<p> Cardiovascular disease continues to be the leading cause of morbidity and mortality in the US and worldwide. Traditional treatments include vascular surgeries, such as angioplasty, stent placement, and vascular graft or vascular reconstruction. Of importance for this dissertation are the outcomes following vascular graft surgeries. More than 50% of vascular grafts fail within the first few years due to maladaptive responses, such as inflammation. There is a critical need to develop improved treatments to the traditional grafting procedures. One proposal to enhance outcomes is the use of cellularized, low modulus, synthetic poly(ethylene) glycol (PEG)-based biomaterials. PEG-based hydrogels have been shown to support the 3D growth and differentiation of vascular cells and may provide structural support for the vessel. A principal concern is that a growing percentage of individuals contain anti-PEG antibodies, including IgG antibodies. T cells are mediators of antibody production and play a major role in angiogenesis and in the development of arthrosclerosis. Therefore, studies to elucidate the T cell-PEG matrix interactions are needed to control and predict maladaptive responses. Here, an established murine D10-IL2, Th2 cell line, was used as a model of T lymphocyte activity to: 1) better understand the influence of PEG on T cell metabolism; 2) determine the consequence of an acute Th2 inflammatory microenvironment on the expression of pro-inflammatory responses in fibroblasts within the 3D matrix; and 3) investigate antigen presenting cell (APC)-independent T cell activation. This research demonstrated that Th2 cells experience a reversible suppression of mitochondrial membrane potential (&Delta;&Psi;m) upon initial exposure to PEG. Data also suggested that T cells were susceptible to APC-independent activation during contact with the PEG matrix, as measured by an increase in IL4 and IL10 expression and the production of inflammatory cytokines (IGFBP-3, CTACK, MIP2, LIX). Additionally, this research led to the development of a bio-degradable PEG-based hydrogel system. This allowed for the investigation of aortic fibroblast cell responses to an acute inflammatory 3D microenvironment and demonstrated that the hydrogel system provided a limited protective barrier during inflammation. This research has public health benefits and has provided an improved understanding of the immunogenic nature of PEG.</p>

<p> Early embryonic cells in many species polarize radially by distinguishing their contacted and contact-free surfaces. Radial polarization is a critical patterning event driven by cell-cell contact and is required for developmental processes, such as the first differentiation event in the early mammalian embryo. The homophilic adhesion protein E-cadherin is required for contact-induced polarity in many cells. However, it is not clear whether E-cadherin functions instructively as a spatial cue, or permissively by ensuring adequate adhesion so that cells can sense other contact signals. In <i>C. elegans,</i> radial polarity begins at the four-cell stage, when cell contacts restrict the PAR polarity proteins to contact-free surfaces. We previously identified the RhoGAP PAC-1 as an upstream regulator that is required to exclude PAR proteins from contacted surfaces of early embryonic cells. PAC-1 is recruited specifically to sites of cell contact and directs PAR protein asymmetries by inhibiting the Rho GTPase CDC-42. How PAC-1 is able to sense where contacts are located and localize to these sites is unknown. We show that HMR-1/E-cadherin, which is dispensable for adhesion, functions together with HMP-1/&alpha;-catenin, JAC-1/p120 catenin, and the previously uncharacterized linker PICC-1/CCDC85/DIPA to bind PAC-1 and recruit it to contacts. Furthermore, we show that ectopically localizing the intracellular domain of HMR-1/E-cadherin to contact-free surfaces of cells recruits PAC-1 and depolarizes cells, demonstrating that HMR-1/E-cadherin plays an instructive role in polarization. Furthermore, we show that radial polarity is defective in embryos lacking HMR-1/E-cadherin. Our findings identify an E-cadherin-mediated pathway that translates cell contacts into cortical polarity by directly recruiting a symmetry-breaking factor to the adjacent cortex.</p>

<p> <i>Candida albicans</i> is one of the most common causes of nosocomial infection that can lead to serious or even fatal illness. <i> C. albicans</i> is naturally resistant to complement activation through its cell-surface displayed mannan, but the resistance can be overcome by anti-mannan antibody. Previous studies have shown that phagocytosis of <i>C. albicans </i> by human neutrophils is promoted by anti-mannan antibody but is not further enhanced by complement. The purpose of this study was to expand the previous study to include human macrophages and mouse neutrophils. First, human macrophages, derived from peripheral blood monocytes, were incubated with <i>C. albicans</i> in the presence of each of the four anti-mannan IgG antibodies (M1g1, M1g2, M1g3, and M1g4) with or without complement. Phagocytosis was determined by microscopy and phagocytic killing by colony forming unit. It was found that each variant had a subclass-specific effect to enhance both phagocytosis and phagocytic killing when compared to no-antibody control (p &lt; 0.001) but addition of complement did not show a synergistic effect. Next, the effect of anti-mannan antibody and complement on phagocytosis of <i> C. albicans</i> by mouse neutrophil-like cells (MPRO) was assessed and results similar to those found with human macrophages were observed. Finally, an alternative method to determine phagocytic killing of <i>C. albicans </i> by human neutrophils was evaluated where respiratory burst values were measured in the presence of anti-mannan antibody and complement. It was found that respiratory burst was highly correlated with phagocytic killing based on colony forming unit (R = 0.652), but the correlation was not statistically significant (p = 0.077). Taken together, these results demonstrate that anti-mannan antibody is required for efficient phagocytosis and phagocytic killing of <i> C. albicans</i> and complement does not appear to enhance antibody-mediated phagocytosis.</p><p>

<p> Women with Type 2 diabetes (T2D) have a 49% increase in breast cancer related mortality compared to women without T2D. Epidemiological studies report that increased endogenous insulin levels and increased insulin receptor (IR) expression are associated with poor survival in breast cancer patients. Therefore, it is essential to investigate the role of endogenous hyperinsulinemia on breast cancer progression. Presented in this thesis are contributions to understanding the effect of insulin in a mouse model of hyperinsulinemia (MKR mouse). First, data is shown that highlights the significant increase in primary MVT-1 tumors and pulmonary metastasis in the MKR mouse compared to Wild Type mice. The studies presented show that the primary tumors from the MKR mice have significantly higher Vimentin protein expression compared to primary tumors from control mice. Next, the studies determine that silencing Vimentin expression in the tumor cells leads to either decreased number of pulmonary metastasis in the hyperinsulinemic mice. The work in this thesis also establishes a novel immunodeficient hyperinsulinemic (Rag/MKR) mouse model that enabled the study of the effects of endogenous insulin on the progression of human cancer cells. The hyperinsulinemia of the Rag/MKR mice promoted a significant increase in tumor growth of MDA-MB-231 and LCC6 cells. The knockdown of the insulin receptor in the LCC6 cells led to primary tumors that were significantly smaller in both the hyperinsulinemic Rag/MKR and Rag/WT control mice compared to the tumors from the LCC6 control cells. Finally, it is shown for the first time that the knockdown of the IR promotes a reversal of the epithelial-mesenchymal phenotype by repressing mesenchymal markers and re-expressing epithelial markers in the LCC6 insulin receptor knockdown tumors. The data presented in this thesis highlight a potential contribution to the understanding of the role of insulin in the setting of hyperinsulinemia and provide potential targets for therapy to improve survival in women with breast cancer and hyperinsulinemia.</p>

<p> The molecular mechanisms governing gamma-globin expression in a subset of fetal hemoglobin (alpha₂:gamma₂; HbF) expressing red blood cells (F-cells) and the mechanisms underlying the variability of response to hydroxyurea induced gamma-globin expression in the treatment of sickle cell disease are not completely understood. To explore molecular differences in these conditions, a serum-free <i>in vitro</i> culture system suitable for large scale production of erythroblasts derived from primary human hematopoietic progenitors is optimized. The culture system recapitulates steady-state adult erythropoiesis and can support erythroid differentiation with the addition of cytotoxic hydroxyurea. Using this system, intra-person clonal populations of erythroblasts derived from bone marrow common myeloid progenitors were evaluated for molecular factors associated with gamma-globin expression. Data demonstrate that the level of fetal hemoglobin produced in F-cells negatively correlates with expression of BCL11A, KLF1 and TALL With the addition of hydroxyurea, successful induction of gamma-globin includes a further reduction in BCL11 A, KLF1 and TALI expression along with a decrease in SOX6 expression. These data suggests that expression changes in this transcription factor network modulate gamma-globin expression in F-cells during steady state erythropoiesis and after induction with hydroxyurea.</p>

<p> Meiosis is a highly conserved specialized cell division that occurs in many organisms, including budding yeast and mammals. Meiosis divides the chromosome number of the cell in half to create gametes for sexual reproduction. A single round of chromosome duplication is followed by two rounds of chromosome segregation, Meiosis I (homologs segregate) and Meiosis II (sister chromatids segregate). Proper segregation at Meiosis I requires that homologs are connected by both crossovers and sister chromatid cohesion. Crossovers are formed by the repair of double strand breaks (DSBs) preferentially by the homolog. The choice of repair template is determined at the time of strand invasion, which is mediated by two recombinases, Rad51 and the meiosis-specific Dmc1. Rad51 is necessary for Dmc1 to function properly but its strand exchange activity is inhibited both by Dmc1 and Mek1, a meiosis-specific kinase, which is activated by DSBs. Mek1 suppresses interaction between Rad51 and its accessory factor Rad54 in two ways. First, phosphorylation of Rad54 lowers its affinity for Rad51. Second, phosphorylation stabilizes Hed1, a meiosis-specific protein that binds to Rad51 and excludes Rad54. Although <i>RAD54</i> is not required for wild-type levels of interhomolog recombination, <i> rad54</i>&Delta; diploids exhibit decreased sporulation and spore viability, indicating the presence of unrepaired DSBs. My thesis tested the idea that Mek1 kinase activity is down-regulated after interhomolog recombination to allow Rad51-mediated repair of any remaining DSBs. </p><p> Meiotic recombination occurs in the context of a proteinaecous structure called the synaptonemal complex (SC). The SC is formed when sister chromatids condense along protein cores called axial elements (AEs) comprised of the meiosis-specific proteins, Hop1, Red1 and Rec8. AEs are brought together by interhomolog recombination, which creates stable connections and the gluing together of the AEs by the insertion of the transverse filament protein, Zip1, in a process called synapsis. Pachynema is the stage of meiotic prophase in which chromosomes are fully synapsed and where interhomolog recombination has proceeded to the double Holliday junction (dHJ) stage. </p><p> Meiotic progression requires transcription factor <i>NDT80</i>, a middle meiosis transcription factor required to express >200 genes, including the polo-like kinase, CDC5 (required for Holliday junction resolution and SC disassembly) and <i>CLB1</i> (required for meiotic progression). Diploids deleted for <i>NDT80</i> arrest in pachynema with unresolved dHJs. I used an inducible version of <i>NDT80</i> (<i>NDT80-IN </i>) to separate prophase into two phases: pre-<i>NDT80</i>, when interhomolog recombination occurs and post-<i>NDT80</i>, when it is proposed that inactivation of Mek1 allows intersister recombination to repair residual DSBs. <i>RAD54</i> is sufficient to function after interhomolog recombination, as inducing both <i>RAD54</i> and <i>NDT80</i> simultaneously rescues the spore inviability defects observed in <i>NDT80-IN rad54&Delta;</i> diploids. Using an antibody specific for phosphorylated Hed1 as an indicator of Mek1 kinase activity, I showed that Mek1 is constitutively active in <i>ndt80</i>-arrested cells and that induction of <i>NDT80</i> is sufficient to abolish Mek1 activity. Furthermore, inactivation of Mek1 by Ndt80 can occur in the absence of interhomolog strand invasion and synapsis. Mek1 inactivation correlates with the appearance of <i>CDC5</i> and the degradation of Red1. My work demonstrates that the sole target of <i>NDT80</i> responsible for inactivating Mek1 is <i>CDC5</i>. </p><p> Unrepaired DSBs trigger the meiotic recombination checkpoint resulting in prophase arrest, which requires Mek1 and works by sequestering Ndt80 in the cytoplasm. Mek1 also delays meiotic progression in wild-type cells, likely through inactivation of Ndt80. My work shows that Ndt80 in turn negatively regulates Mek1. Based on my observations, as well as published work showing that synapsis results in the removal of Mek1 from chromosomes, I propose that recombination and meiotic progression are coordinated by regulation of Mek1. </p>

<p> Ovarian cancer (OC) takes the lives of nearly 14,000 US women every year. Although platinum is one of the most effective drugs in treating ovarian cancer, the development of platinum resistance is one of the biggest challenges facing patients. I have shown that the long non-coding RNA HOTAIR contributes to platinum-resistant OC and determined the regulators and targets of HOTAIR during the platinum-induced DNA damage response. My published data supports the role of HOTAIR in contributing to DNA damage induced cellular senescence and secretion of pro-inflammatory cytokines leading to cisplatin resistance. My unpublished work (under review) analyzed the interaction of HOTAIR with the PRC2, its known interacting partner. In this study, I developed a novel strategy blocking HOTAIR-PRC2 interaction and resensitized ovarian tumors to platinum in mouse studies. The results offer a pre-clinical proof of concept for targeting long non-coding RNAs as a therapeutic approach and may represent a strategy to overcome chemotherapy resistance in tumors exhibiting high expression of HOTAIR, a frequent observation in high grade serous OC.</p>

<p>When a plant emerges from the soil, it faces a critical developmental transition from utilizing stored energy to grow rapidly toward the light, to developing chloroplasts and beginning photosynthesis. While it is known that this process involves massive transcriptional reprogramming of the nuclear and plastidial genomes, the connections between chloroplast development and nuclear light signaling events are not well understood. One very promising target for investigating these connections is HEMERA (HMR), a dual-localized regulatory protein that is found in both nuclei and chloroplasts. HMR was previously identified as pTAC12, an essential component of the plastid-encoded RNA polymerase complex responsible for transcription of chloroplast photosynthetic genes. In the nucleus, HMR acts within the phytochrome signaling pathway as a transcriptional co-activator of a subset of growth-relevant genes in response to light, to regulate the elongation of the embryonic stem, or hypocotyl. HMR’s combination of roles in the nucleus and chloroplasts are dramatically demonstrated by the phenotypes of the hmr mutant, with a long hypocotyl and albino leaves when grown in the light. </p><p>While the functions of HMR in each compartment have been studied separately, the mechanisms by which the HMR protein is targeted to each compartment have not yet been determined. To address this, I characterized the localization signals of HMR with a combination of in vitro approaches and characterization of transgenic Arabidopsis lines. These experiments revealed that HMR has a cleavable N-terminal chloroplast transit peptide within its first 50 amino acids, while two predicted nuclear localization signals proved not to be highly functional. Surprisingly, HMR in the chloroplasts and nucleus appeared to both be the same cleaved form of the protein. We thus identified the mature form of HMR by mass spectrometry, finding that it begins from lysine as the result of transit peptide cleavage and possibly additional N-terminal processing. Through GST pull-down assays, we determined that this mature form of HMR was fully capable of interacting light signaling components. However, analysis of transgenic lines showed that expression of mature HMR alone could not complement the long-hypocotyl phenotype of the hmr mutant. Analysis of the transcription of HMR nuclear target genes confirmed that mature HMR lacked nuclear functionality. </p><p>Further investigation revealed that mature HMR does not accumulate within the nucleus, most likely as a result of its nonfunctional nuclear localization signals. However, addition of the transit peptide from the small subunit of Rubisco fully restored nuclear accumulation and function of mature HMR in Arabidopsis. Additional experiments testing the localization of a simple model of dual-targeted proteins with two types of localization signal showed that transit peptides might take priority over nuclear localization signals. These results together suggest an unexpected model of localization where HMR is first targeted to the chloroplasts, and then it is subsequently re-localized to the nucleus, thus connecting its nuclear and plastidial functions. Further investigation of this proposed retrograde plastid-to-nucleus translocation pathway promises to shed additional light on the link between nuclear light signaling events and chloroplast development.</p> / Dissertation

<p> Here we present the results of two distinct projects in the field of cellular mechanics. In the first project, we describe a non-monotonicity in the scaling of force production in actomyosin stress fibers. We develop a continuum mechanical model to explain that non-monotonicity and, using both analytical and numerical techniques, conclude that the scaling is due to an interaction between different physical lengthscales inherent in the actomyosin force-production system. Using singular perturbation methods, we study the model further to make predictions about the physical conditions under which a cell can break symmetry. In the second project, we explore how lipid flow in the plane of the plasma membrane contributes to membrane translocation during cell migration. By numerically solving the Stokes equations, we quantify the magnitude of the force necessary to generate this flow, and analyze how the presence of transmembrane protein obstacles influences the resulting front-to-rear membrane tension gradient. We make several analytic estimates of the mechanical importance of this membrane tension for cell motility.</p>

<p> Chronic alcohol induced liver disease (ALD) comprises of a spectrum of disease stages progressing from fatty liver, steatohepatitis, fibrosis, to cirrhosis that may eventually lead to death. Although, the early stages of ALD are reversible, 40% of the patients develop advanced stage liver disease characterized by significant hepatic fibrosis and cirrhosis, for which, currently, liver transplantation is the only curative approach. However, the number patients waiting for liver transplantation far exceeds the meager number of available donors resulting in premature mortality of such patients. Hence, there is an urgent need for therapies for not only prevention and early intervention to stop the disease progression, but also to effectively regenerate the remaining healthy liver so that the patient can be reasonably functional before they can fully recover with a liver transplantation. Thus, any biologically natural modulator that can effectively prevent the progression of ALD until the donor liver is available for transplantation would be desirable even if it cannot completely cure the disease. </p><p> Thymosin &beta;4 (T&beta;4) is an immune modulating natural peptide secreted by thymus gland that is known to prevent inflammation and fibrosis, and promote wound healing and regeneration in the eye, skin and heart. Previous work from our laboratory has also shown that T&beta;4 protects against carbon tetrachloride induced acute liver injury in rat. However, not much is known of the role of T&beta;4 in alcoholic liver injury. Therefore, in this dissertation research, the role of T&beta;4 was investigated in acute on chronic ethanol and lipopolysaccharide (LPS) induced hepatic oxidative stress, inflammation, and fibrosis in an in vivo mouse model, as well as its regenerative potential was studied in chronic ethanol fed mice after partial hepatectomy. Furthermore, the underlying molecular mechanism by which T&beta;4 exerts its action, particularly on fibrosis was examined using human hepatic stellate cells (HSC), the main fibrogenic cells of the liver. </p><p> Based on the well accepted two-hit model for ALD, in the hepatocytes, ethanol acts as the first hit and is oxidized to acetaldehyde, the highly toxic first metabolite of ethanol oxidation by alcohol dehydrogenase (ADH) and ethanol-inducible cytochrome P450 2E1 (CYP2E1) leading to the generation of reactive oxygen species (ROS), resulting in oxidative stress. On the other hand, ethanol-induced leaky gut results in the release of endotoxin (LPS) that acts as the second hit and activates nuclear factor Kappa B (NF?B) in the Kupffer cells and the subsequent production of the pro-inflammatory cytokines that propagates liver inflammation. ROS and the pro-inflammatory cytokines act as fibrogenic stimuli for the activation of HSC and their trans-differentiation from quiescent lipid storing phenotype to activated myofibroblasts that express fibrogenic genes and proliferate and migrate to the site of injury and form a fibrous scar, resulting in fibrosis. This is essentially due to the fact that the quiescent HSC exhibit up-regulated adipogenic gene, peroxisome proliferator-activated receptor gamma (PPAR&gamma;), and down-regulated fibrogenic gene, methyl CpG binding protein (MeCP2), whereas the reverse is true upon their activation to myofibroblasts. </p><p> The experimental results showed that T&beta;4 reduced the ethanol and LPS induced levels of ROS by increasing the levels of the antioxidants, glutathione and superoxide dismutase. It also inhibited the nuclear translocation of NF&kappa;B by blocking the phosphorylation of the inhibitory protein I&kappa;B and thereby prevented the up regulation of pro-inflammatory genes, TNF-&alpha;, IL-1&beta;, and IL-6. T&beta;4 also prevented the activation of HSC by up-regulating miRNA 132, thus suppressing MeCP2, that coordinately reversed the down-regulated adipogenic gene, PPAR&gamma;, and the up-regulated fibrogenic genes (&alpha;-smooth muscle actin, PDGF-&beta; receptor, collagen 1, and fibronectin), and fibrosis. Moreover, T&beta;4 also promoted liver regeneration after partial hepatectomy in chronic ethanol fed mice by increasing hepatocyte growth factor and its receptor, c-Met; &alpha;-fetoprotein; proliferation markers, proliferating cell nuclear antigen and Ki-67 as well as the liver progenitor cell marker, cytokeratin 19.</p><p> Furthermore, it was discovered that in human HSC cultures, T&beta;4 prevented PDGF-BB induced fibrogenesis and also abolished PDGF-BB induced HSC proliferation and migration by blocking the phosphorylation of Akt by preventing the binding of Akt to actin. Moreover, experiments with two bioactive peptides of T&beta;4, the amino terminal peptide (1-15 aa) and the actin binding peptide (17-23 aa) revealed that T&beta;4 exerts most of its anti-fibrotic actions <i> via</i> its actin binding domain.</p><p> In conclusion, these data suggest that T&beta;4 has antioxidant, anti-inflammatory, anti-fibrotic and hepatic regenerative potential against alcoholic liver injury. </p>