Doctor of Philosophy (PhD), University of Toledo, 2012, College of Medicine

Folic acid is an essential water soluble vitamin required for nucleic acid and protein biosynthesis. The first trimester of pregnancy needs constant folate transport from maternal circulation to support rapid fetal cell division, growth and proliferation. Folate receptor α mediates the transplacental folate transport and facilitates normal embryonic growth. Female sex hormones, estrogen and progesterone regulate the complicated process of embryogenesis. Estrogen and progesterone through their receptors regulate folate receptor α, but the role of androgens that increase during first trimester in folate receptor α regulation is unknown. We show that androgen/androgen receptor increases folate receptor α mRNA and protein expression. We utilized folate receptor α promoter deletion and mutant constructs to identify the androgen receptor binding sites. We show that androgen receptor and CAAT enhancer binding protein α bind the folate receptor α promoter regions and cause promoter activation. Co-immunoprecipitation studies show that androgen receptor and CAAT enhancer binding protein α interact in placental trophoblast cells. These results suggest that androgen receptor and CAAT enhancer binding protein α interact, bind the folate receptor α promoter and regulate folate receptor α gene in cancer and placental trophoblast cells. In normal prostate, androgen-bound androgen receptor activates its target genes by binding to cis androgen response elements. This is the classical mechanism of androgen receptor action. In prostate cancer, androgen receptor exerts its growth promoting effects by tethering to other transcription factors. We identified Elk-1 transcription factor that needs androgen receptor for its transcriptional activation. Co-immunoprecipitation and mammalian two-hybrid assays show that Elk-1 interacts with N-terminal domain of androgen receptor. Microarray analysis and gene validation show that Elk-1 and androgen receptor association regulates genes involved in (open full item for complete abstract)

Committee: Manohar Ratnam (Committee Chair); Ivana De La Serna Ph.D. (Committee Member); Cynthia Smas Ph.D. (Committee Member); Robert Trumbly Ph.D. (Committee Member); Lirim Shemshedini Ph.D. (Committee Member); Beata Lecka-Czernik Ph.D. (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy in Medical Sciences (Ph.D.), University of Toledo, 2005, Graduate School

Folate receptor (FR) is a GPI-anchored cell surface protein. Three iso-forms of FR have been identified and characterized, FRa, FRb and FRg/FRg'. Folate receptor is capable of transporting folate/antifolate compounds and folate conjugates into the cell functions in transport of folates into the cell. Folate receptor a is found on certain normal epithelial cells and highly expressed in various cancer tissues including non-mucinous ovarian cancers, uterine endometrial endometrioid adenocarcinoma, ependynomas, menigioma and some colon and breast cancers. In normal tissues, FRa is restricted on the apical (luminal) aspect of the polarized epithelial cells, thus rendering it inaccessible through the circulation. However, it is accessible through the circulation in malignant tissues owing to the hypervascularization seen in these tissues. Folate compounds or conjugates can be effectively taken up into the cell by means of FRa due to its ability to shuttle back and forth between the cell membrane and endosomal compartment. Folate receptor mediated targeting has been shown to be highly specific and effective and dependent on the density of FR on the cell membrane. However, the expression of FR in tumors is highly variable among tumors of certain type and heterogeneous within a tumor. Therefore, an optimal expression of FR in malignant tissues will help optimize the effectiveness of FR-mediated targeting. We have found that both in cell culture and in tumor xenograft mouse model, Dexamethasone (Dex) can up-regulate the FRa gene in Hela cells and this is significantly enhanced when either valproic acid (VPA) or trichostatin A (TSA), both HDAC inhibitors, is used in conjunction with Dex. In addition, in Hela cells co-transfected with a FRa promoter luciferase construct and an expression vector for either PRa or PRb, the progestin R5020 can up-regulate the FRa promoter up to ten-fold. Furthermore, in non-responsive cells such as T47D, co-treatment of R5020 with TSA can up-regul (open full item for complete abstract)

Committee: Manohar Ratnam (Advisor) Subjects: Biology, Molecular

Master of Sciences (Engineering), Case Western Reserve University, 2022, Biomedical Engineering

The knockdown of immune checkpoint proteins, such as VISTA, can allow for the reversal of an immunosuppressed tumor microenvironment back to a pro-inflammatory and immuno-active state, causing inhibition of T-cell suppression and activation of innate myeloid antigen presenting cells. Here, we evaluated whether a folate-targeted nanoparticle could enhance VISTA siRNA delivery, leading to robust reprogramming and activation of an anti-tumor immune response. Flow cytometry studies have indicated that cells of multiple murine triple negative breast cancer and melanoma models that express VISTA also express Folate Receptor β (FRβ), indicating that FRβ targeting of a lipid nanoparticle (LNP) can potentially allow for enhanced uptake of the LNP by immune cells. Uptake of the targeted nanoparticle by the tumor and resulting expansion of activated immune populations, coupled with decreased systemic effects, indicate that IT administration of a FRβ targeted nanoparticle in a murine melanoma model can facilitate effective reprogramming of the tumor immune landscape.

Committee: Efstathios Karathanasis PhD (Advisor); Horst von Recum PhD (Committee Member); Vijay Krishna PhD (Committee Member); Steven Eppell PhD (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2014, Photochemical Sciences

Nowadays, our society is facing numerous ecological, chemical, and medical challenges that could be met with new scientific discoveries. Among these scientific studies are the qualitative and quantitative analytical determinations of chemical and biological species associated with such challenges. Design and synthesis of chemical sensors capable of operating in a complex media are of crucial importance due to their potential application in the environmental and biological processes. Fluorescence based sensors are gaining wide popularity for chemical trace detection due to their high sensitivity and low cost. Chemical sensors that operate based on specific analyte-induced changes in fluorescence appear particularly attractive because they offer the potential for detecting low analyte concentrations. The present work describes the application of different types of fluorescence-based sensors designed to distinguish various structurally similar analytes, analyte mixtures as well as specific analytes in a complex media such as blood serum and urine. Here, we describe efficient methods for sensing several analytes of interest such as ovarian cancer biomarker, cancer associated nitrosamines, drug-related amines with potential for abuse and pyrophosphate in real-time PCR monitoring. Our work is a contribution to the development of practical laboratory procedures tackling challenges of our society.

Committee: Pavel Anzenbacher PhD (Advisor); George Bullerjahn PhD (Committee Member); Andrew Torelli PhD (Committee Member); Andrew Layden PhD (Committee Member) Subjects: Analytical Chemistry

Doctor of Philosophy, The Ohio State University, 2007, Pharmacy

One of the lingering challenges in cancer therapy is to selectively destroy malignant cells and minimize the toxicity to normal tissues. The field of therapeutic targeting has thus been attractive with the ultimate goal of developing anti-cancer agents that work like “magic bullets”. Herein, we explore therapeutic approaches through targeting to CD37 and folate receptor, two promising cellular surface markers that can be utilized for antibody and liposome-based targeted therapies. In the first part, a novel recombinant CD37-targeted small modular immunopharmaceutical (CD37-SMIP) and nanoscale liposomal particles were used to target CD37 molecule. We first demonstrated specific expression of CD37 surface antigen on B but not T cells. Crosslinking the CD37 resulted in dose and time dependent apoptosis by CD37-SMIP in CLL B cells. In addition, CD37-SMIP induced antibody dependent cellular cytotoxicity but not completment dependent cytotoxicity in CLL cells. In vivo therapeutic efficacy of CD37-SMIP was demonstrated in a Raji cell xenograft mouse model. These findings provide strong justification for CD37 as a therapeutic target and introduce SMIP as a novel class of targeted therapies for B cell malignancies. Furthermore, immunoliposomes specific to CD37 were constructed to potentiate the effect of CD37-SMIP. CD37-immunoliposomes specifically bound to CD37+ cells and induced apoptosis without the need for a crosslinking secondary antibody. These data suggest that CD37-immunoliposomes can act as a novel formulation design for CD37-SMIP, and may also be utilized for delivery of therapeutic agents. In the second part, folate receptor (FR) was investigated as a cellular target for liposomal delivery. In the first study, PEG-cholesterol and folate-PEG-cholesterol were synthesized for PEGylation and FR targeting. These two compounds were incorporated into the lipid bilayer of FR-targeted liposomal doxorubicin. The study suggested that cholesterol is a viable bilayer anchor f (open full item for complete abstract)

Committee: Robert Lee (Advisor) Subjects: Health Sciences, Pharmacy

Doctor of Philosophy in Medical Sciences (Ph.D.), University of Toledo, 2007, College of Graduate Studies

FR quantitatively recycles, within minutes, between the cell surface and endocytic compartments via a Cdc42-regulated endocytic pathway. In this study, we investigated the molecular mechanism by which FR recycling is regulated by PMA since its effects on physiological processes are known to stimulate cell signaling through the second messenger diacylglycerol. Phorbol-12-myristate-13-acetate causes a dose dependent increase in the number of receptor molecules residing on the cell surface by inhibiting internalization. Moreover, the release of the intracellular calcium stores which is triggered by the ionophore cyclopiazonic acid results in a doubling of cell surface FR thereby simulating the effect of PMA. Since intracellular calcium is needed for externalization of FR, the PMA effect is probably mediated by a classical PKC isoforms. Western blot of total MA104 cell lysates and quantitative real time RT-PCR show that PKCa is the only classical PKC that can be detected in these cells. Overexpression of wild-type PKCa results in an increase in PMA-induced FR externalization relative to the control cells overexpressing GFP. Knockdown of PKCa using retrovirus or overexpression of a dominant negative PKCa mutant or a constitutively active PKCa which is targeted to the membrane but not specifically to rafts abrogate the PMA effect on FR recycling. These data further suggest that PKCa, targeted to specific membrane microdomains, is one of the key players in mediating the phorbol ester effect on FR recycling in MA104 cells. In order to identify other candidate proteins involved in FR recycling, we purified FR-rich rafts using an immobilized biotinylated folate probe. Among the proteins identified, RACK1 mediates the PMA effect on FR recycling in MA104 cells. The association of RACK1 with FR-rich rafts was further confirmed by electron microscopy in situ in MA104 cells. Though RACK1 has been reported to be present in the cytosol and in caveolae, no evidence for its associatio (open full item for complete abstract)

Committee: Manohar Ratnam (Advisor) Subjects:

Doctor of Philosophy in Medical Sciences (Ph.D.), University of Toledo, 2005, College of Graduate Studies

The folate receptor (FR) type Beta, which is expressed in about 70% of acute myelogenous leukemia (AML) and can be selectively up-regulated by all-trans retinoic acid (ATRA), is a promising target for therapeutic intervention in AML. Using KG-1 and MV4-11 AML cells and recombinant 293 cells, we show that the histone deacetylase (HDAC) inhibitors Trichostatin A (TSA), valproic acid (VPA) and FK228 potentiate ATRA induction of FR-Beta. ATRA and/or TSA did not induce de novo FR synthesis in any of a variety of FR-negative cell lines tested. These results indicate that the combination of ATRA and innocuous HDAC inhibitors may be expected to facilitate selective FR-Beta-targeted therapies in AML. Mechanistic studies showed that (1) TSA did not alter the effect of ATRA on the expression of retinoic acid receptors (RARs) Alpha, Beta or Gamma, (2) ATRA increased the association of RAR Alpha with the basal FR-Beta promoter; (3) the enhancement of the ATRA effect by TSA was associated with increased acetylation of the core histones H3 and H4 at or near the basal FR-Beta promoter, (4) TSA caused a striking ATRA-dependent increase in Sp1 binding at the promoter without a further change in the association of RAR Alpha or an increase in Sp1 expression; (5) TSA further decreased the availability of putative repressor AP-1 protein(s). We further demonstrate that FR-Beta selectively mediated cell growth inhibition by the (6S) diastereoisomer of dideaza tetrahydrofolate in a manner that was potentiated by ATRA and HDAC inhibition. This study also found that ATRA induced apoptosis in MV4-11 cells but increased FR-Beta expression in the surviving cells. These surviving cells resistant to ATRA induced apoptosis can mimic the residual cells refractory to ATRA differentiation therapy. Thus this cell line can serve as a model cell line to establish FR-Beta-targeted therapy for minimal residual disease in APL. Sublines of MV4-11 cells that have uniform cell populations were isolated. Prelim (open full item for complete abstract)

Committee: Manohar Ratnam, Ph.D. (Advisor) Subjects: Biology, Molecular