No description available.

No description available.

No description available.

<p>Telomeres are protein-DNA structures at the ends of eukaryotic chromosomes. The DNA portion is comprised of double-stranded and single-stranded G-rich repetitive DNA. The protein portion is anchored by the "shelterin complex" composed of six proteins. Inappropriate DNA repair and telomere length dysregulation result in cell cycle arrest, genome instability, and carcinogenesis. Thus, this DNA/protein structure protects telomere ends and regulates telomere length.</p><p>The shelterin component TRF1, a double-stranded telomeric DNA binding protein, was found to bind accessory protein PinX1 at mitosis. Given this, I investigated the effect of reducing PinX1 level on cell cycle progression and apoptosis. I found that reducing PinX1 expression with shRNA, as assessed by immunoblot, led to delayed entry into mitosis and elevated levels of apoptosis in human cells. These results indicated that PinX1 plays an important role in mitosis progression and cell viability.</p><p>Intriguingly, binding of PinX1 to TRF1 at mitosis increased the stability of the latter. Moreover, PinX1 binds to the same site on TRF1 as the protein TIN2, which can suppress degradation of TRF1 by inhibiting poly ADP-ribosylation of TRF1 by the enzyme tankyrase. Collectively, these results suggested that TIN2 might be released from TRF1 to promote the binding of PinX1 on TRF1 at mitosis. Given that proteins are often regulated in the cell cycle by phosphorylation, I investigated whether TIN2 was phosphorylated at mitosis. To this end, I performed phospho-proteomic analysis of human TIN2, which revealed two phosphorylated residues, serines 295 and 330. Both sites were phosphorylated specifically during mitosis, as detected by two independent approaches, namely Phos-tag reagent and phosphorylation-specific antibodies. Phosphorylation of serines 295 and 330 appeared to be mediated, at least in part, by the mitotic kinase RSK2 in vitro and in vivo. The identification of these specifically timed post-translational events during the cell cycle demonstrates the mitotic regulation of TIN2 by phosphorylation. However, as expressing non-phosphorylatable mutants of TIN2 failed to reveal any overt phenotypes, the consequences of these phosphorylation events remain to be determined.</p><p>Lastly, the TRF1-related double-stranded telomeric DNA binding protein, TRF2, was shown to associate with another shelterin component, POT1. POT1 forms heterodimer with TPP1 to bind single-stranded telomeric DNA. Previous research found that mutations of POT1 with reduced binding affinity to either TRF2 or to TPP1 cause distinct phenotypes. To determine whether similar separation-of-function mutants could be generated to dissect the function of POT1s in mice, which are encoded by two genes, Pot1a and Pot1b, I screened a panel of substitution mutants of mPOT1a for loss of binding to mTRF2 and mTPP1. These studies revealed that mPOT1a does not bind mTRF2, but the association with mTPP1 could be disrupted.</p><p>In summary, the described studies have shed insight into the complexity of shelterin regulation, and in particular, highlighted protein-protein interactions and post-translational modifications.</p> / Dissertation

<p>Every day, cells face agents that generate lesions in genomic DNA, which can interfere with the processes of DNA replication and gene expression. These lesions can range from small abasic sites to alkylated bases to large proteins frozen on the DNA and can be caused by both endogenous and exogenous agents. These lesions must be repaired to maintain genomic stability, and multiple pathways exist to perform the necessary repairs or to bypass the damage. These pathways have been discovered and studied using a variety of experimental techniques, both in vitro and in vivo. While these studies have contributed valuable information about many of cellular processes, there are still gaps in the DNA repair field.</p><p>The goal of this study is to bridge some of those gaps by constructing a system to introduce DNA containing a site-specific lesion into Escherichia coli cells at high enough levels to monitor the lesion's fate in vivo and in real time. This system combines two separate DNA molecules to simplify the introduction of a site-specific lesion. The first molecule is the DNA from bacteriophage &#955;, a virus that is able to infect E. coli cells at a high level of efficiency. A typical commercial packaging reaction can yield titers of approximately 1.0 x 109 plaque forming units (PFU)/mL. However, bacteriophage &#955; has a large genome of approximately 48.5 kb, which makes it a difficult substrate for extensive cloning and manipulation. In contrast, cloning and manipulation of a small plasmid (~4 kb) is a much simpler endeavor, and small plasmids have been used previously to produce DNA containing a site-specific lesion. The problem with using a plasmid occurs when attempting to introduce it into cells, as the process of transformation is not very efficient and can cause unintended consequences in the cells. This new system allows for the incorporation of the lesion into the plasmid, which is then integrated into a bacteriophage &#955; vector, &#955; Kytos. The combination of these two molecules produces bacteriophage &#955; DNA containing a site-specific lesion, which can infect the cells at high efficiency, allowing the fate of the DNA to be monitored in real-time. Interestingly, repair of a single EthenoA lesion after infection appears to be a very inefficient process. Even if the repair system is activated by induction with methyl methanesulfonate (MMS) or if individual repair proteins are overexpressed, little to no repair occurs. As methylation occurs upon injection, it does appear that the DNA is exposed to proteins in the cell, including any repair proteins present. These results indicate that other processes, perhaps replication or transcription, are required to repair a single EthenoA lesion in vivo.</p> / Dissertation

No description available.

<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>