<p> Bronchopulmonary dysplasia (BPD) is a serious respiratory complication for the preterm newborn characterized clinically by prolonged respiratory distress and histologically by alveolar simplification and decreased pulmonary vasculature. The development of BPD is well linked to oxidative stress suffered by the newborn as a result of a preterm fetal-neonatal transition, supplemental oxygen, infection, increased inflammation, and mechanical ventilation. Damage suffered by oxidative stress may be through direct mechanisms or through alteration of redox&not;sensitive pathways involved in cell death, cell survival, differentiation, and proliferation. Redox&not;sensitive modifications regulating protein function and redox-sensitive pathways have mainly been ascribed to oxidative modification of cysteine thiols. As their modification is critical for protein function, maintenance of the thiol redox status is crucial. Thioredoxin-1 (Trx1) functions in maintenance of thiol redox homeostasis, and its redox activity is intimately linked to antioxidant, cytoprotection, proliferation responses, and cytoprotection. While Trx1 targets of redox regulation have been identified, we hypothesize that additional protein may be redox regulated and that Trx1 target profiles may change during oxidative stress. Therefore a novel immunoprecipitation approach, identified as the substrate trap approach, was developed to identify Trx1 targets. The following demonstrates the use of the substrate trap approach for identification of Trx1 redox targets and further application of the approach to identify alterations in target profiles in response to oxidative stress. Use of nuclear targeted substrate trap was successfully employed to enrich from nuclear Trx1 targets. As a final component the characterization of the Trx1 system in mouse from late embryonic development through the first week of life animals were exposed to room air or hyperoxia (model of BPD). Characterization indicates impairment of the Trx1 system in response to hyperoxic injury. As Trx1 is known to regulate proliferation, cell death, survival, differentiation pathways, impairment of the Trx1 system during early neonatal development may potentiate hyperoxic injury and alterations in lung development. Better understanding of Trx1 interactions occur through the substrate trap in a physiological model of BPD will help elucidate redox-signaling pathways involved in BPD pathogenesis.</p>

<p> The use of epigenetic modifying drugs such as DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) is becoming more common in the treatment of cancer. Currently, there is a profound interest in determining predictive biomarkers for patient response and the efficacy of known and novel drugs. There are likely distinct &ldquo;epigenetic profiles&rdquo; defined by the location and abundance of DNA methylation patterns and histone modifications. Here we propose to investigate the response of a selected subset of genes to particular DNMTi and HDACi treatments, in two human cancer cell lines, colorectal carcinoma HCT-116 and liver adenocarcinoma HepG2. In this study we identified unique epigenetic profiles based on microarray and bioinformatics derived epigenetic data that are predictive of the response to epigenetic drug treatment. Microarray studies were used to identify re-activated genes common in two different cancer cell types treated with epigenetic drugs. Bioinformatics data was compiled on these genes and correlated against re-expression to construct the genes&rsquo; &ldquo;epigenetic profile&rdquo;. We then verified the response of the select group of genes in HCT-116 and HepG2 upon treatment at varying concentrations of epigenetic drugs and illustrated that selective reactivation of the target gene. Additionally, two novel genes were introduced and one selectively activated over another. </p><p> Further research would prove invaluable for the medical and drug development communities, as a more extensive model would certainly be of use to determining patient response to drug treatment based on their individual epigenetic profile and leading to more successful novel drug design.</p>

<p> Sphingolipids (SL) are essential components of eukaryotic cells involved maintaining membrane integrity. They are important for membrane trafficking and function in signaling as messenger molecules. SLs are synthesized <i> de novo</i> from very long chain fatty acids (VLCFA) and sphingoid long-chain bases (LCB), which are amide-linked to form ceramide and further processed by addition of various head-groups. Little is known concerning the regulation of VLCFA levels and how cells coordinate their synthesis with the availability of LCBs for SL synthesis. Here I show that Elo2, a key enzyme of VLCFA synthesis, is controlled by signaling of the guanine nucleotide exchange factor Rom2, initiating at the plasma membrane. This pathway controls Elo2 phosphorylation state and VLCFA synthesis. My data identify a regulatory mechanism for coordinating VLCFA synthesis with SL metabolism and link signal transduction pathways from the plasma membrane to the regulation of lipids for membrane homeostasj.</p>

<p> <i>Chromobacterium violaceum</i> (CV) produces a violet color pigment known as Violacein. It has been reported that violacein has anticancer activity. This compound is produced by CV a gram-negative facultatively anaerobic bacterium found in soil and water environmental samples. The purpose of this study was to determine the effect of purified violacein on select cancer cell lines. Violacein used in this study was purified from CV strain (14N23), a strain isolated from environmental samples collected in the Tennessee Copper Basin. The previous reports used a crude extract preparation of violacein; thus, it was of interest to determine the effect of the pure compound on cancer cell growth was similar to that of the crude extracts. The compound purified following the method of Mehta, et al. was exposed to cancer cells and cell death assessed using the Alamar Blue procedure. It was found that violacein had no effect on A549, BT549, and PC3 cancer cell growth; however, there was a significant effect on Colo-320 cancer cells. It was concluded that further studies are required to assess the effect of violacein on enzymes and proteins involved in the cancer cell apoptotic pathways. Such studies will explain why cancer cell death was observed in certain cancer cells and not others.</p>

<p> Angiogenesis, the formation of new blood vessels, requires the reorganization of microtubules (MT), which are polar cytoskeletal structures, consisting of a free&#129;-plus end and a minus&#129;-end that is free or anchored. CAMSAP2 and CAMSAP3 have been shown to bind and stabilize MT minus&#129;-ends; yet, this activity&#129;'s contribution to MT organization and directional migration is unknown. To investigate this contribution, we performed live&#129;-cell imaging of polarized HUVECs expressing CAMSAP2 or CAMSAP3. Our results show that CAMSAP2 and CAMSAP3 localized to the trailing edge of cells. Pharmacologic disassembly of MTs resulted in CAMSAP reorganization to the leading edge. MCAK expression is not sufficient for CAMSAP reorganization, but may recruit CAMSAP to the MT minus&#129;-end. MT growth dynamics analysis revealed that CAMSAP2 and CAMSAP3 promoted dynamic MT growth. These results suggest that CAMSAP2 and CAMSAP3 protect MTs against MCAK&#129;]mediated disassembly and also function to nucleate new, dynamic MTs at the leading edge.</p>

<p> Most of the colon cancer patient tumors progress to metastases, despite undergoing surgical resection or adjuvant chemotherapy. Predicting which patients will progress to metastases has been extremely challenging. There is an urgent need to identify early novel prognostic biomarkers that can early on predict the patient outcome. Vitamin C has been shown to have a pro-oxidant effect on cancer that enhances tumor growth and survival. Vitamin C is transported into mammalian cells via two isoforms of sodium-dependent vitamin C transporters (SVCTs), SVCT1 and SVCT2. The expression and clinical implications of SVCTs in tumor tissues could help us investigate its prognostic value in predicting patient outcome. In this report, we performed immunohistochemistry to determine SVCT1 and SVCT2 expression on primary tumors of 178 colon cancer patients. Colon cancer cells selectively expressed SVCT2 but not SVCT1. Moreover, poorly differentiated and metastatic tumors correlated with higher SVCT2 expression. Furthermore, increased SVCT2 expression was associated with shorter progression-free survival in patients with no or little lymph node invasion. We confirmed that SVCT2 could be an early stage prognostic biomarker that can predict colon cancer disease progression and survival.</p><p>

<p> Manipulating the phenylpropanoid (Pp) pathway has been of great focus to bio-engineers as this pathway is responsible for production of many compounds that are important to human health for their known antioxidant, anti-viral, anti-inflammatory, anti-allergenic and vasodilatory properties. The secondary by products of the Pp pathway are important for the physiological well-being of the plant as it contributes to plant&rsquo;s ability to tolerate changing environment. Plant bio-engineering, involves manipulating gene expression of proteins that regulate functional proteins which are known to attribute to stress tolerance. Our research focused on one such regulatory protein called the 14-3-3 lambda (14-3-3&lambda;) protein and its effects on anthocyanin production in two different plants: a plant model <i>Arabidopsis thaliana </i> (<i>A. thaliana, Columbia-0</i>), and a naturally drought tolerant resurrection plant <i>Selaginella lepidophylla</i> (<i> S. lepi</i>). Due to their structural characteristics the family of 14-3- 3 proteins bind to many different client proteins and hence can function as signaling factors in eukaryotes. Anthocyanins are anti-oxidants produced in plants that alter plants physiology to resist stress. The goal of this study was to establish which nodes in the anthocyanin synthesis pathways are influenced by 14-3-3&lambda; in both <i>A. thaliana</i> and <i>S. lepi </i>. Data from this study established the steps in the Anthocyanin pathway that 14-3-3&lambda; affects to alter anthocyanin production during normal hydration and drought stress states. Based on our published studies and experimental data we have identified that the 14-3-3&lambda; isoform is playing a significant role in the anthocyanin pathway during drought stress. Using a reverse genetics approach, the amounts of secondary anthocyanin metabolites produced in a 14-3-3&lambda; knockout mutant were compared to the wild-type <i> A. thaliana</i> during normal hydration and drought conditions. Analytical techniques such as high performance liquid chromatography (HPLC) and liquid chromatography-Mass Spectrometry (LC-MS/MS) in combination with open access databases were used for metabolite profiling. The metabolite profile lead to candidate metabolites that differed between the drought-treated and hydrated groups in the knockout mutants and wild-type. Identification of these metabolites determined the nodes of Pp pathway that were affected by 14-3-3&lambda;, namely the enzymes chalcone synthase and chalcone isomerase. These findings in <i> A. thaliana</i> were expanded in the naturally drought resistant plant <i> S. lepi</i> using similar analytical approaches employed in <i> A. thaliana</i>. The results proved that 14-3-3&lambda; affects biosynthesis of anthocyanin during drought stress in <i>A. thaliana</i> and <i> S. lepi</i> in a similar manner, hence suggesting a similar role of 14-3-3&lambda; in the production of anthocyanins in both the plants.</p><p>

<p> The eukaryotic cytoskeleton is composed of varying proteinaceous filaments and is responsible for intracellular transport, cell proliferation, cell morphogenesis, and cell motility. Microtubules are one of three cytoskeletal components and have a unique polymer structure. The hollow cylinders undergo rapid polymerization and depolymerization events (<i>i.e.</i> dynamic instability) to promote assembly at the leading edge of the cell and disassembly in the rear of the cell to drive the cell front forward and facilitate directional migration. High-resolution light microscopy and automated tracking allow visualization and quantification of microtubule dynamics (<i>i.e.</i> growth speeds and growth lifetimes) during time-lapse imaging. These techniques were used to understand how the physical environment influences molecular control of endothelial cell morphology. The ultimate goal of this work is to test hypotheses relevant to vascular development and diseases associated with endothelial cell angiogenesis &ndash; defined as the development of new blood vessels from pre-existing vessels. Angiogenesis is of particular relevance because it is a commonality underlying many diseases affecting over one billion people worldwide, including all cancers, cardiovascular disease, blindness, arthritis, and Alzheimer's disease.</p><p>

<p> <i>Wolbachia</i> are gram-negative, obligate, intracellular bacteria infecting a majority of insect species and filarial nematodes. In both insects and nematodes <i>Wolbachia</i> are primarily transmitted through the female germ line. <i>Wolbachia</i> carried by filarial nematodes are the cause of the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. In order to combat these diseases, we created a <i>Wolbachia</i>-infected Drosophila cell line that enabled high throughput screening for novel potent anti-<i> Wolbachia</i> compounds. Of the 36,231 compounds screened in house, 8 compounds dramatically reduced <i>Wolbachia</i> titer both in the cell and nematode based screen. Significantly, we discovered that the albendazole metabolite, albendazole sulfone, reduces <i>Wolbachia</i> titer in Drosophila melanogaster and the filarial nematode <i>Brugia malayi </i> perhaps by directly targeting <i>Wolbachia</i> FtsZ. Using the <i>Wolbachia</i>-infected cell line, we discovered that in addition to vertical germ line transmission, <i>Wolbachia</i> are efficiently transmitted horizontally via cell-to-cell transmission. We show that horizontal transfer is independent of cell-to-cell contact, can efficiently take place within hours, and uses both host cell phagocytic and clathrin/dynamin-dependent endocytic machinery. Modifications to our high-throughput screen in combination with genome-wide RNA interference (RNAi) identified host factors that influence <i> Wolbachia</i> titer. When these host factors were tested in <i> Drosophila melanogaster in vivo</i> we found that maintenance of <i>Wolbachia</i> titer relies on an intact host Endoplasmic Reticulum (ER) associated degradation (ERAD) system. These data, in combination with electron microscopy studies, demonstrated that <i>Wolbachia</i> is intimately associated with the host ER and suggested a previously unsuspected mechanism for the potent ability of <i>Wolbachia</i> to prevent RNA virus replication. To examine the impact of nutritional on <i>Wolbachia </i> titer, Drosophila were fed sucrose- and yeast-enriched diets. These conditions resulted in increased and decreased <i>Wolbachia</i> titer in Drosophila oogenesis, respectively, and that somatic TOR and insulin signaling mediate the response of the yeast-enriched diet on <i>Wolbachia </i>. Taken together, these studies provide initial insights into the molecular and cellular interactions between <i>Wolbachia</i> and its insect and nematode hosts.</p><p>

<p> Progression through the mammalian cell cycle is a tightly regulated process that allows cells to replicate their genomes and divide properly. In growth factor-deprived conditions or in response to stress, the cell will exit the cell cycle either reversibly through quiescence, or permanently via senescence. Studies have shown that the SIN3 family of proteins plays a crucial role in these cell cycle exit processes. SIN3 proteins are highly conserved, and exist in mammals as two family members: SIN3A and SIN3B, which function as flexible scaffolding proteins to assemble co-repressor complexes. Our laboratory has recently implicated SIN3B as a critical mediator of each of these cell cycle exit processes. However, its mechanism of action and the consequences of its disruption pertaining to cancer progression have not been comprehensively elucidated. Here we demonstrate that SIN3B is required for the induction of senescence in a mouse model of prostate cancer, and thus prevents the progression to aggressive and invasive carcinoma. In addition, through interaction analysis, we uncovered a novel and robust association between SIN3B and the DREAM complex. The DREAM complex, comprised of p107/p130, E2F4/5, DP1 and the MuvB core complex, is responsible for the repression of hundreds of cell cycle-related transcripts during quiescence. We determined that the deletion of <i>SIN3B</i> resulted in the derepression of DREAM target genes during quiescence, but was not sufficient to allow quiescent cells to resume proliferation. However, the ectopic expression of APC/C^CDH1 inhibitor EMI1 was sufficient for <i>SIN3B</i> deleted cells, but not wild-type cells, to reenter the cell cycle. These studies demonstrate a critical role for SIN3B in the senescence and quiescence programs, and provide important mechanistic insight into the molecular pathways that exquisitely regulate cell cycle exit.</p><p>