<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> 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> 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>

<p> Throughout development, canonical Wnt signaling contributes to the formation and maintenance of a wide array of cells, tissues, and organs. Dys-regulated Wnt signaling during embryonic development is implicated in developmental defects known as neurochristopathies, including craniofacial and heart defects, as well as defects in neural development. Due to its roles in stem cell maintenance and self-renewal, tissue homeostasis, and regeneration, aberrant Wnt signaling in adult tissues can result in various forms of cancer, including colorectal cancer, breast cancer, lung cancer, and gastro-intestinal cancer, among others. Dys-regulated Wnt signaling is also implicated in other pathologies including bone disease, and metabolic diseases, such as Type II diabetes. Our lab has previously identified a novel crosstalk between canonical Wnt signaling and ephrin signaling. Ephrin signaling occurs through the interaction of ephrin ligands and Eph receptor tyrosine kinases, and is bidirectional. Due to the roles of ephrin signaling in tissue development and maintenance, aberrant ephrin signaling is implicated in many diseases including bone remodeling diseases, diabetes, and cancer. The molecular mechanism of the crosstalk between canonical Wnt signaling and ephrin-B signaling remains unknown. &beta;-catenin is a key intracellular effector of canonical Wnt signaling that transduces the signal to the nucleus, where &beta;-catenin interacts with the TCF/LEF transcription factors and activates transcription of target genes. Due to its central role in transducing the canonical Wnt signal to the nucleus, we predict that ephrin-B signaling antagonizes canonical Wnt signaling by affecting the stability and/or sub-cellular localization of &beta;-catenin, or the interaction between &beta;-catenin and TCF/LEF transcription factors. By employing mouse ephrin-B constructs in human cell lines, we show that the canonical Wnt - ephrin-B crosstalk is conserved between frogs and mammals. We also found that ephrin-B antagonism of canonical Wnt signaling is likely independent of ubiquitin proteasome system (UPS)-mediated degradation of &beta;-catenin. Furthermore, confocal immunofluorescence microscopy revealed that overexpression of ephrin-B in HEK293T cells treated with lithium chloride (LiCl) seems to promote membrane localization of &beta;-catenin, particularly at the apical Z sections. These results suggests that re-localization of &beta;-catenin to the cell membrane may contribute to the ephrin-B antagonism of canonical Wnt signaling.</p>

<p>Many RNA viruses, including enteroviruses, remodel host ER membranes to form platforms with unique lipid components to assemble replication complexes and synthesize new viral RNA. Cholesterol is a critical component of cellular membranes regulating fluidity and being indispensable for proper assembly and function of membrane based protein-lipid complexes. Here we show that enteroviruses harness the clathrin mediated endocytosis (CME) pathway to transfer free cholesterol from the plasma membrane to the viral replication organelles (VROs). We show that cholesterol is responsible for regulating viral protein processing and facilitates viral RNA synthesis, and disrupting CME causes cellular cholesterol pools to be stored in lipid droplets obstructing the transfer to VROs and inhibiting viral replication. In contrast, we found that the presence of excess intracellular cholesterol, as in cells lacking caveolins or those from patients with Niemann-Pick disease, stimulates viral replication. We demonstrate that, the redistribution of free cellular cholesterol and the cellular recycle dynamics are affected during infection. The CME rate of uptake does not change during the initial 2 hours of infection while the rate of cellular endosomal recycling is inhibited resulting in a net decrease of free cholesterol at the plasma membrane, and facilitating the access and active transfer of cholesterol from enriched internal cellular compartments to VROs. Our findings indicate that cholesterol is critical for enteroviral replication and that CME has an important role in the enteroviral life cycle and in the host cellular cholesterol homeostasis. </p>

<p> COPD is characterized by an abnormal regulatory T cell (Treg) response with a shift towards a Th1 and Th17 cell responses. However, it is unclear if the function of Treg cells is impaired by smoking and in COPD. In addition, the miRNA profile of Treg cells in COPD is unknown and whether miRNA deregulation contributes to COPD immunopathogenesis. We set the objective to study Treg cell function isolated from peripheral blood of patients with COPD versus controls and to compare their miRNA profiles. We also were interested in exploring the function of some of the differentially expressed Treg cell miRNAs. We assessed the Treg cell function by observing their suppressive activity on autologous effector T cells and analyzed their miRNA expression initially by microarray analysis then conducted real time RT-PCR validation for selected miRNAs. In Silico target gene analysis for the validated miRNAs suggested that miR-199-5p is particularly relevant to Treg cell physiology so its function was investigated further using CCD-986Sk and MOLT-4 cells. We found no difference in Treg cell function between COPD and controls but we were able to identify 6 and 96 miRNAs that were differentially expressed in COPD versus control Treg cells. We confirmed that miR-199a-5p was repressed by approximately 4 fold in Treg cells of COPD patients compared to cells in healthy smokers. Importantly, miR-199a-5p had significant overrepresentation of its target genes in the Treg cell transcriptome, with many targets associated with the TGF-&beta; activation pathway. We also confirmed the function of miR-199a5p in an in-vitro loss-of-function cell model running TaqMan&reg; arrays of the Human TGF-&beta; Pathway. These findings suggest that the abnormal repression of miR-199a-5p in patients with COPD compared to unaffected smokers may be involved in modulating the adaptive immune balance in favor of a Th1 and Th17 response.</p>

<p> The yeast vacuole is a dynamic organelle that is functionally analogous to the mammalian lysosome and serves as a model for the study of membrane fusion and fission. Mechanisms of membrane fission and fusion dynamics have been well conserved from yeast to humans. However, the regulatory mechanisms that govern cellular fission and fusion dynamics remain poorly understood. Our lab has previously established that Env7 is a conserved yeast palmitoylated protein kinase that localizes to the yeast vacuole and negatively regulates vacuole membrane fusion during budding and hyperosmotic stress. Phosphorylation of Env7 is dependent on another vacuolar membrane kinase, Yck3, and is essential to Env7 stability and negative regulation of vacuolar membrane fusion. In this study, we aim to further our understanding of the role Env7 plays at the vacuole by 1) characterizing the phosphorylation of Env7 as a function of cell cycle using cell cycle arrest and synchronization techniques, and 2) generating functional biochemically tagged Yck3 to be used in interaction and phosphorylation assays with Env7. Cell cycle arrest and synchronization techniques have not previously been established in our lab. Here, we report reliable protocols of inducing cell cycle arrest using &alpha;-factor mating pheromone and Hydroxyurea. Results show that Env7 is hyperphosphorylated when cell cycle is arrested at G₁ phase using &alpha;-factor mating pheromone. In both cell cycle arrest approaches, vacuoles show significant increase in fragmentation, and Env7 remains localized to the membrane of fragmented vacuoles. In cell culture synchronized with &alpha;-factor, Env7 shows an increase in phosphorylation between S-phase and G₂, with decreased phosphorylation in M and G₁. We were successful in engineering biochemically tagged Yck3 and established that the expressed 6XHis-Yck3 is functional and able to restore phosphorylation of Env7 <i>in vivo</i>. We also established that overexpressed 6XHis-Yck3 localized correctly to the vacuolar membrane. These tools will be used in future studies on interactions and regulation of membrane fusion.</p><p>

<p> My research using the model plant <i>Arabidopsis thaliana </i> is focused on unraveling the signal transduction pathways involved in elicitor-mediated plant defense, particularly defense pathways involved in resistance to fungal pathogens. I have isolated a group of related genes, the <i>ATL</i> family, which appear to play a direct role in defense against fungal pathogens. Previous research has shown that ATL proteins can be induced by chitin and they are involved in basal resistance to the fungal pathogens. </p><p> ATL9, an <i>Arabidopsis</i> RING zinc finger protein, is an E3 ubiquitin ligase that can be induced by chitin and is involved in basal resistance to the fungal pathogen, <i>Golovinomyces cichoracearum</i> (<i>G. cichoracearum</i>). In order to understand the expression and regulation of ATL9, I studied the expression pattern of <i>ATL9</i> and the functions of its different protein domains. Using a p^ATL9:<i> GUS</i> transgenic <i>Arabidopsis</i> line I found that ATL9 is expressed in different tissues in <i>Arabidopsis</i> at various developmental stages and that GUS activity was induced rapidly upon wounding. Previous research in our lab also showed that ATL9 is a short-lived protein within plant cells and it is degraded via the ubiquitin-proteasome pathway. Protein prediction software indicated that ATL9 contains two transmembrane domains (TM), a RING zinc-finger domain, and a PEST domain. Data from confocal microscopy and western analysis indicate that both the PEST domain and the RING domain have effects on ATL9 degradation. To study the importance of these domains in ATL9's function, I constructed a series of deletion mutants and generated transgenic <i>Arabidopsis</i> plants. As expected, transgenic <i> Arabidopsis</i> containing the deletion constructs showed that both the RING domain and the TM domains are important to its resistance phenotype against <i> G. cichoracearum</i>. Interestingly, the PEST domain was also shown to be significant for the resistance to fungal pathogens. Additionally, I discovered that ATL9 can bind the defense related proteins FBS1, PCC1, and PDF1.2 directly and degrade them via the proteasome. Finally, I propose a hypothesized mechanism is proposed describing the function of ATL9 and its possible interaction with other proteins in enhancing the plant defense response.</p><p>

<p> Over half of all pregnancies worldwide occur in malaria endemic regions. Placental malaria, a serious condition caused by the malaria parasite <i> Plasmodium falciparum</i>, occurs when malaria-infected red blood cells adhere to the tissue of the placenta, with potentially devastating consequences for both mother and infant. Placental malaria infections are responsible for approximately 30% of preventable low birth weight newborns, 20% of stillbirths, and 200,000 infant deaths per year in Africa alone. Placental malaria infection is mediated by VAR2CSA, a <i>P. falciparum</i> protein that is expressed by the parasite only when in a pregnant woman, and translationally repressed outside of pregnancy. However, the mechanisms by which this repression and expression occur or, indeed, how the parasite senses when its host is pregnant are unknown. Elucidation of the genetic determinants of this specific translational regulation could provide insight for therapeutic development for placental infection. Additionally, further study of overall translation and its pharmacologic inhibition under &ldquo;normal&rdquo; circumstances may help identify novel therapies for malaria in general.</p><p> Utilizing a novel <i>in vitro</i> translation system derived from <i>P. falciparum</i> cultures, I have shown that synthesis of VAR2CSA is repressed under normal conditions, and that multiple elements in the 5&rsquo; untranslated region of the <i>var2csa</i> gene contribute to this repression. Further, this repression occurs only in <i>P. falciparum </i>, and not mammalian <i>in vitro</i> translation systems, indicating a <i>P. falciparum</i>-specific mechanism of inhibition. Importantly, I have found that circulating factors present in maternal serum during the first and second trimesters of pregnancy relieve repression of VAR2CSA translation, identifying two placental enzymes as candidate factors. Both enzymes serve to alter the pH of the microenvironment and, in fact, increasing pH in the <i>P. falciparum</i> <i>in vitro </i> translation system mimics the increase in VAR2CSA production induced by pregnant serum. Separately, I have utilized this <i>in vitro</i> translation system to identify inhibitors of translation among clinically approved antimalarial drugs and found that none utilize this mechanism of action. Importantly, this disproved the recent assertion that mefloquine inhibits translation, while also underscoring the therapeutic potential for targeting the translational apparatus as a novel and orthogonal mechanism of action. </p><p>