainly centered on the use of peptides for cancer treatment that are cost effective, specific and non-toxic to target both Her-2 and VEGF. Treatment with conformational B-cell epitopes affords the possibility of generating an enduring immune response and eliciting protein reactive high affinity peptide antibodies. We have designed conformational peptides of the pertuzumab epitope and also peptides that mimic the VEGF binding sites. With the use of in vitro assays and two in vivo tumor models (transgenic and transplantable), we have shown that combination treatment aimed at targeting the HER-2/neu and VEGF signaling pathways produce additive effects with no problems of cardiotoxicity. We also showed that combining these peptide mimics with standard chemotherapy produces increase su (open full item for complete abstract)

Committee: PRAVIN KAUMAYA PhD (Advisor); JESSE KWIEK PhD (Committee Member); NICANOR MOLDOVAN PhD (Committee Member); MARSHALL WILLIAMS PhD (Committee Member) Subjects: Microbiology

Doctor of Philosophy, The Ohio State University, 2010, Pharmacy

Cancer is the leading cause of death worldwide. Despite advances in tumor biology and novel cancer therapies, cancer remains a global health problem. It is therefore imperative to search for new ways to improve cancer therapy and to further investigate tumor biology to identify molecular mechanisms that can be exploited to develop new therapies. Drug delivery to tumors is necessary for chemotherapy to be effective. Tumor vasculature consists of abnormal vessels which limit drug perfusion into tumor tissues. We hypothesized that targeting integrin receptors on endothelial cells with synthetic disintegrins would increase tumor vascular permeability and improve anti-cancer effects of cyclophosphamide. We synthesized disintegrins by conjugating integrin-binding peptides containing arginine-glycine-aspartate to ends of 3.4, 6 or 10 kDa polyethylene glycol polymers. The largest synthetic disintegrin, G10, inhibited endothelial cell attachment in vitro. G10 also increased tumor vascular permeability of EMT6 tumors to Evans Blue dye, which binds albumin, compared to its control. A single intratumoral dose of G10 alone or in combination with a single systemic dose of cyclophosphamide reduced tumor growth compared to respective controls. These data indicate that a divalent integrin-targeting agent of sufficient size can increase tumor vascular permeability and improve effectiveness of a chemotherapeutic agent. We also examined effects of the peptidyl-proline isomerase (PPIase), Protein Never in Mitosis Gene A Interacting-1 (PIN1), on tumor growth. PIN1 is the only PPIase that specifically recognizes and isomerizes the prolyl bond between cis and trans of phosphorylated Ser/Thr-Pro motifs. PIN1 is overexpressed in many human cancers and is associated with poor prognosis. As expected, stable PIN1 depletion (KD) using lentivirally-delivered shRNA reduced EMT6 mammary carcinoma and Lewis Lung Carcinoma (LLC1) growth in culture. In contrast, EMT6 and LLC1 tumors lacking PIN1 grew (open full item for complete abstract)

Committee: Dale Hoyt Ph.D. (Advisor); Terry Elton Ph.D. (Committee Member); Ching-Shih Chen Ph.D. (Committee Member); Jack Yalowich Ph.D. (Committee Member) Subjects: Molecular Biology; Oncology; Pharmaceuticals; Pharmacology; Pharmacy Sciences

Doctor of Philosophy, The Ohio State University, 2008, Microbiology

Rationally designed peptide mimics that can substitute for protein biological activity are good candidates for a new generation of therapeutic drugs. Peptides have higher tissue penetration, relatively low cost production and can be easily modified for improved bioavailability. The formation of new blood vessels, angiogenesis, is required for cancer tumor growth and metastasis. Since vascular endothelium growth factor (VEGF) and VEGFR-2 is the most significant interaction for angiogenesis activation; we designed conformational peptides that can mimic the VEGF binding site. The inhibitory potential of peptide mimic was evaluated in several angiogenesis model assays such as HUVEC proliferation, tube formation in Matrigel and migration. The VEGF conformational peptide mimic, VEGF-P3(CYC), was synthesized with two extra cysteine residues at positions 80 and 92 , which upon cyclization constrain the peptide in a loop conformation, in order to better mimic the anti-parallel twisted structure in the VEGF native protein structure. This constrained peptide demonstrated the best angiogenic inhibitory effects in in vitro assays and significantly delayed tumor growth in VEGF-/+/Neu2-5-/+ transgenic animal model when compared to VEGF-P3(CYC), non-cyclic peptide, and VEGF 102-122, natural sequence peptide. Peptides in which the amino acid sequence and the chirality are reversed are termed retro-inverso peptide. We synthesized and tested the angiogenesis inhibitory effects of retro-inverso peptide VEGF-RI-P4(CYC) which demonstrated some degree of anti-angiogenesis effects in vitro. Chimeric VEGF peptide mimics incorporating promiscuous T-cell epitope were synthesized and used to elicit antibodies in rabbits. The conformational peptide VEGF-P3(CYC) showed to be the best mimitope of VEGF, generating antibodies with tighter affinity to VEGF. Preliminary results showed that the anti-MVF-VEGF 102-122 delayed tumor growth in VEGF-/+/Neu2-5-/+ transgenic mice. However, anti-MVF-VEGF-P3(C (open full item for complete abstract)

Committee: Pravin Kaumaya PhD (Advisor); Neil Baker PhD (Committee Member); Clay Marsh MD (Committee Member); Marshall Williams PhD (Committee Member) Subjects: Microbiology

Doctor of Philosophy, The Ohio State University, 2007, Integrated Biomedical Science

Head and neck squamous cell carcinoma (HNSCC) is the most common form of oral cancer. Current standard therapy involves surgical resection of the primary tumor followed by radiotherapy and/or chemotherapy. Each year 3-7% patients succumb to a second primary tumor at. Clinical evidence supports a contribution of sustained inflammation, reactive species (RS) and their sequelae in carcinogenesis. Studies in Chapter 2 focused on elucidating mechanisms by which RS facilitate HNSCC tumorigenesis. The purpose of this investigation was to assess the effects of reactive oxygen and nitrogen species on selected parameters of the HNSCC tumorigenic phenotype i.e. nuclear factor (NF)kappaB activation, sustained cell proliferation, and production of proinflammatory and proangiogenic proteins. Results showed that reactive oxygen (H2O2) and for the first time reactive nitrogen (SIN1) activated NFkappaB in HNSCC cells. Notably, TNF, H2O2, NOC18 and SIN1 increased IL-8, VEGF and EGFR protein levels. Our results indicate RS mediate HNSCC development by activation of NFkappaB followed by increased growth factor production. N-acetyl-cysteine (NAC) has previously been identified as a potentially effective chemopreventative agent. As a precursor to L-cysteine, the rate limiting reactant in glutathione (GSH) synthesis, it possesses ability to increase cellular stores of the antioxidant GSH, and inhibit matrix metalloproteinases (MMPs). MMPs are a family of enzymes noted for their ability to degrade the extracellular matrix (ECM) and their overexpression has been linked to the ability of tumors to invade, metastasize and release growth factors. Chapter 3 studies evaluate the effect of NAC treatment on RS induced activation of NFkappaB and activator protein (AP)-1 transcription factors and on the induction of MMP-2, MMP-9, tissue inhibitors of MMPs (TIMP)-1 and TIMP-2. Results demonstrated no significant response with respect to the transcription factors NFkappaB or AP-1, MMP-2 and -9 mRNA or (open full item for complete abstract)

Committee: Susan Mallery (Advisor) Subjects: Biology, Cell