Doctor of Philosophy, The Ohio State University, 2022, Biomedical Engineering

Epithelial-Mesenchymal Transition (EMT) is the transdifferentiation of epithelial cells to a mesenchymal phenotype, in which cells lose epithelial-like cell-cell adhesions and gain mesenchymal-like enhanced contractility and mobility. The Transforming growth factor-β1 (TGF-β1) driven signaling cascade drives the biochemical changes in intracellular gene concentrations. EMT is crucial for tissue regeneration and is also implicated in pathological conditions, such as cancer metastasis. Cancer cells exploit EMT to evade apoptosis and migrate through the circulatory system, from the primary tumor to a distant organ, where they metastasize and colonize into the new tissue. Before tumor arrival, tumor-mediated signals help prime this secondary environment to develop favorable conditions for metastasis. In this thesis, I develop a multisytem multicellular model that incorporates biochemical and mechanical signaling to characterize epithelial tissue dynamics during EMT. Furthermore, I develop a model to represent the tumor environment, including cellular and extracellular conditions for tissue dynamics during Pre-Metastatic Niche (PMN) formation. Additionally, I also take a spatial approach to investigating EMT by developing a supervised machine learning regression pipeline that performs image analysis on immunofluorescence images of cells treated with TGF-β1 to quantify various stages of EMT progression.

Committee: Seth Weinberg (Advisor); Rengasayee Veeraraghavan (Committee Member); Aleksander Skardal (Committee Member); Thomas Hund (Committee Member) Subjects: Biomedical Engineering; Biomedical Research

Doctor of Philosophy, University of Akron, 2022, Chemistry

Methanotrophic bacteria absorb methane and oxidize it as their sole source of carbon and energy. Almost all methanotrophic bacteria contain an extensive network of Intracytoplasmic membrane (ICM). The ICMs contain particulate methane monooxygenase (pMMO), which is the initial enzyme in the metabolism of methane. Due to the accumulation of high lipid content in the form of ICM and the formation of polyhydroxybutyrate (PHB), there is a growing interest in utilizing these bacteria to convert the ICM and PHB to biofuel. Structural aspects of the ICM have been characterized by transmission electron microscopy. However, the dynamics and functional role of ICMs remain elusive. A rapid fluorescence microscopy method to visualize ICMs in situ using lipophilic dyes was developed in Chapter III. The extent to which ICM formation occurs in cells depends on the concentration of copper. The ICM formation was visualized and quantified in type I methanotroph Methylotuvimicrobium alcaliphilum (comb. Nov. 20Z) by tracking the bulk copper conversion spectroscopically and by live single-cell confocal imaging. Both methods showed a lag phase prior to the increase in ICM amounts over time. During the ICM formation, there was a significant amount of cell to cell heterogeneity. Further, rapid in-vivo quantification of the PHB method was developed to determine the conditions that enhance the PHB accumulation in methanotrophs. A rapid and cost-effective single cell PHB analysis through fluorescence microscopy by staining via Nile Blue A (NBA) in type II methanotroph Methylocystis sp. Rockwell was described in Chapter IV. NBA stained both the outer membrane of the cell and individual granules of PHB, distinctly but not the ICMs. The ICMs in Methylocystis. sp. Rockwell resides peripheral to the inner membrane whereas PHB is present in the cytoplasmic region. Methylocystis sp. Rockwell accumulated PHB when grown in ammonium mineral slats (AMS) medium, regardless of nitrogen or carbon stress. PH (open full item for complete abstract)

Committee: Adam Smith (Advisor); Sailaja Paruchuri (Committee Member); Nic Leipzig (Committee Member); Chrys Wesdemiotis (Committee Member); Yi Pang (Committee Member) Subjects: Biology; Chemistry

PhD, University of Cincinnati, 2022, Medicine: Molecular, Cellular and Biochemical Pharmacology

The Transforming Growth Factor Beta (TGFß) Family consists of over 30 secreted growth factor ligands, which signal using seven type I and five type II serine/threonine kinase receptors. Given the large number of ligands compared to the number of receptors, there is a significant overlap of multiple ligands utilizing the same receptors. However, one ligand, Anti-Mullerian Hormone (AMH), is the only ligand in the TGFß family with its own unique type II receptor, Anti-Mullerian Hormone Receptor 2 (AMHR2). AMH promotes Mullerian duct regression during male fetal sexual differentiation and regulation of folliculogenesis in women. However, during fetal growth, loss of function mutations in AMH or AMHR2 have been linked to the development of Persistent Mullerian Duct Syndrome (PMDS), where males are born with normal external male genitalia, but also the presence of a uterus and fallopian tubes internally. Alternatively, more recent studies have shown that AMH also plays a role in the development of Polycystic Ovary Syndrome (PCOS) in women. Moreover, the role of AMH in female fertility is becoming more widely accepted. As such, AMH has potential for use in fertility therapies. Prior to the work outlined in this thesis, there was little understanding of how AMH binds to AMHR2. Part of this gap stems from the fact that no structural information about AMH or AMHR2 was available. Therefore, in this work we, solved the structure of AMH bound to the extracellular domain of AMHR2 and characterized the molecular interactions between these proteins. Overall, this work highlights how AMH engages AMHR2 using a modified paradigm of receptor binding facilitated by modifications to the three-finger toxin fold of AMHR2. Furthermore, through mutational studies, we identified residues in both AMH and AMHR2 that are key to the AMH/AMHR2 interaction. In conclusion, understanding these elements contributing to the specificity of binding will help in the design of agonists or antagonis (open full item for complete abstract)

Committee: Thomas Thompson Ph.D. (Committee Member); Terry Kirley Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member); Jo El Schultz Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member) Subjects: Biochemistry

Master of Science, The Ohio State University, 2021, Biomedical Engineering

Cathepsin B is a lysosomal cysteine protease that has a variety of functions intracellularly in healthy cells. However, in certain physiological processes, such as wound healing, and in several disease states, like cancer, cathepsin B is secreted extracellularly and is involved in matrix degradation. Cathepsin B's extracellular proteolytic activity is thought to play an important role in cancer progression and metastasis. Measuring cathepsin B's extracellular activity has been proven difficult. Gene and protein expression levels are often not representative of the true extracellular activity of cathepsin B, because a variety of factors influence the activity such as pH, various inhibitors, and the amount that is secreted extracellularly. Thus, multiple cathepsin B-cleavable fluorescent peptide biosensors have been created to specifically measure the activity of cathepsin B. But, these biosensors have been designed to be used in-solution, which is not always representative of the environment cancer cells experience in vivo. We redesigned a cathepsin B-cleavable fluorescent peptide biosensor, so it can be conjugated into poly(ethylene glycol) (PEG) hydrogels. Cathepsin B activity then can be measured in three-dimensional (3D) matrices that are more representative of the in vivo microenvironment. In addition to investigating the regulation of extracellular cathepsin B activity of cancer cells, this cleavable, fluorescent peptide can be used in other ways in the future. We demonstrate that a similar fluorescent peptide for matrix metalloproteinases (MMPs), another enzyme contributing to the cleavage of the extracellular matrix, conjugated to a cancer drug has the potential to be used as a drug delivery platform. Another further goal for the cathepsin B biosensor is to perform a drug screen on encapsulated patient tissue to examine the impact of cancer drugs on cathepsin B activity to inform personalized treatment decisions. Overall, designing a cathepsin B-cleavable b (open full item for complete abstract)

Committee: Aleksander Skardal (Committee Member); Jennifer Leight (Advisor) Subjects: Biomedical Engineering; Biomedical Research; Polymer Chemistry; Polymers

PhD, University of Cincinnati, 2021, Medicine: Molecular Genetics, Biochemistry, and Microbiology

The transforming growth factor beta (TGFß) family consists of over 30 dimeric ligands that play distinct roles throughout biology, from development to homeostasis. These processes include wound-healing, reproductive health, adipogenesis, muscle and cardiac maintenance, and bone growth, among others. Three classes of TGFß have emerged: the TGFßs, the activins and the bone morphogenetic proteins (BMPs), separated based on sequence identity, receptor binding repertoires and biological functions. To signal, each ligand forms a hexameric complex with four receptors: two of the type II variant, of which there are five available, and 2 of the type I variant, of which there are seven available. The ratio of ligands to signaling receptors creates a receptor utilization bottleneck, where key differences in each ligand/receptor generate specificity and dictate interaction partners. In addition, different mechanisms of receptor assembly have emerged for the TGFß and BMP classes, where the positional assembly of ligand/receptor complex differs fundamentally. However, prior to our work, little structural information existed characterizing activin/receptor interactions, particularly for the type I receptor. Here, we present both biochemical and structural studies extending our understanding of the activin class. We have solved 4 unique structures within the activin class: 1) the structure of apo-GDF11, 2) the structure of GDF11 in complex with ActRIIB and Alk5, 3) the structure of GDF11 in complex with an ActRIIB-Alk4 fusion, and 4) the structure of ActA in complex with the ActRIIB-Alk4 fusion. Through this work, we presented the first structure of an activin class member bound by both the cognate type II and type I receptors, which bind independently. A composite interface is built from surfaces on both ligand monomers, where the majority of receptor ß4ß5 loop is engaged and a conserved knob-in-hole moiety is used to anchor the binding. Furthermore, through these studies, we have (open full item for complete abstract)

Committee: Thomas Thompson Ph.D. (Committee Chair); Andrew Herr Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); John Monaco Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member) Subjects: Biochemistry

PhD, University of Cincinnati, 2021, Medicine: Cancer and Cell Biology

Hematopoietic stem cells (HSCs) regenerate the hematopoietic tissue during stress hematopoiesis, requiring that HSCs come out of quiescence and re-fill the depleted pool, as well as self-renew to maintain a healthy HSC pool. Injuries associated with stress hematopoiesis include myeloablative chemotherapy, infection/inflammation and allogeneic bone marrow transplantation. However, self-renewal and differentiation must be tightly controlled. Too much differentiation results in an exhaustion of the HSC pool and bone marrow failure, while too much self-renewal can lead to leukemogenesis. The transforming growth factor ß (TGFß) signaling pathway has been well-studied to regulate HSCs during steady state hematopoiesis. However, very little is known about its role in regulating HSCs during stress hematopoiesis. Moreover, its role in HSC regulation is both context-dependent and concentration dependent. Using a mouse model to conditionally overexpress an active form of the main TGFß ligand, aTGFß1, we found that increased aTGFß1 caused very little impact in the hematopoietic system during steady state hematopoiesis, driving a very mild neutropenia and bone marrow dysplasia during aging. However, aTGFß1 overexpression prevented the murine hematopoietic system from recovering to normal levels of peripheral blood leukocyte and platelet levels after chemotherapeutic stress using 5-fluorouracil. Moreover, during the recovery phase aTGFß1-overexpressing mice had increased HSC cell cycling and DNA double strand breaks compared to controls. Separately, acute inflammation-driven stress caused by polyinosinic:polycytidilic acid (pIC) drove pancytopenia, bone marrow dysplasia and splenomegaly, but expanded the hematopoietic stem and progenitor (HSPC) pool long after the short-term effects of pIC had resolved, phenotypes similar to the human bone marrow failure myelodysplasia. Mechanistically, we found that aTGFß1 overexpression altered the HSC transcriptome after pIC-driven str (open full item for complete abstract)

Committee: Marie-Dominique Filippi Ph.D. (Committee Chair); Paul Andreassen Ph.D. (Committee Member); Jose Cancelas-Perez M.D. (Committee Member); H. Leighton Grimes Ph.D. (Committee Member); Gang Huang Ph.D. (Committee Member); Damien Reynaud (Committee Member) Subjects: Health Sciences

Master of Science (MS), Wright State University, 2019, Microbiology and Immunology

Cardiac resident macrophages have recently been reported to facilitate electrical conduction by interacting with cardiomyocytes via connexin 43 (Cx43) hemichannels. Cx43 is critical for impulse propagation and coordination between muscle contractions. Cardiomyocyte electrophysiology can be altered when coupled with non-cardiomyocyte cell types such as M2c tissue-resident macrophages. Our hypothesis is that: M2c biochemical autocrine signals such as transforming growth factor beta (TGF-β1) and interleukin-10 (IL-10) introduced into a co-culture with cardiomyocytes and macrophages will upregulate Cx43 expression and maintain the macrophages in an M2c anti-inflammatory phenotype mimicking cardiac resident macrophages. Consequently, these enhanced interactions between cardiomyocytes and macrophages will intensify the fluorescence of the membrane potentiometric dye, Di-8-ANEPPS. In the co-cultures, a ratio of 1:50 IL-10(p)-GFP+ Raw264.7 macrophages to HL-1 cardiomyocytes treated with both IL-10 and/or TGF-β1 exhibited a higher fluorescence with Di-8-ANEPPS in areas where macrophages are interacting with cardiomyocytes. Suppressor of cytokine signaling 3 (SOCS3) down regulated fluorescence of the membrane potentiometric stain; SOCS3 is known to interfere with TGF-β1, but not IL-10 signaling pathways. To confirm the cellular interactions via gap junctions, Flow Cytometry was performed using a monoclonal anti-Cx43 antibody conjugated with Alexa Fluor® 488 to compare Cx43 expression in the co-cultures treated with TGF-β1 and IL-10, to those without treatment. These results suggest that TGF-β1 and IL-10 increase Cx43 expression while maintaining macrophages in the M2c phenotype by secreting these autocrine signals. TGF-β1 and IL-10 signaling enhance macrophage interactions with cardiomyocytes in co-culture via Cx43, theoretically raising the cellular resting membrane potential, leading to a more excitatory cardiomyocyte.

Committee: Nancy J. Bigley Ph.D. (Advisor); Thomas L. Brown Ph.D. (Committee Member); Dawn P. Wooley Ph.D. (Committee Member) Subjects: Immunology

Doctor of Philosophy, Case Western Reserve University, 2018, Pathology

Human cell transformation models have provided critical insight into our understanding of the events that drive cancer development and progression. However, these models have largely ignored the contributions of cytokines that originate outside a cancer cell in the surrounding tumor microenvironment (TME). Although tumor cells exploit cytokine signaling to promote growth, invasion, and metastasis, the response of normal and premalignant cells to cytokines in the developing TME remains unclear. Continued refinement of human cell transformation models will expand our understanding of the tumor-suppressive mechanisms dismantled by developing cancer cells and lead to the identification of novel targets for future cancer therapies. The studies described here refined a transformation model that begins with normal human mammary epithelial cells (HMEC) isolated from reduction mammoplasties to investigate the mechanisms regulating the divergent responses of normal and transformed cells to the cytokine Oncostatin M (OSM). Using normal HMEC, we revealed that OSM-induced senescence requires a novel link between OSM/Signal Transducer and Activator of Transcription 3 (STAT3) signaling and Transforming Growth Factor-ß (TGF-ß)/SMAD3 signaling. Notably, constitutive c-MYC expression dismantled STAT3/SMAD3-induced senescence and cooperated with OSM to drive transformation, epithelial-mesenchymal transition (EMT), and invasion. We then utilized this HMEC model to examine how OSM/STAT3 signaling and TGF-ß/SMAD3 signaling cooperate in transformed HMEC. In contrast to normal cells, OSM did not engage senescence in transformed HMEC and instead induced the EMT-inducing transcription factor (EMT-TF) Snail, epithelial–mesenchymal plasticity, and cancer stem cell (CSC) properties. Inhibiting TGF-ß-mediated signaling or suppressing expression of SMAD3 and SMAD4, but not SMAD2, abrogated OSM/STAT3-induced plasticity and CSC properties in transformed HMEC. Finally, my studies lead to the discove (open full item for complete abstract)

Committee: Mark Jackson Ph.D. (Advisor); Hannah Gilmore M.D. (Committee Chair); Analisa DiFeo Ph.D. (Committee Member); Clive Hamlin Ph.D. (Committee Member); William Schiemann Ph.D. (Committee Member); George Stark Ph.D. (Committee Member); Zhenghe Wang Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Cellular Biology; Genetics; Health Sciences; Medicine; Molecular Biology; Pathology

Doctor of Philosophy, Case Western Reserve University, 0, Molecular Medicine

Breast cancer (BC) is the most diagnosed cancer amongst women in the United States and accounts for approximately 40,000 deaths per year. The nonreceptor kinase, c-Abl, regulates numerous aspects of normal mammary epithelial cell (MEC) physiology, however we found c-Abl expression to be downregulated during breast cancer progression. Importantly, the loss of c-Abl expression correlated with worse relapse free survival rates for BC patients. Interestingly, as BCs progress they lose sensitivity to the cytostatic actions of the pleiotropic cytokine, transforming growth factor ß (TGF-ß), as well, and instead utilize it for disease progression. These findings suggest that TGF-ß and c-Abl are both essential for the regulation of normal MECs. Overexpression of a constitutively active c-Abl mutant in aggressive triple negative BC (TNBC) cells attenuated oncogenic TGF-ß signaling and instead coupled TGF-ß to a novel Smad2/Smad1/5/8 pathway that reactivated p53 expression to induce a p21-dependent senescence. The novel c-Abl->TGF-ß->Smad2/Smad1/5/8->p53->p21 signaling axis robustly inhibited tumorigenesis of our aggressive TNBC xenograft model. Our findings further established that p53 and c-Abl become discordantly expressed in TNBCs. The ratio at which c-Abl and p53 are expressed predicts the levels of which the mitotic kinase, TTK, is expressed in TNBC. TTK is known to regulate the cellular localization of c-Abl in response to oxidative stress. This mechanism is particularly relevant because of the predominantly cytoplasmic localization c-Abl is found in certain TNBC cells and of the survival signaling c-Abl exerts while in the cytoplasm. Although, we found inhibition of TTK kinase activity or depletion of its expression sensitized TNBC cells to activated c- Abl, it was not due to alterations in the cellular localization of c-Abl. Instead, we found mutant p53 directly bound c-Abl in the cytoplasm and regulated its function in TNBCs. Finally, we found (open full item for complete abstract)

Committee: William Schiemann (Advisor); Mark Jackson (Committee Chair); Thomas Egelhoff (Committee Member); Alex Almasan (Committee Member); Nathan Pennell (Committee Member) Subjects: Biology; Molecular Biology; Oncology

PhD, University of Cincinnati, 2016, Medicine: Molecular Genetics, Biochemistry, and Microbiology

BMPs represent the largest subfamily of the greater TGF-ß superfamily of signaling ligands. Importantly, BMPs have been shown to play critical roles in development and disease, ranging from tissue differentiation, proliferation, and homeostasis to atherosclerosis, CVD, pulmonary arterial hypertension, cancer, and kidney fibrosis. For these reasons, the BMPs and the proteins that modulate their function are promising targets to treat these various pathologies. The DAN-family represents the largest collection of BMP antagonists in vertebrates, with 7 unique members. Prior to our work, little mechanistic and structural information existed characterizing the DAN-BMP interaction. Here, we present studies extending the molecular and biochemical characterization of the DAN-family. We have solved 3 unique structures within the DAN-family: 1) the structure of Gremlin-2/Grem2/PRDC a strong BMP inhibitor, 2) the structure of NBL1, a moderate BMP inhibitor, and 3) the structure of Gremlin-2 bound to the ligand GDF-5. Through this work, we have classified the binding epitope on Gremlin-2 necessary for binding to BMP ligands, including BMP2, BMP7, and GDF5. Furthermore, through our work on NBL1, we have began to classify variability across the DAN-family in terms of function, characterizing several features and regions accounting for their range in activity. Most importantly, the structure of Grem2-GDF5 reveals numerous features unique to the DAN-family as compared to other BMP antagonist families. This structure reveals two Grem2 dimers binding perpendicularly to the ends of GDF5, forming an H-like structure. Comparison to the unbound Grem2 structure reveals a dynamic N-terminus that undergoes significant transition upon binding, leading to simultaneous binding of Type I/Type II ligand interaction sites. Binding studies show that DAN family members can interact with BMP-Type I receptor complexes, whereas Noggin outcompetes the Type I receptor for ligand binding. Interestingly, G (open full item for complete abstract)

Committee: Thomas Thompson Ph.D. (Committee Chair); Andrew Herr Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); Mark Rance Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, The Ohio State University, 2014, Biochemistry Program, Ohio State

Myofibroblasts are the primary cellular mediators of wound-healing that provide contractile force required for wound contraction, closure, and restoration of tissue integrity. De novo expression of smooth muscle alpha-actin (SMA) is a physiologically relevant process required for assembly of thin filaments and generation of contractile force by myofibroblasts. Chronic myofibroblast differentiation and associated excessive secretion of type I collagen paves the way to the emergence of fibrocontractive disorders that eventually lead to cardiopulmonary remodeling and dysfunction. Transforming growth factor beta-1 (TGF beta-1) is the major profibrotic agonist that drives myofibroblast differentiation in healing wounds. Characterization of transcriptional control of the SMA gene has led to the identification of cis-regulatory DNA elements within the 200 bp SMA proximal core promoter as well as corresponding trans-acting proteins that modulate SMA gene output. Dynamic interplay between transcriptional activators and repressors is implicated as the major means for governing SMA gene transcription during TGF beta-1-mediated myofibroblast differentiation. Based on previous biochemical and molecular biological studies, we hypothesized that the purine-rich element (Pur) binding proteins alpha and beta (Pur alpha and Pur beta) may negatively modulate the DNA-binding activities of serum response factor (SRF) and TGF beta-1-regulated Smad3 transcriptional activators with SMA promoter DNA during the process of myofibroblast differentiation. Comparison of SMA protein expression in human pulmonary fibroblasts (hPFB) cultured on a rigid plastic substrate or softer substrates prepared from native or denatured forms of type I collagen indicated that SMA expression was highly suppressed on collagenous substrates and was associated with an enhanced ability of Pur alpha/beta repressor proteins to interact with the SMA promoter. These results indicated that Pur alpha/beta may play (open full item for complete abstract)

Committee: Arthur Strauch PhD (Advisor); Daren Knoell PhD (Committee Member); Beth Lee PhD (Committee Member); Susheela Tridandapani PhD (Committee Member) Subjects: Biochemistry; Cellular Biology; Molecular Biology

MS, University of Cincinnati, 2007, Medicine : Epidemiology (Environmental Health)

Objectives: 1) Determine the prevalence of subclinical coronary atherosclerosis (CA) in patients with systemic sclerosis (SSc) 2) evaluate serum proinflammatory HDL (piHDL) and transforming growth factor-Β1 (TGF-Β1) levels as potential novel markers of atherosclerotic risk in SSc. Methods: Cross-sectional study of 17 patients with SSc and 17 matched controls. Measurements included coronary calcium score; lipid profile; serum levels of high-sensitivity C-reactive protein, homocysteine, piHDL, and TGF-Β1. Results: Coronary calcium was found in 12 participants (9/17 SSc, 3/17 controls, p=0.03). The mean CCS in subjects with SSc was significantly greater compared with controls (p = 0.003). Mean values for serum homocysteine and TGF-Β1 levels were significantly greater in patients with SSc (p < 0.05). Five patients with SSc (29%), but no controls had detectable levels of piHDL (p=0.06). Conclusions: Rates of subclinical CA are greater in patients with SSc compared with controls. These findings should be confirmed in a larger study.

Committee: Dr. Joel Tsevat (Advisor) Subjects: Health Sciences, Immunology

Master of Science, The Ohio State University, 2011, Mathematics

Fully understanding the development of a cancer within an organism requires detailed understanding on interactions taking place within the microenvironments of individual cells. Cancer formation requires not only growth of cancerous epithelial cells but also changes to the immune and mesenchymal cells in the neighborhood of the tumor. Without environmental changes tumor restructuring and angiogenesis needed for a tumor to fully develop, the cancer would not pose a severe threat. A primary mechanism for controlling said angiogenesis and stoma restructuring is in the cytokine family of transforming growth factor beta. In this paper the activity of the regulatory gene Ets2 and its effects on transforming growth factor beta through macrophage and fibroblasts will be studied via a system of ordinary differential equations. The ODEs will demonstrate the potential for Ets2 to act as an intermediate in an autocatalytic pathway that can drive TGFb to heightened levels necessary for the promotion of tumorigenesis.

Committee: Avner Friedman (Advisor); Michael Ostrowski (Committee Member) Subjects: Mathematics

Doctor of Philosophy, Case Western Reserve University, 2006, Pharmacology

The 15-30% of human breast cancers that have upregulated HER2/ErbB2/Neu are highly aggressive and resistant to traditional treatments, resulting in poor prognosis. To identify novel therapeutic targets, we derived the transcriptomes associated with tumor progression in two independent mouse models of ErbB2/Neu-induced tumorigenesis. From MMTV-Neu mice, we identified 324 candidate genes unique to ErbB2/Neu-induced tumors relative to wild-type mammary glands. A subset of these genes also changed expression levels in preneoplastic mammary glands, indicating a pivotal role early in ErbB2/Neu-initiated tumorigenesis. Downregulation of several known transforming growth factor (TGF)-β target genes in the ErbB2/Neu molecular signature suggested attenuation of the TGF-β signaling cascade in these tumors. Analysis of TGF-β-Receptor-I/ALK5 by western blot and immunohistochemistry confirmed that Smad-dependent TGF-β signaling was inactive in these tumors. Although absent in most of the tumor, colocalization of phosphorylated Smad2 and Activin-Receptor-IB/ALK4 at the tumor periphery suggested functional Activin signaling at the leading edge of these tumors. Collectively, these data indicate intrinsic TGF-β pathway suppression in ErbB2/Neu tumors via loss of TGF-β-Receptor-I/ALK5. Recent studies have shown that pregnancy can accelerate ErbB2/Neu tumor development, inducing a susceptible cell population in MMTV-Neu mammary glands. The stochastic pattern of tumor development in multiparous MMTV-Neu mice suggests additional events are required for ErbB2/Neu oncogenesis. It remains unclear whether such events are genetic or reflective of the dynamic, pregnancy-associated hormonal control of the gland. Bitransgenic mice generated by breeding MMTV-Neu mice with a model of ovarian hyperstimulation developed multifocal mammary tumors in an accelerated, synchronous manner compared to virgin MMTV-Neu animals. This synchrony of tumor development suggests that trophic maintenance of the mamm (open full item for complete abstract)

Committee: Ruth Keri (Advisor) Subjects:

Doctor of Philosophy, Case Western Reserve University, 1990, Biochemistry

The expression of transforming growth factor beta (TGFβ) mRNA and the effects of exogenous TGFβ on the growth of glioblastoma cells were investigated. All of the glioblastoma cell lines and normal glial cells tested expressed TGFβ mRNA transcripts. The level of TGFβ1 and TGFβ2 mRNA varied among the cell lines. Normal glial cells expressed as much TGFβ1 mRNA as several of the glioblastoma cell lines. The half-life of both TGFβ1 and TGFβ2 mRNA was at least 25 hours. TGFβ treatment transiently increased the steady state levels of TGFβ1, TGFβ2, and PDGF-B mRNAs. The effects of exogenous TGFβ on the growth of human glioblastoma cell lines were assayed. In three of eight glioblastoma cell lines tested, growth rates in monolayer culture were decreased 30%-75% by TGFβ in a defined, serum-free medium, but not in serum-containing media. TGFβ did not affect the anchorage independent growth of these cells, as measured by colony growth in soft agar. The morphologies of the three cell lines that were growth-inhibited by TGFβ were dramatically altered by TGFβ. The effects of TGFβ on one glioblastoma cell line, A1207, were studied more compl etely. TGFβ caused these cells to round up, detach from the substrate, and grow in spheroids over a 2-5 day period. TGFβ decreased the attachment of these cells to fibronectin, laminin, and collagen by about 20%. As measured by flow cytometry, the level of the α4 integrtin on the cell surface was decreased, while the levels of β1, α2 and α5 integrins subunits were not affected by TGFβ treatment. TGFβ affected the tenascin mRNA and protein levels in A1207 cells. Tenascin mRNA was increased 8-fold after 36 hours of TGFβ treatment, a time corresponding to the morphological changes. Tenascin secretion into the medium was increased by about 10-15 fold as compared to control cells after 72 hours of TGFβ exposure. Tenascin was shown to interfere with the attachment of A1207 cells to fibronectin. These results are consistent with the hypothesis that in (open full item for complete abstract)

Committee: David Goldthwait (Advisor) Subjects: Biology, Molecular