College of Agriculture & Natural Resources

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The collections in this community comprise faculty research works, as well as graduate theses and dissertations.

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    Lineage reprogramming of tumor-infiltrating cytotoxic T lymphocytes using protein stem cell transcription factors
    (2015) BhaduriHauck, Anjuli Lucija; Xiao, Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Adoptive cell transfer therapy (ACT) is one of the most promising immunotherapies against cancer. However, this treatment regimen requires the expansion of a small population of effector cells, known as tumor infiltrating lymphocytes, into the billions in order to overcome the immunosuppressive tumor microenvironment. The cytotoxic T lymphocytes (CTLs) within this invading immune cell population are the most critical components to kill the growing cancer cells. Nonetheless, the rapid expansion of already exhausted tumor-infiltrating cytotoxic T lymphocytes (TICTLs) may further push them to a terminally differentiated state that reduces their proliferative response upon antigen stimulation. Recently, induced pluripotent stem cells (iPSCs) generated from TICTLs have been suggested as a way to create a renewable source of rejuvenated tumor-specific CTLs, but retroviral reprogramming is inefficient, and can lead to an increased chance of tumorigenesis. To improve the expansion of TICTLs, we used transient protein exposure to SOX2, OCT4, and NANOG (SON) in order to push these exhausted TICTLs to a less differentiated stage, preferably stem cell-like memory CTLs (Tscm). These three transcription factors were transiently delivered using a nuclear protein delivery system. We found only the TICTLs treated with SON (STICTLs) exhibited an increased proliferation rate and extended survivability, independent of additional cytokines and antigen stimulation both in vitro and in vivo; effector CTLs did not respond to the SON regimen. These highly proliferative STICTLs could be associated with up regulation of certain genes related in cell cycle control, such as cyclin D1. Though these STICTLs still express a T cell receptor (TCR), as well as many critical downstream components, they were unable to elicit a reaction against antigen exposure. Though clearly not iPSCs, it is possible that the SON treatment had pushed the TICTLs into a state similar to an early double negative thymocyte. Our findings indicate that TICTLs are uniquely responsive to protein SON compared to naïve and effector CTLs; suggesting TICTLs may also be sensitive to regulation by other more lineage specific transcription factors, thus present new avenue for cancer immune therapy.
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    MOLECULAR MECHANISMS UNDERLYING CADHERIN-6B INTERNALIZATION IN PREMIGRATORY CRANIAL NEURAL CREST CELLS DURING THEIR EPITHELIAL-TO-MESENCHYMAL TRANSITION
    (2015) Padmanabhan, Rangarajan; Taneyhill, Lisa A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The generation of migratory cells from immotile precursors occurs frequently throughout development and is crucial to the formation and maintenance of a functioning organism. This phenomenon, called an epithelial-to-mesenchymal transition (EMT), involves the disassembly of intercellular adhesions and cytoskeletal rearrangements in order to promote migration. Importantly, aberrant EMTs and cell migration can lead to devastating human conditions including cancer metastasis and fibrosis. How cells accomplish EMT to become migratory is still an unanswered question in the biomedical field. To this end, we use chick neural crest cells as an in vivo model to elucidate the molecules and pathways that regulate EMT and migration. Neural crest cells are a population of embryonic cells that are originally stationary within the dorsal neural tube but later migrate to form a variety of adult derivatives, such as the craniofacial skeleton, skin pigment cells and portions of the heart. To facilitate EMT, chick premigratory neural crest cells lose intercellular contacts mediated, in part, by the transmembrane cell adhesion protein Cadherin-6B (Cad6B). While Cad6B mRNA is transcriptionally repressed in premigratory neural crest cells, loss of Cad6B protein does not directly follow and instead occurs ~90 minutes later, just prior to migration. This rapid depletion of Cad6B is all the more striking given that the half-life of most cadherins, including Cad6B, is ~6-8 hours in vitro. As such, unique post- translational mechanisms must exist to remove Cad6B from premigratory neural crest cell plasma membranes to facilitate neural crest EMT. Since cadherins are known to be downregulated through internalization mechanisms (e.g., endocytosis, macropinocytosis) in other in vitro systems, the hypothesis of this dissertation is that Cad6B is internalized, and that this process plays a critical function to enable neural crest EMT. To this end, we document the existence of Cad6B cytoplasmic puncta in cultured cells, cultured neural crest cells and transverse sections of chick embryos. We subsequently identified a p120-catenin binding motif in the Cad6B cytoplasmic tail and demonstrated its functionality through site-directed mutagenesis, revealing a role in enhancing Cad6B internalization and reducing the stability of membrane-bound Cad6B. Furthermore, we uncover for the first time that Cad6B is removed from premigratory cranial neural crest cells through cell surface internalization events that include clathrin-mediated endocytosis and macropinocytosis. Both of these processes are dependent upon the function of dynamin, and inhibition of Cad6B internalization abrogates neural crest cell EMT and migration. Collectively, our findings provide a molecular blueprint for how cadherins are dynamically regulated during the formation of migratory cell types required for normal embryonic development and tissue repair as well as those generated during human diseases and cancers. Importantly, our research is multi-disciplinary, integrating cell biology and physiology to reveal how a cellular event, the active downregulation of a membrane protein, results in a physiological event, neural crest EMT and migration.
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    METABOLIC CHANGES ASSOCIATED WITH ANDROGEN INDEPENDENT GROWTH IN A MOUSE MODEL OF PROSTATE CANCER
    (2014) Martin, Philip Lloyd; Samal, Siba; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    PTEN and TP53 loss are common molecular alterations in aggressive prostate cancer that progresses to castrate resistant prostate cancer (CRPC). PTEN/TP53 loss contributes to regulation of self-renewal and differentiation in prostate progenitor cells, the presumptive tumor and metastasis initiating cells for prostate cancer. TP53 plays an important role in regulating normal cellular metabolism, and loss of function is responsible for metabolic alterations in tumor cells, including increased aerobic glycolysis. We use a novel model of Pten/Tp53 deleted prostate cancer to investigate properties of tumor and metastasis initiating cells, and metabolic alterations that contribute to the evolution of CRPC. We employed a genetically engineered mouse model of Pten-/-Tp53-/- prostate cancer to develop an orthotopic model derived from a clonal cell line from the parental heterogeneous prostate carcinoma. We used histopathology and immunohistochemistry to characterize the orthotopic primary tumors and metastases. We performed metabolomic screening followed by focused analysis of HK II enzyme levels, activity, and cellular distribution in androgen replete and androgen deprived tumors. We also compared HK II levels in primary and metastatic human prostate cancer. Tumor heterogeneity was due to transformation of tumor and metastasis initiating cells with biphenotypic potential capable of basal and luminal differentiation. There was epithelial-to-mesenchymal transition (EMT) in cells of the luminal lineage. The model was capable of androgen independent growth, which influenced the differentiation of metastasis initiating cells. CRPC had increased reliance on glycolysis with increased cytoplasmic and mitochondrial-associated HK II. These metabolic adaptations afforded CRPC increased ability to withstand metabolic stress. HK II levels in human metastases were markedly increased compared to primary tumors. Pten/Tp53 loss in prostate cancer contributes to lineage plasticity in both tumor and metastasis initiating cells, contributing to heterogeneity observed in primary tumors and metastases. Increased glycolysis due to increased total and mitochondrial HK II is a metabolic adaptation that contributes to the evolution of aggressive disease, with progression to androgen independence, providing increased energy and carbon precursors for anabolic processes. Mitochondrial bound HK II blocks apoptosis and contributes to survival in the androgen deprived environment. Targeting this metabolic adaptation may provide improved treatment for this deadly disease.
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    CHARACTERIZATION OF LIVER X RECEPTORS IN PROSTATE CANCER CHOLESTEROL METABOLISM AND PULMONARY IMMUNE RESPONSE
    (2011) Trasino, Steven E.; Lei, David K; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Liver x receptors (LXRs) are central regulators cholesterol homeostasis and the innate immune response. As modulators of inflammation and cholesterol metabolism LXRs might diminish dyslipidemia and inflammation related pathologies caused by high fat (HF) diets or obesity. There is also data demonstrating that LXRs can protect against progression of prostate cancer (PCa), but little is known about the cholesterol modulating effects of LXRs in transformed cells. The goal of this project is to characterize the cholesterol modulating properties of LXRs in two models of PCa and the anti-inflammatory properties of LXRs in swine bronchial alveolar macrophages (AMs). This project will also examine whether the anti-inflammatory and lipid lowering properties of the dietary probiotic bacteria Lactobacillus casei (L. casei), can interact with the LXR axis in AMs. Studies in two PCa cell lines, LNCaP and PC-3, revealed that LXR ligands regulate the LXR responsive genes, ABCA1 and ABCG1 through the LXR&beta isoform and not LXR&alpha in PC-3 cells, but only ABCG1 in LNCaP. LXR- ABCA1 mediated reverse cholesterol transport (RCT) resulted in a decrease in plasma membrane lipid raft cholesterol domains in PC-3 cells, suggesting a potential anti-cancer axis for LXR activation. Studies in LNCaP and PC-3 cells also demonstrated that soy isoflavones can activate transcriptional activation of ABCA1 and ABCG1 in LNCaP and PC-3 cells through the LXR&beta isoform, but did not lead to an increase in RCT. Metabolic and anti-inflammatory studies of LXR in AM from Ossabaw pigs fed either a control (C) diet, HF, HF plus L casei (HFPB) or L. casei alone (CPB) diet revealed that AM from HF fed pigs had significantly higher concentrations of cholesteryl-esters (CE) compared with AM from control (C) diet fed pigs. Ex-vivo activation of LXR with the LXR ligand T0901317 opposed LPS mediated upregulation of IL-1&beta , IL-6, IL-8 and IL-10 mRNA levels in AM from HF, HFPB and CPB fed pigs. Finally, it was observed that LPS stimulation lead to significant inhibition of LXR transcription of LXR&alpha, ABCA1, ABCG1, cholesterol 25 hydroxylase (CH25H) and PPAR&gamma in AM. This effect was abrogated by L. casei for ABCA1, CH25H and PPAR&gamma mRNA expression