The infections agent of Lyme disease, Borrelia Burgdorferi is a complex microorganism with a highly diverse genome. One of the most remarkable aspects of the B. burgdorferi genome is the large number of sequences encoding predicted or known lipoproteins, including outer-surface proteins. The B. burgdorferi genome encodes no recognizable toxins. Instead, this extracellular pathogen causes pathology by migration through tissues, adhesion to host cells, and evasion of immune clearance. Inflammation elicited by infection with B. burgdorferi depends on the ability of the spirochete to survive in the mammalian host, as well as the immune response that arises upon the interaction of the bacterium with phagocytic, T and other cell types. Innate immune responses are critical in recognition and clearance of pathogens, and also play an important role in the outcome of adaptive immune responses. The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns (PAMPs) and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. In this study we demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK also controls tumor necrosis factor production and TLR-mediated macrophage responses to B. burgdorferi. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete. MCJ is a newly identified member of the DnaJ protein family of cochaperones that contains unique features different than the normally described DnaJ proteins. However, there is little known about its function and the role it plays in different cells and systems. It has been previously shown that MCJ is required for the repression of the ABCB1 drug transporter expression in breast cancer cells, and that this repression is mediated through the control of c-Jun protein stability. We were therefore interested in determining the role that MCJ plays in macrophages in response to B. burgdorferi antigens. We now provide evidence that MCJ controls inflammatory responses of macrophages through the regulation of c-Jun protein stability, and the expression and release of the inflammatory cytokine TNF, through the regulation of the expression of TNF converting enzyme (TACE) inhibitor tissue inhibitor of metalloproteinase 3 (TIMP-3).

Lymphatic filariasis is a human parasitic disease which causes great physical suffering, while also having a substantial economic impact on the peoples of the tropics and sub-tropics. In order to collect accurate epidemiological data which are crucial for designing effective control programs, reliable diagnostic and taxonomic information is needed. The studies described here were designed to obtain DNA sequence data from specific coding and non-coding loci of the parasite genome, which could augment existing morphological, biochemical, and ecological data, to test hypotheses concerning diagnostic and taxonomic classification. Highly repeated DNA elements, members of the Hha I repeat family, were cloned, sequenced and the data employed in a phylogenetic analysis of species, sub-species and distinct geographic isolates of human filarial parasites from the genus Brugia. Comparative analysis reveals that although the 322 base pair (bp) repeat sequences between Brugia pahangi and Brugia malayi are nearly 90% identical overall, there is a small 70 bp region which contains enough divergence to clearly distinguish between these two major species. Nucleotide differences in this and other regions were exploited to draw distinctions between repeats cloned from B. timori, B. patei and various geographic isolates of B. malayi which differ in biological characteristics such as host range, vector preference and periodicity. In addition to the Hha I repeats, the gene encoding a prominent, stage-specific surface antigen from the animal parasite, Brugia pahangi was also cloned and sequenced. Homologous sequences were obtained from the related human pathogen Brugia malayi and a comparative analysis initiated. The results show that the protein coding, flanking and intervening sequences are highly conserved between the two species. In addition to its utility as a taxonomic and phylogenetic tool, the highly conserved nature of this protein sequence makes it a potential candidate for recombinant vaccine development.

No description available.

<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> Extracellular vesicles (EVs) are small, lipid compartments that function in the long-distance transport of proteins, nucleic acids and other metabolites. In mammals, EVs are important vehicles of intercellular communication and play a crucial role in modulating immune responses. Plant cells also secrete EVs, particularly in response to infection, but the contents of these vesicles have not been analyzed and their function is unknown. To better understand plant EVs and their roles in defense and signaling, I first pioneered methods for isolating and purifying EVs from the intercellular wash of <i>Arabidopsis </i> leaves. Secondly, I examined the protein and RNA contents of purified EVs. Proteomic analyses revealed that <i>Arabidopsis</i> EVs are enriched for defense- and stress-related proteins. Consistent with this finding, EV secretion was enhanced in response to biotic stress. Furthermore, a collaboration with Blake Meyer&rsquo;s laboratory at the Donald Danforth Plant Science Center identified several species of small RNA in purified EV samples, as well as a surprising enrichment for tiny RNAs (tyRNAs) 10 to 17 nt long. Finally, by examining the interactions between <i>Arabidopsis</i> EVs and the plant pathogenic fungus <i>Colletotrichum higginsianum</i>, I discovered that plant EVs associate with fungal structures and affect developing fungal morphology. Combined, my research represents a significant advancement in the field of plant EV research. It provides strong evidence for the involvement of plant EVs in the immune response and suggests they are capable of trafficking proteins and RNAs into invading pathogens.</p><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>