Browsing by Subject "Biochemistry"
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Item The Analysis of Pyridyloxobutyl RNA Adducts in Rats Treated With 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone(2009-04-08) Muzic, JohnTobacco-specific nitrosamines are potent carcinogens formed from parent compounds such as nicotine during the curing process of tobacco. The cytochrome P450 enzyme family metabolically activates these nitrosamines, which then attach pyridyloxobutyl (POB) groups to DNA bases. The formation of POB-DNA adducts in tissues of rats treated with tobacco-specific nitrosamines have been demonstrated in previous studies. These adducts can potentially lead to mutations in DNA which promote the formation of tumors. However there exists no data on the formation of POB-RNA adducts. RNA adducts could be important in carcinogenesis, and could potentially be a more reliable biomarker than DNA adducts for tobacco-specific nitrosamine exposure. The objective of our project was to chemically characterize and quantify POB-RNA adducts in the tissue of rats treated with a nitrosamine compound, specifically 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). RNA was isolated from rat liver treated with NNK over a course of 20 weeks and from untreated liver. Combined liquid chromatography-mass spectrometry and high performance liquid chromatography were used to identify RNA adducts. The results indicate the presence of POB-RNA adducts in the treated livers. Further work will involve synthesizing standards by reacting NNK with nucleosides and confirming the structure of adducts with NMR. The results of this study will confirm the presence of RNA adducts due to nitrosamine exposure and provide insight into the utility of RNA adducts as a biomarker used for chemopreventative strategies.Item ATP Rapid Testing in Health Care Settings(2012-04-18) Elias, AaronInfection Prevention in hospitals is an integral part of what makes hospitals effective at total patient care. Environmental surfaces in patient rooms in hospitals include counters, tray tables, nursing equipment, and many other high touch surfaces, all of which can carry bacteria and harmful substances, and can cause secondary infections if they are not cleaned properly. This study was conducted to test how well these high-touch environmental surfaces are cleaned using a rapid test, as opposed to standard microbiological methods. This rapid test measured the amount of ATP found on a surface (given in RLUs), in order to estimate the amount of contamination. In general, most environmental surfaces in different departments across Fairview Health Services met cleanliness standards. A standard must be made for this type of testing in order to know what ATP levels are acceptable. It was also determined that using this ATP rapid test is a viable way to check how effective patient rooms are being cleaned.Item Communication in Membrane Repair(2015-06) Mahling, Ryan WThe force generated by muscle cells places a high amount of stress on their plasma membranes creating lesions which must be effectively repaired in order for the cell to survive. Multiple proteins have been implicated in the membrane repair process, one of which is dysferlin, a seven C2 domain containing protein. Of the seven C2 domains within dysferlin, only the C2A domain exists in two isoforms and has been suggested to be the Ca2+ sensor within dysferlin. Mutations within dysferlin have been found to cause several types of muscular dystrophies including Limb-Girdle muscular dystrophy, Myoshi Myopathy and Distal Anterior Compartment Myopathy. In vivo studies have revealed that after membrane rupture, dysferlin interacts with multiple proteins including annexin A2. In order to gain a better understanding of how this system functions, this author used methods including differential scanning calorimetry (DSC) and spectroscopy (fluorescence and circular dichroism) to examine both isoforms of the C2A domain and annexin A2. All three proteins were found to be marginally stable suggestive of a system that is highly capable of information propagation. From this, a picture emerges where mutations within dysferlin could result in a dramatic shift in the conformational ensemble available to the protein, which would interfere with its ability to properly interact and communicate with the other members of the membrane repair machinery. This would result in the loss of the ability to properly repair the membrane.Item Designing a Screening Method for Organic Seed Treatments(2009-04-08) Lis, DmitriyCertified organic crop producers have limited options available for controlling seed related diseases. I have developed a screen that could be utilized to test various plant extracts as a possible seed treatment. In the initial screen, I tested paper birch (Betula papyrifer) and staghorn sumac (Rhus typhina) extracts against 4 plant pathogens: Fusarium solani, Phytophthora sojae, Rhizoctonia solani, and Pythium spp. The highest concentration of the sumac extract (25.0 micrograms/ml) reduced the growth of R. solani by 67%, F. solani and Pythium spp. by 100%, and P. sojae by 80%. The highest concentration of the sumac extract (25.0 micrograms/ml) reduced the growth of F. solani and Pythium spp. equal to or greater than the commercial seed treatment fungicides. The highest concentration of the paper birch extract (25.0 micrograms/ml) reduced the growth of R. solani by 47%, Pythium spp. by 40%, and P. sojae by 40%. Neither the sumac or the paper birch extract reduced the germination of soybean seeds.Item Detection, diagnostics, and characterization of virus-like organisms and conformational disease-like proteins in plants(2016-10) Bratsch, Sara AnnThe projects in this thesis all investigated virus-like organisms in agronomically important plants. Chapter one consists of reports describing viruses identified in new hosts or locations. Tobacco rattle virus (TRV) was identified in symptomatic Phryma leptostachya L, a native perennial, from plants in an uncultivated habitat and suggests that TRV may be endemic to North America. Since TRV is the causal organism of corky ringspot of potato this study raises the possibility that native perennial plants could serve as a potential reservoir to cause disease in potato. Canna yellow mottle virus was isolated for the first time from symptomatic Canna indica in Kenya. Cut flowers are a major agronomic crop of Kenya and growers should plant only virus indexed plants to limit losses from virus infection. Orchid fleck virus was confirmed by microscopy and sequence analysis for the first time in the United States in Phalaenopsis hybrida. Asymptomatic P. hybrida tested by one step reverse transcription polymerase chain reaction (RT-PCR) did not yield the expected product while a two step RT-PCR, creating cDNA first, yielded the expected product. This indicates the one step RT-PCR diagnostic test can yield false negatives for asymptomatic plants. Chapter two describes the production of polyclonal antibodies for the detection of Orchid fleck virus (OFV). OFV is a mite transmitted virus and has been reported word wide. The previous project identified a need for a reliable, inexpensive method to detect OFV in plants use for propagation, breeding, conservatories, or virus indexing projects. Polyclonal antibodies were produced in rabbits against Escherichia coli expressed OFV phosphoprotein and matrix protein. The resulting antiserums were assayed in PTA-ELISA and DAS-ELISA. OFV phosphoprotein antisera in PTA-ELISA readily differentiated between healthy and OFV infected orchid (Phalaenopsis hybrida) tissue. OFV matrix antisera in PTA-ELISA detected bacterially-expressed protein but did not differentiate between healthy or OFV infected tissue. The OFV phosphoprotein antiserum can be used by in PTA-ELISA to reliably detect OFV. Chapter three describes the molecular and biological characterization of a new Nepovirus causing a leaf mottling disease in Petunia hybrida. The sequence of the majority of the genome was determined by next generation sequencing and the sequence of remainder of the genome was obtained using a 5’ RACE amplification and RT-PCR using poly-A tail and virus specific primers. Due to phylogenetic relationship and sufficient genome dissimilarity to characterized viruses I propose the name of Petunia Chlorotic Mottle Virus for a new Nepovirus. The fourth chapter describes characterization of filamentous virus-like particles in members of the Asteraceae plant family including sunflower, chrysanthemum, coneflower, gerbera daisy, and zinnia. The filaments were 7-10 nanometers in diameter and could exceed 3,000 nm in length. The N-terminal sequences of the major proteins associated with purified filaments from several species were nearly identical and shared homology with the kunitz soybean trypsin inhibitor (KTI) family of proteins. CID MS/MS sequencing of the major proteins of purified sunflower filaments also shared homology with a KTI sequence. A Western blot using antiserum prepared against recombinant sunflower KTI protein labeled the observable protein bands from sunflower filaments. Filaments composed of a major protein of KTI have been found across the Asteraceae family but have not been observed in dandelion, thistle, or lettuce.Item Developing a Disulfide Replacement Picture of APOBEC3G(2010-04-21) Biermann, MitchThe human protein APOBEC3G (A3G) interferes with HIV infection by acting as a cytidine deaminase, an enzyme that induces numerous mutations in HIV’s genetic material that ultimately destroy it. But A3G is only successful at this for a time. The HIV protein viral infectivity factor (Vif) destroys A3G. Developing a way to mask A3G from Vif is a major therapeutic goal. Uncovering the three-dimensional structure of A3G is crucial to rational drug design. The catalytic C-terminal domain of A3G has been solved, but the crucial Vif-interacting N-terminal domain remains invisible to medicinal chemists. A major obstacle toward this goal is the N-terminal domain’s poor solubility. Here we explore a novel technique, disulfi de replacement, in which pairs of cysteine residues are incorporated into the protein at hypothetically close positions and checked for disulfi de bonding. We isolated a model peptide containing a disulfi de bond from its reduced form, and we observed an engineered disulfi de from the Ctd of A3G at two residues known to be spatially close. However, the sensitivity of the approach in digested peptide samples must be improved. We would like to acknowledge Yongjian Lu, Takahide Kono, the Chemistry Department Mass Spectrometry Facility, and all the other members of the Matsuo lab for their support.Item Developing a Quantitative Method for Determining Lipofuscin Content in Mouse Retinal Pigment Epithelium: An Age Comparison of Wild Type and Immunoproteasome Knockout Mice(2012-04-18) Lee, CynthiaAging of post-mitotic cells is associated with the accumulation of lipofuscin, which can lead to deleterious changes in the body and increased susceptibility to certain diseases. This study focused on measuring lipofuscin in the aging retina and determining whether the absence of immunoproteasome affected this age-related process. We hypothesize that lipofuscin increased with aging and the absence of immunoproteasomes. To test our hypothesis, RPE cells from mice of different ages, including wild type and immunoproteasome knockout strains, were used. The cells were processed for lipid extracts containing lipofuscin. The lipid extracts were then used to measure lipofuscin content using fluorescence spectroscopy. Here we developed an optimal method for measuring lipofuscin, including homogenization with PBS buffer, and extraction under dark condition. It was found that fluorescence intensity increases with age in knockout mice, but decreases with age in wild type mice. The intensity was also observed to be higher in knockout compared with wild type. Intensity-average-emission-maximum (IAEM) values were found to vary within different age groups. Our method of quantifying lipofuscin could detect differences in content between retinas from mice of different ages and between strains. The higher content of lipofuscin in KO mice supports our hypothesis. Varied IAEM suggests different fluorescent species developed with aging. This study is important as it might contribute to the aspect of aging theory and, thus, provide insight to studies associated with degenerative diseases, such as age macular degeneration (AMD).Item Direct Reprogramming of Human Cell Lines in vitro Towards a β-Cell Phenotype(2018) Ryan, Nicholson;Type 1 diabetes results from the loss of insulin producing β-cells of the pancreas, causing dependence on exogenous insulin for those affected. β-cell transplantation has been a promising treatment method, but lack of donor cells and requirement for repeated treatments currently limit this approach. Alternative treatments for type 1 diabetes are investigating reprogramming cell types toward a β-cell-like phenotype to generate a reliable source of insulin-secreting cells for transplant therapies. The pancreatic transcription factors Pdx1, Ngn3, and MafA have been combined in an adenoviral vector (Ad-PNMa) and used to reprogram rodent cells to generate insulin-secreting cells both in vivo and in vitro. However, studies examining similar reprogramming of human cell lines have met with little success to date. Here, we attempt to reprogram a human hepatocarcinoma cell line with the Ad-PNMa vector, and human pancreatic epithelial ductal (HPDE) cell lines with the Ad-PNMa vector and new Ad-PN and Ad-PNMb vectors expressing human Pdx1, Ngn3, and MafB. It was found that qRT-PCR detected weak insulin gene expression in Ad-PNMa infected HepG2 cells. Infection of HPDE cells with Ad-PNMb vector caused approximately 64% of cells to express the three vector cargo genes when treated at a concentration of 1.7x1010 ifu/ml. No insulin production was detected in HDPE cells following vector treatments, indicating that expression of these genes is not sufficient to induce insulin expression in HPDE cells. It may be possible to reprogram the HPDE cells to a β-cell phenotype with the expression of additional genes.Item The Effect of Estrogen Depletion on Skeletal Muscle Metabolism(2009-04-08) Kosir, Allison M.Skeletal muscle plays a significant role in altering metabolic activity of the body by influencing blood lipid profiles and insulin sensitivity through enzymatic processes. Hormones such as estrogen have been shown to effect skeletal muscle function. Research indicates the metabolic influence of skeletal muscle may be altered by the depletion of estrogen. Through its role in energy metabolism and expenditure, skeletal muscle likely influences the development of cardiovascular risk factors; including hypertension, diabetes, dyslipidemia, and obesity. To study the effects of estrogen depletion on skeletal muscle metabolic activity, seventeen female C57BL/6 mice have been randomly divided into two groups for the duration of 60 days; ovariectomized (OVX) and control. Circulating concentrations of total cholesterol, high–density lipoprotein, and glucose were measured prior to surgery and at 20, 40, and 60 days post-surgery. Pre- and post-trial measurements of body composition were measured on the EchoMRI. Body weight was measured weekly and food intake was calculated during a one week period. Twenty-four hour cage activity was monitored in activity chambers at 50 days. Enzymatic activity of skeletal muscle will be measured to assess oxidative capacity and rates of fatty-acid breakdown. Early results indicate that the OVX mice are gaining weight and have increased total cholesterol over baseline values. I hypothesize that estrogen-depletion will increase circulating cholesterol and glucose levels, decrease circulating high-density lipid, increase body mass and percent body fat, decrease cage activities, and decrease skeletal muscle oxidative capacity and lipid metabolism through reduced enzymatic activity.Item Exosome-Mediated Protection Against Oxidative Stress: A Connection Between NRF2 And Exosomes(2016-12) Rust, BrittneyThe nuclear factor erythroid 2 (NF-E2)-related factor 2 (NRF2) transcription factor has been widely studied for its role as the master regulator of the response to oxidative stress. NRF2 has many mechanisms for protection including upregulation of antioxidant genes, metabolism genes, and proteolysis genes. Furthermore, it was found that a deeply conserved set of NRF2 target genes encode for proteins loaded into exosomes [1]. Exosomes are extracellular microvesicles that are secreted by cells in response to a variety of stimuli, including many stressors. Exosomes serve as a transport mechanism for proteins, RNA, DNA, and other molecules. Therefore, we hypothesized that exosome loading and release is a mechanism used by NRF2 to protect neighboring cells from stress in a non-autonomous manner. In an effort to validate that NRF2 regulates exosomes, I explored the following: (1) exosome release before and after NRF2 activation, (2) the RNA and protein content of NRF2-induced exosomes, and (3) the cytoprotective potential of NRF2-induced exosomes. Overall, based on these studies, we concluded that NRF2 activation induces exosome release, and exosomes are generally enriched for cytoprotective protein SQSTM1. However, although NRF2-induced exosomes were cytoprotective in some assays, this response was not robust enough for me to conclude that NRF2-induced exosomes can protect naïve cells from oxidative stress. Thus, exosomes provide a potential mechanism for NRF2 to provide nearby cells with resources to combat oxidative stress, but further research is necessary to determine whether this mechanism is used in vivo.Item Fishing for Function: Identifying Functional Binding Partners of CUG-Binding Protein 1(2009-04-08) Jeschke, Jonathan C.One of the most remarkable distinguishing features of living organisms is their ability to fluidly adapt to changing environmental conditions. The advances in molecular biology over the past 50 years have resolved the general outlines this capacity for adaptation on the scale of molecules. We now view an organism’s ability to adapt as the result of many complex programs of gene expression. As a basic science, the improved resolution of these expression programs has proved invaluable in understanding many clinical pathologies, the most dramatic being cancer. My laboratory work has focused on resolving a process cells use, at the level of messenger RNA (mRNA), to turn off genes before functional proteins are made. This enzymatic process involves the break down of mRNA polymers where the degrading enzymes are recruited by specifi c adaptor proteins to specifi c sets of mRNA. One of these adaptor proteins is CUG-binding protein 1 (CUGBP1) and identifying the mRNA set CUGBP1 targets has been a focus of our lab. The degradation proteins which CUGBP1 targets to the mRNA are only poorly resolved. I am using a yeast-two hybrid screen to identify CUGBP1 binding candidates. These candidates will then be con firmed by co-immunoprecipitation and mRNA a ffinity chromatography. This will allow me to characterize the specifi c mechanism of decay elicited by the function of CUGBP1.Item Fluorescence Analysis of the Sarcolipin:SERCA Protein Complex(2009-04-08) Rubin, John E.We have used fluorescence resonance energy transfer (FRET) to identify physical interactions between the sarcoplasmic reticulum Ca-ATPase (SERCA) and one of its regulatory proteins, sarcolipin (SLN), in cardiac and skeletal muscle. The sarcoplasmic reticulum (SR) is an intracellular membrane network found in muscle cells whose function is to uptake, store, and release calcium. SERCA functions to transport calcium into the SR to induce muscle relaxation. Theoretical models predict that SLN monomers regulate SERCA by binding the SERCA transmembrane domain, but SLN monomers also self-associate to form oligomers. To test these models, we expressed fluorescent fusion proteins of SLN and SERCA in Sf21 insect cells using the baculovirus system. Quantitative binding stoichiometries were determined by FRET measurements using live cell microscopy on plates coated with mollusk “glue” protein. FRET results indicate that (1) SLN monomers self-associate to form dimers and (2) SLN monomers interact with SERCA to form a 1:1 heterocomplex. We propose that SLN monomers compete in equilibrium between SLN oligomerization and SERCA binding.Item Histone methylation in polycomb gene silencing.(2010-08) Joshi, Preeti MadhavPolycomb (PcG) group proteins are chromatin-modifying factors that collaborate to repress gene expression during development. PcG proteins are crucial for silencing during animal embryogenesis, maintenance of stem cell populations and are also implicated in cancer epigenetics. PcG proteins operate in at least three molecular complexes: PHORC, PRC2 and PRC1. Current models suggest that PRC1 is recruited at target loci by PRC2 and is most directly responsible for gene silencing. Our study uses the Drosophila model system to investigate the precise role of PRC2 in PRC1 targeting. One key function of PRC2 is to catalyze methylation of histone H3 on lysine-27. A common model suggests that this creates a binding site for the recruitment of PRC1. The loss of PRC2 leads to disruption in PcG mediated silencing. We wished to assess if PRC1 is recruited at PcG targets by a direct interaction with PRC2 or by binding to the methyl mark. We have used enzyme-dead versions of PRC2 that can stably accumulate at target loci and shown that PRC1 is also found at these targets suggesting that the initial recruitment of PRC1 is independent of PRC2 catalytic function. We have also engineered a heterologous enzyme, called vSET, to create methylated histones in the absence of PRC2. This heterologous enzyme can be used to test for PRC1 accumulation without PRC2.Item Identifying Interactions Between Ethanolamine Utilization Bacterial Microcompartment Cargo and Shell Proteins(2015) Dahlgren, Kelsey;One of the goals of synthetic biology is to engineer metabolic pathways to produce valuable chemical compounds and biofuels. To gain economic advantage over abiotic processes, metabolically engineered processes must attain high fluxes, sustain high yields, and have minimal effect on host growth rates. Major obstacles that must be overcome for the use of synthetic metabolic pathways include diffusion limitations, alternative metabolic routes, toxic intermediates, and inhibitory products. To avoid these problems in their own metabolic pathways, bacteria use bacterial microcompartments (BMCs), organelles composed entirely of protein that contain cargo proteins, functionally related enzymes and auxiliary proteins, within a proteinaceous shell. The ability to group enzymes in a BMC shell that regulates substrate and product transport is a promising tool for synthetic biology. However, knowledge of BMC cargo protein localization mechanisms is unknown, but necessary for the development of BMCs as nanocontainers for biosynthesis or biocatalysis. This study sought to optimize heterologous expression of of ethanolamine utilization (Eut) BMC cargo proteins with the objective of studying interactions between BMC shell proteins and cargo proteins using in vitro pull-down experiments. Results from this study will inform future work on engineering BMCs for synthetic biology applications.Item Integrated Fluorescence Spectroscopy for FRET Analysis of Novel Ionic Strength Sensors in the Presence of a Hofmeister Series of Salts(2019-07) Miller, RobertLiving eukaryotic cells are complex, crowded, and dynamic organisms that continually respond to environmental and intracellular stimuli. In addition, these cells have heterogeneous ionic strength with compartmentalized variation of both intracellular concentrations and types of ions. The underlying mechanisms associated with ionic strength variations that trigger different biological functions and response to environmental cues remain largely unknown. Therefore, there is a need to develop a quantitative method for mapping the compartmentalized ionic strength and their temporal fluctuations within living cells. In this work, we investigate a class of novel ionic- strength sensors that consists of tethered mCerulean3 (a cyan fluorescent protein) and mCitrine (a yellow fluorescent protein) via a linker of varied amino acids. In these protein constructs, mCerulean3 and mCitrine act as a donor-acceptor pair undergoing fluorescence resonance energy transfer (FRET) based on both the linker amino acids and the environmental ionic strength. The energy transfer efficiency and the donor-acceptor distance of these sensors can be quantified noninvasively using integrated fluorescence methods in response to intracellular ionic strength in living eukaryotic cells. We employed time-resolved fluorescence methods to monitor the excited-state dynamics of the donor in the presence and absence of the acceptor as a function of the environmental ionic strength using potassium chloride (KCl, 0–500 mM). Towards mapping out the response to of these sensors towards biologically relevant salts, we carried out time- resolved fluorescence for FRET analysis of these sensors as a function of the Hofmeister series of salts (KCl, LiCl, NaCl, NaBr, NaI, Na2SO4). We also used these results towards technique development for FRET analysis based on time-resolved fluorescence polarization anisotropy. Our results show that the energy transfer efficiency of these sensors is sensitive to both the linker amino acid sequence and the environmental ionic strength. These studies in a controlled environment complement previous steady-state spectroscopy analysis of these sensors in a cuvette with the advantage of the compatibility of our approach with fluorescence lifetime imaging microscopy on living cells.Item Investigating the Possible Role of a Glycosyl Transferase Protein in the Biosynthesis of Long-Chain Hydrocarbons in Shewanella oneidensis(2012-04-18) Bergquist, CarlIn the search for alternative sources of energy, new organisms are being looked at as potential biofuel producers. It has been shown that the longchain hydrocarbons produced by the bacterium Shewanella oneidensis can be broken down into usable fuel. It is known that the enzyme OleA is largely responsible for hydrocarbon synthesis, but other proteins may also play a role. In my thesis work, I deleted the gene SO_3174, which encodes a glycosyl transferase protein and was found to be interrupted in a transposon screen for increased hydrocarbon production, from S. oneidensis MR-1. I then tried to show that deleting SO_3174 resulted in an increase in hydrocarbon production just as the interruption of the gene had. The deletion strain showed an increased fluorescence in the presence of Nile Red dye, a hydrophobic dye that can be used to indirectly detect hydrocarbon levels. However, the deletion strain did not exhibit increased hydrocarbons during direct analysis of nonpolar extractions. These same results were obtained from a strain containing both the SO_3174 deletion and Stenotrophomonas maltophilia OleA. Overall, I found that the protein encoded by SO_3174 most likely does not play a role in hydrocarbon biosynthesis in S. oneidensis.Item Manipulating Tfam expression and mitochondrial capacity to overcome drug resistance in breast cancer cells.(2012-04-18) Zehowski, CherylFor a patient diagnosed with breast cancer, the determination of a treatment is based on a biopsy and the markers that are present in the tumor at the time of diagnosis. There are tumors that are difficult to determine whether or not it will respond to chemotherapy treatment at the time of diagnosis. The research that I have been working on supports the main goal of the Skildum lab that discovering predictive biomarkers in tumor samples will help to determine a personalized and more effective treatment path. To model drug resistant breast cancer in vitro, I work with MCF-7 (sensitive to chemotherapy treatment) and LCC9 (less sensitive to chemotherapy treatment) breast cancer cells. Tfam is the major regulator of mitochondrial DNA replication and transcription. I discovered that the Tfam gene had an increased expression in the LCC9 cells than in the MCF-7 cells. We then decided to look at the contribution of Tfam expression to drug sensitivity in LCC9 cells. Through siRNA transfection, which interferes with the translation of proteins by binding and promoting degradation of the messenger RNA at specific sequences, we successfully knocked down Tfam mRNA expression. Here we show the consequences of Tfam knockdown are decreased mitochondrial DNA copy number and decreased labeling with a mitochondria specific fluorescent dye. From this finding I have begun to test whether Tfam knockdown in LCC9 cells increases their sensitivity to the chemotherapy drug doxorubicin. If LCC9 cells do have increased drug sensitivity after Tfam knockdown, then the Tfam gene could potentially be used as a biomarker of drug resistance in breast cancer patients.Item Regulation of Guanylyl Cyclase-B: Characterizing the Roles of Gö6976, ATP, and P-site Inhibitors(2011-07-19) Lou, XiaoyingGuanylyl cyclases (GC) play important roles in a wide range of organisms. Three transmembrane GC receptors bind natriuretic peptides with varying affinities. C-type natriuretic peptide (CNP) activates guanylyl cyclase-B (GC-B/NPR-B), which stimulates cGMP synthesis. Although the physiological effects of GC receptors have been well characterized, their regulatory mechanisms are not well understood. The inhibitory effects of the indolocarbazole, Gö6976, on GC-B activity were characterized, and data indicated that Gö6976 potently inhibits the GC activity of GC-B via a process that does not require changes in known phosphorylation sites, inactivation of all catalytic sites, or intact cell architecture. As an ATP analog, Gö6976 may inhibit GC-B by blocking the ability of ATP to activate and/or stabilize the receptor. Maximum activation of GClinked natriuretic peptide receptors in broken cell preparations requires natriuretic peptide binding to the extracellular domain and ATP binding to an unknown intracellular region. Additionally, natriuretic peptide activation of GC-B requires that the intracellular domain be phosphorylated on multiple serine and threonine residues. The dual role of ATP as an allosteric activator of GC-B and a substrate for the kinase that phosphorylates GC-B has led to confusion in the field. Enzymatic timecourse experiments conducted in crude membranes at low and high GTP concentrations support the role of ATP as an allosteric regulator of GTP. Previous studies indicated that dephosphorylation explains long-term inactivation of GCs. However, the deactivation and downregulation of a constitutively phosphorylated version of GC-B were identical to those of the wild type receptor, which indicates that these processes are phosphorylation-independent. One explanation for the time-dependent inactivation of GC-B is the binding of product compounds to the GC domain, similarly to how purine site (P-site) inhibitors inhibit adenylyl cyclase. Preliminary studies characterized the effect of 2’d3’GMP and PPi on GC-B activity in order to gain insight into the catalytic mechanisms of GC receptors.Item Role of CD38/cyclic-ADP-ribose in Human Asthma(2009-04-08) Smelter, Dan F.CD38 is a cellular protein found throughout the mammalian body. It is involved in calcium signaling and innate immunity. Its expression is augmented by the presence of multiple contractile agonists that occur in the development of asthma, and it is implied that CD38 expression is involved in the pathogenesis of asthma. However, CD38's role in human asthma has not been described. In my studies, I wanted to determine whether CD38 expression is involved in human asthma. To test this, I studied the effects of TNF-alpha-induced augmented CD38 expression in airway smooth muscle cells obtained from asthmatics compared to non-asthmatics. Also, I tested the intracellular calcium response to contractile agonists in cells from asthmatics and non-asthmatics following exposure to TNF-alpha.