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1
Tisi, Dominic John Guiseppe. "Structural studies on nucleotide binding proteins". Thesis, Birkbeck (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391822.
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Johansson, Kenth. "Structural studies of four nucleotide binding proteins : aldehyde dehydrogenase, NADP-malate dehydrogenase and two deoxynucleoside kinases /". Uppsala : Swedish University of Agricultural Sciences, 2000. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=009416200&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Mutomba, Martha Chengetai. "Guanine nucleotide-binding proteins of Trypanosoma brucei". Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308280.
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Iuga, Adriana. "Solid-state 31P NMR of nucleotide binding proteins". [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=973225238.
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Heurtel, Thuswaldner Sophie. "Nucleotide-binding Proteins in the Plant Thylakoid Membrane". Licentiate thesis, Linköping Department of Biomedicine and Surgery, Linköping University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-7934.
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Worth, Graham Alan. "The energetics of nucleotide binding to RAS proteins". Thesis, University of Oxford, 1992. http://ora.ox.ac.uk/objects/uuid:44524415-2f2b-4601-998c-56110f332153.
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Ras proteins are a special class of proteins that mediate cell growth signals. Their importance lies in the fact that they are products of a proto-oncogene. This means that under certain conditions the gene that determines its structure is altered and a mutant protein results that is involved in the transformation of normal cells to cancer cells. The actual function by which the protein acts in the signal pathway is not known. However it is known that they act as a switch, undergoing a cycle involving the exchange of guaninosine nucleotides in the binding site. This thesis uses computer simulations to study the energetics of this binding, with the long term aim of developing a drug to inhibit the transforming activity of the oncogenic protein. To begin with, a model of the protein based on a crystal structure is built. Using Molecular dynamics the motion of this model is studied. A possible mechanism by which one half of the nucleotide cycle could be induced is investigated, with the result that phosphorylation of the protein may be involved. The main part of the thesis is then devoted to using the free energy perturbation (FEP) method to calculate the difference in Gibbs binding free energy between the nucleotides in the protein. Using histamine as a model, a method of dealing with charged, flexible molecules is developed; namely the inclusion of a reaction field and comprehensive conformational analysis. The results from the associated calculations are seen to be very close to experimental data. The same procedures are then applied to the much more complex ras: nucleotide system with less successful results, the reason for which is mostly due to the restriction of limited computer resources to tackle such a problem. The conclusion is that given the resources and by using the techniques developed in this thesis, this type of calculation is a feasible way to study such systems.
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Bramble, Sharyl Elizabeth. "Guanine nucleotide binding properties and attempted immunopurification of ras protein from dictyostelium discoideum". Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26172.
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One purpose of this study was to determine whether the ras protein from Dictyostelium discoideum (p23) binds guanine nucleotides
like the ras proteins from mammals (p21) and yeast. The other purpose of this investigation was to purify or enrich for p23ras from D. discoideum by immunoaffinity chromatography.
A number of different approaches were used to determine guanine nucleotide binding by p23RAS . A simple filter binding assay, binding to Western blots, and photoaffinity labeling all failed to demonstrate specific binding with lysates of D. discoideum cells. In contrast p21RAS from transformed NIH-3T3 cell lysate was successfully photoaffinity labeled in the presence
of ³²P-α-guanosine 5¹-triphosphate (GTP) suggesting that the technique had been performed correctly. It was concluded that either p23RAS has a very low affinity for guanine nucleotides
such that GTP binding was not detectable in these experiments
or that the ras protein from D. discoideum simply does not bind guanine nucleotides.
The purification of p23RAS from D. discoideum cells was attempted in order to provide a purified protein preparation for guanine nucleotide binding and for reconstitution studies. An anti-ras monoclonal antibody (Y13-259) was used as the ligand for the immunoaffinity chromatography. This approach was not successful in that the ras protein could not be enriched
relative to other proteins because the immunoaffinity columns did not bind p23RAS.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
8
Law, Wing-lun, i 羅永倫. "Expression, purification and preliminary x-ray crystallographic studies of two nucleotide binding proteins". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46939118.
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Akam, Elizabeth Claire. "The activation of guanine nucleotide binding proteins by muscarinic acetylcholine receptor subtypes". Thesis, University of Leicester, 1999. http://hdl.handle.net/2381/29919.
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Agonist-stimulation of human recombinant M1, M2, M3 and M4 receptors, expressed in Chinese hamster ovary cells, was investigated at the level of G protein activation. Functional responses were determined by a number of methods including [35S]-GTPS binding in membranes using both filtration-based and immunoprecipitation-based procedures: Ins(1,4,5)P3 accumulation and 45Ca2+ release from permeabilised cell suspensions; and cAMP accumulation in cell suspensions. M2 and M4 receptors, with equivalent expression levels in this recombinant system, were found only to couple to pertussis toxin-sensitive G proteins with near equal kinetics. Methacholine appeared equipotent when activating the total G protein complement through the M2 and M4 receptors, however, it appeared more potent when activating Gi3/o through the M2 compared to the M4 muscarinic receptor. Using equivalent expression levels of M1 and M3 receptors both the subtypes were found to couple to both pertussis toxin-sensitive and -insensitive G proteins. CHO-M1 and -M3 mediated Ins(1,4,5)P3 generation after pertussis toxin pre-treatment suggested the functional significance of coupling to multiple G protein classes may be in the stimulation of PLC by -subunits derived from Gi-like G proteins. The activation of Gq/11 through the M1 receptor subtype, after methacholine-stimulation, is faster, greater and more potent than that mediated by the M3 receptor subtype, suggesting that the intrinsic activity of the M1 subtype is greater than that of the M3 subtype. The 'partial' agonist pilocarpine also displayed very different G protein activation profiles after stimulation of M1, M2, M3 and M4 receptor subtypes, suggesting that agonists acting at different receptor subtypes may be capable of inducing relatively selective coupling of the occupied receptor to available G proteins. This study therefore concludes that muscarinic receptor subtypes display divergent G protein activation profiles after either 'full' or 'partial' agonist-stimulation.
10
Wadman, Isobel A. "The regulation of human platelet adenylate cyclase by ATP and guanine nucleotide binding proteins". Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241124.
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Wong, Yung Hou. "Molecular basis of the opioid receptor-ligand interactions : the role of guanine nucleotide-binding proteins". Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.303263.
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Sarma, Ranjana. "Investigations of nucleotide-dependent electron transfer and substrate binding in nitrogen fixation and chlorophyll biosynthesis". Thesis, Montana State University, 2009. http://etd.lib.montana.edu/etd/2009/sarma/SarmaR1209.pdf.
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The studies presented in this thesis include studies of nucleotide-dependent conformations of the electron donor protein in nitrogenase and dark-operative protochlorophyllide reductase (DPOR) characterized using small-angle x-ray scattering and x-ray diffraction methods. Nitrogen fixation and chlorophyll synthesis are involved in the reduction of high energy bonds under physiological conditions. Both make use of elegant reaction mechanisms made possible by complex enzyme systems which are evolutionarily related. Nitrogenase reduces nitrogen to ammonia and is a two-component metalloenzyme composed of Fe protein and MoFe protein. For nitrogen reduction, the Fe protein and MoFe protein associate and dissociate in a manner concomitant with hydrolysis of at least two MgATP molecules and enables the concomitant transfer of at least one electron from Fe protein to MoFe protein. During chlorophyll biosysnthesis, the rate limiting step is catalyzed by a two-component metalloenzyme called DPOR. The two components of DPOR are BchL and BchNB proteins and these share high level of sequence similarity with the Fe protein and the MoFe protein, respectively. Based on this sequence similarity and biochemical data available, it is proposed that the reaction mechanism is similar to nitrogenase mechanism in which the components of DPOR associate and dissociate in a nucleotide dependent manner, to enable intercomponent electron transfer. Fe protein and BchL present as unique examples of proteins that couple nucleotide dependent conformational change to enable electron transfer for high energy bond reduction. The present studies have been directed at studying the low resolution studies of MgATP-bound wild-type Fe protein and its comparison to the structure of the proposed mimic, i.e, L127 Delta Fe protein. The studies presented show evidence of the MgATP-bound wild-type Fe protein having a conformation very different from the L127 Delta Fe protein. The chapters also include detailed characterization of the structure of BchL in both MgADP bound and nucleotide-free states which offer detailed insights in the structure based mechanism of BchL, with primary focus on identifying key residues involved in componenet docking and in electron transfer. Together, the studies on the Fe protein and BchL have furthered our understanding of mechanism of electron transfer in these complex enzyme systems.
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Chakrabarti, Partha Pratim. "Biochemical and biophysical analysis of the GTPase activating proteins of the small guanine nucleotide binding protein Rap1 and RheB". [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975809598.
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Adhikari, Anirban. "Regulation of guanine nucelotide exchange in inhibitory G protein alpha subunit by activator of G protein signaling 3 and novel regulatory peptides". Embargoed access until after 12/19/2006, 2005. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=114.
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Mahajan, Shikha. "Protein Profiling of Adenine Nucleoside and Nucleotide Analogs Binding Proteins Using N6-Biotinylated-8-azidoadenosine Analogs as Affinity Based Protein Profiling Probes". Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4139.
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Identification of differential expressions of proteins in proteomic profiles of biological samples shows great potential as a valuable technique for the early diagnosis of various diseases. An important challenge in modern protein profiling approaches is to reduce the complexity of the samples by limiting the number of proteins that need to be evaluated for distinction in the expression between normal and deceased cells. In this research, an affinity based approach for the enrichment of nucleotide and nucleoside binding proteins from a complex cell proteome has been developed. To achieve this goal, new N6-biotinylated-8-azido-adenosine probes (AdoRs) have been designed and synthesized to photolabel the nucleotide and nucleoside binding proteins. These probes contain a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Further, a mass spectrometric protein profiling approach is employed to quantitatively identify small variations in expression of nucleoside and nucleotide binding proteins in samples of interest.
Mouse neuroblastoma N18TG2 cell proteome has been used as a model system for the development of the LC-MS/MS based proteomic analysis of these affinity enriched protein fractions. Upon enrichment, the photolabeled proteome exhibited an approximately four-fold abundance of nucleoside and nucleotide binding proteins over
nonlabeled proteome. The approach was extended to compare the proteomic profiles of nucleotide and nucleoside binding proteins in cancerous (Hey) and non-cancerous (T-80) human ovarian cell proteome. Certain proteins that were not detected in cell lysate were also identified in labeled proteome, thereby demonstrating the strength of our approach in enriching low abundant proteins.
To substantiate the qualitative analysis, we have employed the Stable Isotope Labeling in Amino Acid Cell Culture (SILAC) for the quantitative study of the protein expression in cancerous and non-cancerous human ovarian cells. A modest panel of proteins with differential expressions in these cell lines was identified, a few of which have been correlated to various forms of cancer. Vimentin, stress induced phosphoprotein-1, and heat shock protein 90 that were identified to have altered expressions in these cell lines are among some of the proteins associated with ovarian cancer.
16
Grady, Amanda Ellen. "The regulation of the type 5 cyclic nucleotide phosphodiesterase in airway smooth muscle by metal ions and small molecular weight proteins". Thesis, University of Strathclyde, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366810.
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Meier, Katharina [Verfasser], Jörg [Akademischer Betreuer] [Gutachter] Durner i Brigitte [Gutachter] Poppenberger-Sieberer. "Cyclic nucleotide signaling in Arabidopsis thaliana and the identification of cGMP binding proteins / Katharina Meier ; Gutachter: Jörg Durner, Brigitte Poppenberger-Sieberer ; Betreuer: Jörg Durner". München : Universitätsbibliothek der TU München, 2017. http://d-nb.info/1128819368/34.
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Rudolf, Jana. "Characterisation of XPD from Sulfolobus acidocaldarius : an iron-sulphur cluster containing DNA repair helicase". Thesis, St Andrews, 2007. http://hdl.handle.net/10023/159.
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Lee, Meng-Tse. "Catalytic Mechanisms in Sec7 and Vps9 Domain Exchange Factors for Arf and Rab GTPases: A Dissertation". eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/598.
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Vesicle budding, membrane trafficking, and lipid metabolism depend on the switching of Arf and Rab GTPases from the inactive GDP bound state to the active GTP bound state. However, Arf and Rab GTPases have intrinsic rates of GDP to GTP exchange that are much slower (hours to days) than the time scale of the relevant trafficking processes (seconds or less). In cells, the activation of Arf and Rab GTPases is tightly regulated by guanine nucleotide exchange factors (GEFs) with Sec7 or Vps9 domains, respectively.
Full length Cytohesins, which have a domain architecture consisting of heptad repeats, a Sec7 domain, a pleckstrin homology (PH) domain, and a polybasic motif, have 100-fold lower exchange activity than the isolated Sec7 domain. Insights into the low exchange activity were obtained by structural, biochemical and kinetic analyses. It was found that the Sec7-PH domain linker and a C-terminal amphipathic helix physically block the docking sites for the switch regions of Arf GTPases. Mutations within either element result in partial or complete relief of autoinhibition. Autohibition is also strongly relieved by phosphorylation of protein kinase C (PKC) sites in the polybasic motif of Cytohesin-1 or by phosphoinositide head group-dependent binding of active Arf6.
Despite unrelated folds, Sec7 and Vps9 domains engage cognate GTPases in a strikingly similar manner and supply a critical acidic residue that interacts with an invariant lysine residues from phosphate binding (P) loop of the GTPase in the nucleotide free complex. The key acidic residues have also been proposed to disrupt the Mg2+ binding site; however, it is not known whether disruption of Mg2+ binding contributes to the rate limiting step for nucleotide release. To investigate the kinetic mechanism for catalysis of nucleotide exchange in the absence of autoinhibitory interactions, a detailed stopped flow kinetic analysis of the intrinsic and GEF mediated exchange reactions was conducted for the isolated catalytic cores. Using three different fluorescence methods to monitor Mg2+ dissociation, formation of the nucleotide free intermediate, and subsequent nucleotide binding, the catalytic cores of Cytohesin-1 and Rabex-5 were found to robustly accelerate nucleotide exchange on Arf1 and Rab5, respectively, by at least 105- fold at physiological concentrations of Mg2+. The acceleration of nucleotide exchange was reduced by roughly an order of magnitude at sub-micromolar concentrations of Mg2+. In addition, the Cytohesin-1 and Rabex-5 catalytic cores have similarly high catalytic efficiencies (kcat/KM) as well as high lower limits on both the rate (kcat) and steady state (KM) constants for GDP release at physiological as well as low Mg2+ concentration. The limits on kcat and KM are comparable to the highest values reported for other well characterized GEFs and likely reflect dual requirements of membrane targeting and autoregulatory mechanisms for tight control of catalytic output. These results provide a solid structural and mechanistic foundation for future experiments to investigate the spatial-temporal dynamics of Cytohesin and Rabex-5 activation in cellular contexts.
20
Carmine, Andrea. "On Parkinson's disease and schizophrenia : case control studies, cellular localization and modelling of candidate genes /". Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-671-5.
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Raper, Jayne. "Guanine nucleotide binding protein function in T.B. Brucei". Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305533.
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Gorman, Christine. "The interaction of Ras with Raf and other potential effectors". Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312373.
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Saiu, P. "Structural and functional studies on nucleotide binding to AMP-activated protein kinase". Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/645676/.
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AMP-activated protein kinase (AMPK) is an enzyme that senses and regulates cellular energy balance thus playing a key role in homeostasis. As such it is a target for treatment of metabolic disorders such as type II diabetes. AMPK is a hetero-trimeric complex composed of an α, β and γ subunit. α contains the catalytic kinase domain, β is a scaffolding subunit that enables complex formation and γ monitors cellular energy via nucleotide binding to its CBS domains. AMPK is primarily activated by phosphorylation at Thr-172 on the activation loop in the kinase domain. It exerts its cellular effects via phosphorylation of a range of downstream targets involved in different aspects of energy production & utilization. The aim of this thesis is to characterize the mechanistic basis of energy regulation of mammalian AMPK via structural and binding measurements. Fluorescence studies have been facilitated by the use of N-methylanthraniloyl (mant) labelled AMP and of β-Nicotinamide adenine dinucleotide 2’-phosphate (NADPH) to monitor competition with AMP, ADP and ATP. A number of mutations in the γ subunit, which interfere with the normal function of AMPK and cause Wolff-Parkinson- White (WPW) syndrome, have also assessed for changes in nucleotide binding affinities and potential implications for the regulation of kinase activity. Thermal denaturation experiments are used to investigate the stabilizing effects of nucleotides and other small molecule ligands. This method was used in a low throughput screen against an enriched list of compounds selected from an in silico screen to try to identify novel activators. I have also determined the structure of the regulatory fragment of the enzyme bound to 5-aminoimidazole-4-carboximide riboside monophosphate (ZMP), an intermediate on the biosynthetic route to AMP, the fluorescence reporter mant-AMP and the WPW mutants Arg298\rightarrowGly, Arg69\rightarrowGln and His150\rightarrowArg. The structures of the mutants have revealed that nucleotide binding is impaired due to a reduced affinity for the nucleotides thus affecting the regulation of the kinase.
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Carie, Adam E. "Tumor suppressive effects of the Beta-2 adrenergic receptor and the small GTPase RhoB". [Tampa, Fla.] : University of South Florida, 2008. http://purl.fcla.edu/usf/dc/et/SFE0002330.
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Daumke, Oliver. "Structural and functional analysis of the GTPase activating protein of the small guanine nucleotide binding protein Rap1". [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971950806.
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Thain, Alison. "An analysis of the nucleotide requirements for DNA binding by the human pallomavirus type 16 E2 protein". Thesis, University of Bristol, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245392.
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Wreggett, Keith Allen. "Characterization of the interaction of the D2-Dopamine receptor with a guanine nucleotide-binding protein". Thesis, McGill University, 1985. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=73965.
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Brown, Lesley Ann. "Guanine nucleotide-binding protein function in myocytes isolated from normal, failing and beta-adrenoceptor desensitised myocardium". Thesis, Imperial College London, 1991. http://hdl.handle.net/10044/1/46688.
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Chen, Xi Lin. "Régulation de l'apoptose des lymphocytes T par GIMAP5 (GTPase of Immune Associated Nucleotide Binding Protein 5)". Thèse, Université de Sherbrooke, 2015. http://hdl.handle.net/11143/7593.
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Abstract : Long-term survival of T lymphocytes in a quiescent state is essential to maintain their cell numbers in secondary lymphoid organs. Interaction of the T cell antigen receptor (TCR) with self-peptide/MHC synergizes with IL-7-induced anti-apoptotic signals to promote T cell survival. These extrinsic stimuli are also implicated in T cell metabolism and survival by regulating several signaling pathways including the phosphatidyl-inositol-3 kinase (PI3K)/Akt pathway. In mice and in rats, loss of functional GTPase of the immune associated nucleotide binding protein 5 (GIMAP5) causes peripheral T lymphopenia due to spontaneous death of T cells. The underlying mechanisms responsible for the pro-survival function of GIMAP5 in T lymphocytes remain largely unknown. Previous work from my laboratory has shown that T cells from GIMAP5-deficient rats show reduced influx of calcium (Ca[superscript 2+]) from the extracellular milieu following stimulation of the TCR complex. In this thesis, I characterized the mechanism by which GIMAP5 regulates Ca[superscript 2+] homeostasis, and elucidated the signaling pathways modulated by GIMAP5 to facilitate the survival of T cells. Firstly, I investigated if GIMAP5 prevents apoptotic death of T lymphocytes by affecting the Ca[superscript 2+] buffering capacity of mitochondria, which is required for sustained Ca[superscript 2+] influx via the plasma membrane channels. I observed that mitochondrial Ca[superscript 2+] accumulation following capacitative Ca[superscript 2+] entry is defective in T cells from Gimap5 deficient rats. Disruption of microtubules, but not the actin cytoskeleton, abrogated Ca[superscript 2+] sequestration by mitochondria in T cells from control but not Gimap5 deficient mice. Similarly, mice lacking functional GIMAP5 displayed defective T cell development and Ca[superscript 2+] influx. Furthermore, I observed that the proximal signaling events following TCR stimulation was reduced and was accompanied by defective proliferation in T cells from Gimap5 deficient mice. Additionally, IL-7-induced STAT5 phosphorylation was decreased in CD4[superscript +] T cells from Gimap5 deficient mice. I also showed that loss of functional Gimap5 results in increased basal activation of mammalian target of rapamycin (mTOR), independent of protein phosphatase 2A (PP2A) or AMP-activated protein kinase (AMPK). Instead, the constitutive activation the PI3K pathway contributed to the spontaneous high mTOR activation. Collectively, my observations suggest that the pro-survival function of GIMAP5 in T-lymphocytes may be linked to the regulation of diverse signaling pathways in a context dependent manner. GIMAP5 also facilitates microtubule-dependent mitochondrial buffering of Ca[superscript 2+] following capacitative entry. GIMAP5 is required to integrate the survival signals generated following activation through TCR and IL-7R.Résumé : La survie à long terme des lymphocytes T en état de repos est essentielle pour maintenir leurs nombres dans les organes lymphoïdes secondaires. Le récepteur antigénique des cellules T (TCR) en contact avec les peptides du soi / CMH et en synergie avec l'IL-7 induit des signaux anti-apoptotiques pour favoriser la survie des cellules T. Ces stimuli extrinsèques sont également impliqués dans le métabolisme et la survie des cellules T grâce à la régulation de plusieurs voies de signalisation dont la voie phosphatidyl-inositol-3 kinase (PI3K) /AKT. Chez la souris et chez le rat, la perte de l’activité de GIMAP5 (GTPase of Immune Associated Nucleotide Binding Protein 5), provoque une lymphopénie T périphérique en raison de la mort spontanée des cellules T. Le mécanisme sous-jacent responsable de la fonction de survie de GIMAP5 dans les lymphocytes T reste largement inconnu. Nous avons observé que les cellules de rats déficients en GIMAP5, après stimulation par complexe TCR, montrent un afflux de calcium (Ca[indice supérieur 2+]) réduit provenant du milieu extracellulaire. Dans cette thèse, J’ai caractérisé le mécanisme d’action de GIMAP5 dans la régulation de l'homéostasie du Ca[indice supérieur 2+], ainsi que les voies de signalisation modulées par GIMAP5 pour faciliter la survie des cellules T. Tout d'abord, j’ai étudié si GIMAP5 empêche l’apoptose des lymphocytes T en affectant la capacité des mitochondries à réguler la concentration du Ca[indice supérieur 2+], ce qui est nécessaire pour soutenir l’influx de Ca[indice supérieur 2+]. J’ai trouvé que l’accumulation du Ca[indice supérieur 2+] mitochondrial après l’entrée capacitive de Ca[indice supérieur 2+] est défectueuse dans les cellules T de rat déficientes en Gimap5. La disruption des microtubules, mais pas du cytosquelette d'actine, abroge la séquestration du Ca[indice supérieur 2+] mitochondrial dans les cellules T primaires de rat, mais pas dans les cellules T déficientes en Gimap5. J’ai observé que les cellules T provenant de souris déficientes en Gimap5 démontrent une diminution de l’entrée de Ca[indice supérieur 2+]. De plus, la prolifération des cellules T déficientes en Gimap5 est diminuée suite à la stimulation du TCR. En outre, la phosphorylation de STAT5 induit par l'IL-7 est diminuée dans les cellules T CD4[indice supérieur +] de souris déficientes en Gimap5. Également, la perte de Gimap5 aboutit à une activation accrue de la cible mammalienne de la rapamycine (mTOR), indépendamment de la protéine phosphatase 2A (PP2A) ou de la protéine kinase activée par l'AMP (AMPK). Au lieu de cela, l'activation constitutive de la voie PI3K contribue à une forte activation spontanée de mTOR. Collectivement, la fonction de survie de GIMAP5 dans les lymphocytes T peut être liée à la régulation de différentes voies de signalisation. GIMAP5 facilite la fonction, microtubule dépendant, des mitochondries dans leurs actions de régulation du Ca[indice supérieur 2+] après l’entrée capacitive de Ca[indice supérieur 2+]. GIMAP5 est nécessaire pour intégrer les signaux de survie produits suite à l'activation du TCR et de l’IL-7R, qui pourrait être associée à la régulation de l'activité PI3K / AKT / mTOR.
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Levine, Kara B. "Identification of the Human Erythrocyte Glucose Transporter (GLUT1) ATP Binding Domain: A Dissertation". eScholarship@UMMS, 1999. https://escholarship.umassmed.edu/gsbs_diss/247.
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The human erythrocyte glucose transport protein (GLUT1) interacts with, and is regulated by, cytosolic ATP. This study asks the following questions concerning ATP modulation of GLUT1 mediated sugar transport. 1) Which region(s) of GLUT1 form the adenine nucleotide-binding domain? 2) What factors influence ATP modulation of sugar transport? 3) Is ATP interaction with GLUT1 sufficient for sugar transport regulation?
The first question was addressed through peptide mapping, n-terminal sequencing, and alanine scanning mutagenesis of GLUT1 using [32P]-azidoATP, a photoactivatable ATP analog. We then used a combination of transport measurements and photolabeling strategies to examine how glycolytic intermediates, pH, and transporter oligomeric structure affect ATP regulation of sugar transport. Finally, GLUT1 was reconstituted into proteoliposomes to determine whether ATP is sufficient for the modulation of GLUT1 function in-vitro.
This thesis presents data supporting the hypothesis that residues 332-335 contribute to the efficiency of adenine nucleotide binding to GLUT1. In addition, we show that AMP, acidification, and conversion of the transporter to its dimeric form antagonize ATP regulation of sugar transport. Finally, we present results that support the proposal that ATP interaction with GLUT1 is sufficient for transport modulation.
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Rocha, Aline Marubayashi. "Screening mutacional do gene HINT1 em uma amostra da população brasileira com quadro clínico de CMT recessivo". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17140/tde-06012017-112710/.
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O grande grupo heterogêneo de neuropatias periféricas hereditárias estão entre os casos mais comuns de perda sensitiva e fraqueza muscular em crianças e adolescentes. Pelo menos 84 genes estão envolvidos com neuropatias sensitivo-motoras hereditárias (NSMH), sendo suas formas de herança mais comuns as autossômico-dominantes desmielinizante e axonal e as neuropatias ligadas ao cromossomo X, e as mais raras as autossômicorecessivas desmielinizante e axonal e as formas ainda não classificadas. O gene HINT1, possuinte de 3 exons e localizado no cromossomo 5, codifica a proteína Histidine triad nucleotide binding protein 1, uma variante transcricional (mRNA) regulatória que hidroliza substratos. Recentemente mutações em HINT1 foram também relacionadas à neuropatias axonais com neuromiotonia (ARCMT2-NM), e portanto à CMT. O objetivo deste trabalho foi realizar o screening mutacional do gene HINT1 em uma amostra da população brasileira com quadro clínico de CMT recessivo (CMT2-AR), e foram encontradas 1 mutação silenciosa já previamente descrita, 1 polimorfismo exônico e 1 polimorfismo intrônico, também já conhecidos. Concluiu-se que mutações no gene HINT1 não são portanto responsáveis pela CMT-AR nesta amostra da população brasileira.The large heterogeneous group of inherited peripheral neuropathies are among the most common causes of sensory loss and muscle weakness in children and adolescents. At least 84 genes are involved in inherited sensorymotor neuropathies (NSMH), being the demyelinating and axonal autosomaldominant and the X-linked neuropathies their most common forms of inheritance, and the demyelinating and axonal autosomal-recessive and not yet classified forms the most rare ones. The HINT1 gene, with 3 exons and located on chromosome 5, encodes the protein Histidine triad nucleotide binding protein 1, a regulatory transcriptional variant (mRNA) that hydrolyzes substrates. Recently, mutations in HINT1 were also related to axonal neuropathy with neuromyotonia (ARCMT2-NM), and therefore to CMT. The objective of this study was the mutational screening of the HINT1 gene in a sample of the Brazilian population with clinical recessive CMT (CMT2-AR), and 1 silent mutation previously described, 1 intronic polymorphism and 1 exonic polymorphism, both also known, were founded. It was then concluded that mutations in the HINT1 gene are not responsible for CMT2-AR in this particular sample of the Brazilian population.
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Parikh, Ishita. "Expression and Splicing of Alzheimer’s Disease Risk Gene Phosphatidylinositol-Binding Clathrin Assembly Protein". UKnowledge, 2014. http://uknowledge.uky.edu/physiology_etds/18.
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Recent Genome Wide Association Studies (GWAS) have identified a series of single nucleotide polymorphism (SNP)s that are associated with Alzheimer’s disease (AD). One of the SNPs, rs3851179 (G/A), is near the gene phosphatidylinositol-binding clathrin assembly protein (PICALM). To evaluate whether this SNP is associated with PICALM expression, we quantified PICALM mRNA in 56 brain cDNA samples. Using linear regression analysis, we analyzed PICALM expression relative to rs3851179, AD status, and cell type specific markers. An association was detected between rs3851179 and PICALM, microvessel mRNA, glial fibrillary acidic protein (GFAP) mRNA, and synaptophysin (SYN) mRNA. To gain clarity into other possible SNP mechanisms, we searched brain cDNA for PICALM splice variants. We identified several PICALM splice variants involving exons 13-19. To identify and gain an estimation of relative abundance of splice variants, we PCR-amplified across exons 13-20 in cDNA from six individuals, three rs3851179 GG individuals and three rs3851179 AA individuals. Sequencing the cloned isoforms we found that PICALM lacking exon 13 (delta 13) is the most abundant isoform. Other isoforms detected included deletion of exon 18-19. We targeted the latter part of the gene, exon 17-20, to investigate unequal allelic expression using next generation sequencing. Individuals heterozygous for rs76719109 (n= 35), located in exon 17, were used to study the abundance of G/T allele in cDNA and genomic DNA. When we analyzed the T:G allelic ratio, the variant lacking exons 18 and 19 showed unequal allelic expression (p-value < 0.001) in a subset of individuals. One individual was an outlier, showing overall unequal allelic expression, which maybe be harboring a rare mutation capable of modifying PICALM expression. The PICALM intronic SNP rs588076 was associated with delta 18-19 isoform splicing (p-value < 0.001). In conclusion, this study gained a greater insight into the role of AD genetics in PICALM expression and splicing.
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Ye, Zhengmao Ting Jenny P. Y. "Modulation of innate immunity by nucleotide binding biochemical and functional characterization of a CATERPILLER/NLR protein, Monarch-1/NLRP12 /". Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1407.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.Title from electronic title page (viewed Apr. 25, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology." Discipline: Microbiology and Immunology; Department/School: Medicine.
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Johnson, Reed Findley. "Characterization of the 3' terminal 42 nucleotide host protein binding element of the mouse hepatitis virus 3' untranslated region". Texas A&M University, 2003. http://hdl.handle.net/1969/125.
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Kshetri, Man B. "N-TERMINAL DOMAIN OF rRNA METHYLTRANSFERASE ENZYME RsmC IS IMPORTANT FOR ITS BINDING TO RNA AND RNA CHAPERON ACTIVITY". Kent State University Honors College / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1621007414429417.
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Wiles, Natasha Shawn. "Identification of Regulatory Binding Sites and Corresponding Transcription Factors Involved in the Developmental Control of 5'-nucleotidase Expression in Dictyostelium discoideum". Diss., Virginia Tech, 2005. http://hdl.handle.net/10919/27810.
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Gene regulation is a critical aspect of normal development, energy conservation, metabolic control, and responses to environmental cues, diseases and pathogens in eukaryotic organisms. In order to appropriately respond to environmental changes and advance through the life cycle, an organism must manage the expression levels of a large number of genes by utilizing available gene regulation mechanisms. The developmental control of 5â -nucleotidase (5nt) expression in the model system Dictyostelium discoideum has provided a focal point for studies of gene regulation at the level of transcription.
In order to identify temporally-regulated control elements within the promoter of the 5nt gene, 5â and internal promoter deletions were designed and fused to the luciferase and lacZ reporter genes, and reporter enzyme activity was measured in cells from the slug stage of development. The results from these experiments enabled the identification of a 250 bp region of the promoter, which was used as a template for subsequent site-directed mutagenesis experiments. These experiments involved altering 6-12 bp regions of the promoter by substitution. Twelve mutagenized promoters were fused to the luciferase and lacZ reporter genes, and activity was measured at the slug stage of development to more precisely locate cis-acting temporally-regulated control elements. In addition, cAMP induction experiments were performed on amoebae transformed with the mutagenized promoters to identify control elements within the promoter influenced by the presence of cAMP. The regions between -530 and -560 bp and -440 and -460 bp from the ATG translation start site.
In order to evaluate the functions of the cis-acting promoter control elements, electromobility gel shift assays were performed to identify specific DNA-protein interactions on the 5nt promoter. These assays enabled the detection of a 0.13 Rf and 0.33 Rf binding activity to specific sites of the promoter. After characterization of these binding activities, both proteins were purified by a series of column chromatography techniques and characterized after mass spectrometry. The proteins purified were identified as formyltetrahydrofolate synthase and hydroxymethylpterin pyrophosphokinase. These enzymes function in the biosynthetic pathway of tetrahydrofolate and the production of folate coenzymes. The specific interactions of these enzymes with the 5nt promoter suggest these proteins may also function in regulating 5nt expression.Ph. D.
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Sanghvi, Rashesh. "In Silico Analysis Shows That Single Aminoacid Variations In Rhesus Macacque Fcγreceptor Affect Protein Stability And Binding Affinity To IgG1". Digital Archive @ GSU, 2013. http://digitalarchive.gsu.edu/biology_theses/47.
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Rhesus macaques are a widely used animal model of human diseases and related immune responses. Fc receptors (FcRs) mediate the interaction between antibody molecules and innate killing mechanisms, consequently eliminating the pathogen. In rhesus macaques, FcRs are highly polymorphic. To evaluate the potential influence of FcgR polymorphisms on the interaction with antibody molecules, we performed in silico analysis using SIFT, Provean, nsSNPAnalyzer, I-Mutant, MuSTAB and iPTREE-STAB web servers. V20G in FcγRI, I137K in FcγRII and I233V in FcγRIII were further analyzed structurally using FOLD-X, AMMP and Chimera to calculate changes in folding and interaction energy and for structure visualization. Results from our analysis suggest that the selected variations destabilize protein structure. Additionally, Q32R increases the binding affinity of FcγRI, whereas A131T decreases the binding affinity of FcγRII towards IgG1. Together, our results indicate that these substitutions might influence effector and regulatory mechanisms resulting from antibody/FcR interactions.
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Haupt, Melina. "Investigations on the nucleotide binding domain of KdpB by NMR spectroscopy and solution structure of an [alpha]4[beta]7 [alpha-4-beta-7] integrin antagonist". [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972764356.
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Lin, Chien-Ling. "Studies on the Regulation of Cytoplasmic Polyadenylation Element-Binding Protein: A Dissertation". eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/583.
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Post-transcriptional regulation of gene expression sits at the core of proteomic complexity; trans-acting factors that regulate RNA localization and translation capacity are thus indispensible. In this thesis, I present studies of the cytoplasmic polyadenylation element binding protein (CPEB), a sequence specific RNA-binding protein important for cell cycle progression and neural synaptic plasticity. I focus on CPEB because the activity of RNA-binding proteins affects the destiny of their mRNA substrates. As presented in Chapter II, CPEB, though mostly cytoplasmic at steady state, shuttles between the nucleus and the cytoplasm. Surprisingly, the RNA recognition motifs are essential for the nuclear localization. CPEB associates with the polyadenylation machinery in both compartments, suggesting it is involved in both nuclear mRNA processing and cytoplasmic translational regulation. Moreover, the nuclear translocalization is critical to relay a tight translation repression on CPE-containing mRNAs. Chapter III focuses on the regulation of CPEB dimerization. CPEB dimerizes through the RNA-binding domains to inhibit its own RNA binding ability in a cell cycle-dependent manner. By dimerizing, CPEB has enhanced binding to protein destruction factors so that robust active degradation occurs in the later cell cycle. The degradation of CPEB is required for translation activation of a subset of mRNAs and cell cycle progression. In addition, dimerization protects cells from being overloaded with excess CPEB. In sum, the localization and dimerization status of CPEB is dynamic and highly regulated; they in turn regulate the activity of CPEB, which results in responsive translation control. These studies provide a strong foundation to decipher CPEB-mediated gene expression.
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Xia, Yu [Verfasser]. "The role of nucleotide-binding oligomerization domain containing protein 2 (Nod 2) in coxsackievirus B3 induced myocarditis in mice model / Yu Xia". Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2014. http://d-nb.info/1064113559/34.
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Juno, Jennifer. "Contribution of guanine nucleotide binding protein beta polypeptide 3 (GNB3) and lymphocyte activation gene 3 (LAG-3) to HIV susceptibility and immune dysfunction". Permanyer, 2010. http://hdl.handle.net/1993/23948.
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Host genetics play an important role in regulating susceptibility to infectious diseases, including the human immunodeficiency virus (HIV). A polymorphism in a G protein signaling gene (GNB3) previously associated with rapid HIV disease progression is found at high frequencies among African populations, yet its impact on HIV acquisition and disease progression is unknown. The GNB3 gene is located on chromosome 12 near the CD4 gene, as well the gene encoding the regulatory protein lymphocyte activation gene 3 (LAG-3). The goal of this thesis was to characterize the impact of GNB3 genotype on risk of HIV acquisition and disease progression, as well as the relevance of LAG-3 expression to immune exhaustion during HIV infection. Because G proteins are involved in HIV entry and replication in T cells, polymorphisms affecting G protein signaling, such as GNB3 C825T, could dramatically alter susceptibility to HIV infection, viral replication and rates of disease progression. Similarly, the expression of the inhibitory protein LAG-3 could, like other exhaustion markers, mediate increasing immune dysfunction during chronic infection. Both GNB3 and LAG-3 could represent targets for therapeutic intervention to slow disease progression or restore lymphocyte function among HIV-infected individuals.
Surprisingly, our studies showed that GNB3 genotype was not associated with the risk of HIV acquisition in either a female sex worker or perinatal transmission cohort. Disease progression and immune activation among healthy and HIV-infected women were also independent of GNB3 genotype. While the RNA splicing events typically associated with the presence of the GNB3 825T allele could not be detected among cohort participants,
ii
differences in LAG-3 expression were observed between women of differing GNB3 genotypes.
In this cohort, LAG-3 expression on T cells, NK cells and iNKT cells in the peripheral blood was significantly increased among HIV+ women compared to healthy controls, and was not decreased by antiretroviral therapy. The increase in LAG-3 expression was greatest on NK and iNKT cells, an innate lymphocyte subset capable of rapid and robust cytokine production upon stimulation with CD1d-restricted lipid ligands. Lymphocytes derived from the female genital mucosa, the site of HIV acquisition in the female sex worker cohort, expressed significantly higher levels of LAG-3 compared to peripheral blood, suggesting a role for LAG-3 in regulating mucosal immunity, particularly on double negative (CD4-CD8-) T cells.
Finally, we demonstrated that iNKT cells derived from HIV-infected women exhibited significantly lower IFN production compared to healthy controls upon lipid stimulation, which inversely correlated with iNKT LAG-3 expression. Lipid stimulation of PBMC from HIV+ and ARV-treated women also demonstrated perturbations in the secretion of multiple cytokines and chemokines, suggesting that iNKT function is not restored following ART. Together, these data imply that LAG-3 may play an important role in regulating iNKT function during chronic HIV infection. Blocking LAG-3 signaling could therefore restore components of innate immunity that are not improved by current ART, as well as alter HIV susceptibility at the female genital tract, making LAG-3 an attractive target for future therapeutics and viral eradication strategies.
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Straza, Michael W. "A Tale of Two ARFs: Tumor Suppressor and Anti-viral Functions of p14ARF: A Dissertation". eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/472.
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Animals have evolved complicated and overlapping mechanisms to guard against the development of cancer and infection by pathogenic organisms. ARF, a potent tumor suppressor, positively regulates p53 by antagonizing p53’s negative regulator, MDM2, which in turn results in either apoptosis or cell cycle arrest. ARF also has p53-independent tumor suppressor activity. The CtBP transcriptional co-repressors promote cancer cell survival and migration/invasion. CtBP senses cellular metabolism via a regulatory dehydrogenase domain, and is a target for negative regulation by ARF. ARF targets CtBP to the proteasome for degradation, which results in the up regulation of proapoptotic BH3-only proteins, and p53-independent apoptosis. CtBP inhibition by ARF also up regulates PTEN, reducing cancer cell motility, making CtBP a potential therapeutic target in human cancer.
The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and is cytotoxic to cancer cells from a wide variety of tissues. MTOB induced apoptosis was independent of p53, and correlated with the de-repression of the pro-apoptotic CtBP repression target Bik. CtBP over-expression, or Bik silencing, rescued MTOB-induced cell death. MTOB did not induce apoptosis in mouse embryonic fibroblasts (MEFs), but was increasingly cytotoxic to immortalized and transformed MEFs, suggesting that CtBP inhibition may provide a suitable therapeutic index for cancer therapy.
In human colon cancer cell peritoneal xenografts, MTOB treatment decreased tumor burden, and induced tumor cell apoptosis. To verify the potential utility of CtBP as a therapeutic target in human cancer the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Targeting CtBP with a small molecule like MTOB may thus represent a useful and widely applicable therapeutic strategy in human malignancies.
ARF has long been known to respond to virally encoded oncogenes. Recently, p14ARF was linked to the innate immune response to non-transforming viruses in mice. Therefore a wider role for the ARF pathway in viral infection was considered. Previous studies linking p53 to multiple points of the Human Immunodeficiency Virus-1 (HIV-1) life cycle suggested that ARF may also play a role in the HIV life cycle. In this study the interdependency of ARF and HIV infection was investigated. ARF expression was determined for a variety of cell types upon HIV infection. In every case, ARF levels exhibited dynamic changes upon HIV infection-in most cases ARF levels were reduced in infected cells. The impact of ARF over-expression or silencing by RNAi on HIV infection was also examined. Consistently, p24 levels were increased with ARF overexpression, and decreased when ARF was silenced. Thus ARF and HIV modulate each other, and ARF may paradoxically play a positive role in the HIV life cycle.
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Belk, Jonathan Philip. "A Characterization of Substrates and Factors Involved in Yeast Nonsense-Mediated mRNA Decay: A Dissertation". eScholarship@UMMS, 2002. https://escholarship.umassmed.edu/gsbs_diss/65.
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Many intricate and highly conserved mechanisms have evolved to safeguard organisms against errors in gene expression. The nonsense-mediated mRNA decay pathway (NMD) exemplifies one such mechanism, specifically by eliminating mRNAs containing premature translation termination codons within their protein coding regions, thereby limiting the synthesis of potentially deleterious truncated polypeptides. Studies in Saccharomyces Cerevisiae have found that the activity of at least three trans-acting factors, known as UPF1, UPF2/NMD2, and UPF3is necessary for the proper function of the NMD pathway. Further research conducted in yeast indicates that the degradation of substrates of the NMD pathway is dependent on their translation, and that the sub-cellular site of their degradation in the cytoplasm.
Although most evidence in yeast suggests that substrates of the NMD pathway are degraded in the cytoplasm while in association with the translation apparatus, some mammalian studies have found several mRNAs whose decay appears to occur within the nucleus or before their transport to the cytoplasm has been completed. In addition, study of the mammalian TPI mRNA found that this transcript was unavailable as a substrate for the NMD pathway once it had been successfully exported to the cytoplasm, further supporting the notion that the degradation of mammalian substrates of the NMD pathway occurs in association with the nucleus, or during export from the nucleus to the cytoplasm.
To determine if yeast cytoplasmic nonsense-containing mRNA can become immune to the NMD pathway we examined the decay kinetics of two NMDS substrate mRNAs in response to repressing or activating the NMD pathway. Both the ade2-1 and pgk1-UAG-2nonsense-containing mRNAs were stabilized by repressing this pathway, while activation of NMD resulted in the rapid and immediate degradation of each transcripts. These findings demonstrate that nonsense-containing mRNAs residing in the nucleus are potentially susceptible to NMD at each round of translation.
The remainder of this thesis utilizes protein overexpression studies to gain understanding into the function of factors related to the processes of nonsense-mediated mRNA decay and translation in Saccharomyces cerevisiae. Overexpression of a C-terminal truncated form of Nmd3p was found to be dominant-negative for cell viability, translation and the normal course of rRNA biogenesis.
Overexpression studies conducted with mutant forms of the nonsense-mediated mRNA decay protein Upf1p, found that overexpression of mutants in the ATP binding and ATP hydrolysis region ofUpflp were dominant-negative for growth in an otherwise wild-type yeast strain. Furthermore, overexpression of the ATP hydrolysis mutant of Upf1p (DE572AA), resulted in the partial inhibition of NMD and a general perturbation of the translation apparatus. These results support previous studies suggesting a general role for Upf1p function in translation.
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Cook, Neil James. "Le gmp cyclique et la phototransduction chez les vertebres". Strasbourg 1, 1986. http://www.theses.fr/1986STR13147.
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Madera, Dmitri. "Cooperating Events in Core Binding Factor Leukemia Development: A Dissertation". eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/532.
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Leukemia is a hematopoietic cancer that is characterized by the abnormal differentiation and proliferation of hematopoietic cells. It is ranked 7th by death rate among cancer types in USA, even though it is not one of the top 10 cancers by incidence (USCS, 2010). This indicates an urgent need for more effective treatment strategies. In order to design the new ways of prevention and treatment of leukemia, it is important to understand the molecular mechanisms involved in development of the disease.
In this study, we investigated mechanisms involved in the development of acute myeloid leukemia (AML) that is associated with CBF fusion genes. The RUNX1 and CBFB genes that encode subunits of a transcriptional regulator complex CBF, are mutated in a subset (20 – 25%) of AML cases. As a result of these mutations, fusion genes called CBFB-MYH11 and RUNX1-ETO arise. The chimeric proteins encoded by the fusion genes provide block in proliferation for myeloid progenitors, but are not sufficient for AML development. Genetic studies have indicated that activation of cytokine receptor signaling is a major oncogenic pathway that cooperates in leukemia development. The main goal of my work was to determine a role of two factors that regulate cytokine signaling activity, the microRNA cluster miR-17-92 and the thrombopoietin receptor MPL, in their potential cooperation with the CBF fusions in AML development.
We determined that the miR-17-92 miRNA cluster cooperates with Cbfb-MYH11 in AML development in a mouse model of human CBFB-MYH11 AML. We found that the miR-17-92 cluster downregulates Pten and activates the PI3K/Akt pathway in the leukemic blasts. We also demonstrated that miR-17-92 provides an anti-apoptotic effect in the leukemic cells, but does not seem to affect proliferation. The anti-apoptotic effect was mainly due to activity of miR-17 and miR-20a, but not miR-19a and miR-19b.
Our second study demonstrated that wild type Mpl cooperated with RUNX1-ETO fusion in development of AML in mice. Mpl induced PI3K/Akt, Ras/Raf/Erk and Jak2/Stat5 signaling pathways in the AML cells. We showed that PIK3/Akt pathway plays a role in AML development both in vitro and in vivo by increasing survival of leukemic cells. The levels of MPL transcript in the AML samples correlated with their response to thrombopoietin (THPO). Moreover, we demonstrated that MPL provides pro-proliferative effect for the leukemic cells, and that the effect can be abrogated with inhibitors of PI3K/AKT and MEK/ERK pathways.
Taken together, these data confirm important roles for the PI3K/AKT and RAS/RAF/MEK pathways in the pathogenesis of AML, identifies two novel genes that can serve as secondary mutations in CBF fusions-associated AML, and in general expands our knowledge of mechanisms of leukemogenesis.
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Redekar, Neelam R. "Genome and Transcriptome Based Characterization of Low Phytate Soybean and Rsv3-Type Resistance to Soybean Mosaic Virus". Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/75118.
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Soybean is a dominant oilseed cultivated worldwide for its use in multiple sectors such as food and feed industries, animal husbandry, cosmetics and pharmaceutical sectors, and more recently, in production of biodiesel. Increasing demand of soybean, changing environmental conditions, and evolution of pathogens pose challenges to soybean production in limited acreage. Genetic research is the key to ensure the continued growth in soybean production, with enhanced yield and quality, while reducing the losses due to diseases and pests. This research is focused on the understanding of transcriptional regulation of two economically important agronomic traits of soybean: low seed phytic acid and resistance to Soybean mosaic virus (SMV), using the 'transcriptomics' and 'genomics' approaches. The low phytic acid (lpa) soybean is more desirable than conventional soybean, as phytic acid is an anti-nutritional component of seed and is associated with phosphorus pollution. Despite the eco-friendly nature of the lpa soybean, it shows poor emergence, which reduces soybean yield. This research is mainly focused on addressing the impact of lpa-causing mutations on seed development, which is suspected to cause low emergence in lpa soybeans. The differences in transcriptome profiles of developing seeds in lpa and normal phytic acid soybean are revealed and the biological pathways that may potentially be involved in regulation of seed development are suggested. The second research project is focused on Rsv3-type resistance, which is effective against most virulent strains of Soybean mosaic virus. The Rsv3 locus, which maps on to soybean chromosome 14, contains 10 genes including a cluster of coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) protein-encoding genes. This dissertation employed a comparative sequencing approach to narrow down the list of Rsv3 gene candidates to the most promising CC-NB-LRR gene. The evidence provided in this study clearly indicates a single CC-NB-LRR gene as the most promising candidate to deliver Rsv3-type resistance.Ph. D.
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Ertekin, Ozlem. "The Effect Of Indole Acetic Acid, Abscisic Acid, Gibberellin And Kinetin On The Expression Of Arf1 Gtp Binding Protein Of Pea (pisum Sativum L. Cv. Araka)". Master's thesis, METU, 2007. http://etd.lib.metu.edu.tr/upload/12608902/index.pdf.
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ADP Ribosylation Factor 1 (ARF1) is a universal small GTP binding protein
which has an important role in vesicular trafficking between endoplasmic
reticulum and Golgi. ARF1 is a basic component of Coat Protein I (COPI) vesicles
which have functions in both formation of coatomer complex and recruitment of
cargo proteins. In this study, the expression ARF1 was analyzed in pea (P. sativum
L. cv. Araka) grown at different developmental stages. Because of the differential
hormonal levels at corresponding stages, the effects of hormones on ARF1
expression were also studied.
The results of present research show that ARF1 expression in embryos and 2 days
grown plants after germination is lower when compared to 6 days grown plants. In
order to see the hormonal effect, 3 weeks old plants were supplied with 50µM of each hormone for 3 times on alternate days. Protein extraction, cell fractionation,Western blot was carried out and immunoblot analysis was conducted with AtARF1 polyclonal antibodies. It was shown that, in pea shoots, abscisic acid and gibberellin increases the inactive GDP bound ARF1 by hydrolyzing ARF-GTP through activating ARFGTPase activating protein (ARF-GAP) or partially inhibiting ARF-Guanine Nucleotide Exchange Factor (ARF-GEF). In roots, ARF-GDP (cytosolic fraction), ARF-GTP (microsomal fraction) and total amount of ARF1 (13.000 x g supernatant fraction) were down regulated by ~11, ~19 and ~11 fold respectively with the application of gibberellin
and by ~11, ~7 and ~3 fold respectively with the application of abscisic acid
when compared to control plants. These results indicate the importance of plant hormones in the regulation of ARF1 in pea.
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Laine, Jennifer M. "Protein Ligand Interactions Probed by NMR: A Dissertation". eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/617.
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Molecular recognition, defined as the specific interactions between two or more molecules, is at the center of many biological processes including catalysis, signal transduction, gene regulation and allostery. Allosteric regulation is the modification of function caused by an intermolecular interaction. Allosteric proteins modify their activity in response to a biological signal that is often transmitted through the interaction with a small effector molecule. Therefore, determination of the origins of intermolecular interactions involved in molecular recognition and allostery are essential for understanding biological processes. Classically, molecular recognition and allosteric regulation have been associated to structural changes of the system. NMR spectroscopic methods have indicated that changes in protein dynamics may also contribute to molecular recognition and allostery. This thesis is an investigation of the contributions of both structure and dynamics in molecular binding phenomena.
In chapter I, I describe molecular recognition, allostery and examples of allostery and cooperativity. Then I discuss the contribution of protein dynamics to function with a special focus on allosteric regulation. Lastly I introduce the hemoglobin homodimer, HbI of Scapharca inaequivalvis and the mRNA binding protein TIS11d.
Chapter II is the primary focus of this thesis on the contribution of protein dynamics to allostery in the dimeric hemoglobin of scapharca inaequivalvis, HbI. Thereafter I concentrate on the mechanism of adenine recognition of the Tristetraprolin-like (TTP) protein TIS11d; this study is detailed in Chapter III. In Chapter IV I discuss broader impacts and future directions of my research.
This thesis presents an example of the use of protein NMR spectroscopy to probe ligand binding. The studies presented in this thesis emphasize the importance of dynamics in understanding protein function. Measurements of protein motions will be an element of future studies to understand protein function in health and disease.
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Isobe, Yuu. "Direct evidence for the age-dependent demise of GNAS-mutated cells in oral fibrous dysplasia". Kyoto University, 2019. http://hdl.handle.net/2433/242351.
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Pagano, John M. Jr. "RNA Recognition by the Caenorhabditis elegans Embryonic Determinants MEX-5 and MEX-3: A Dissertation". eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/486.
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Post-transcriptional regulation of gene expression is a mechanism that governs developmental and cellular events in metazoans. In early embryogenesis, transcriptionally quiescent cells depend upon maternally supplied factors such as RNA binding proteins and RNA that control key decisions. Morphogen gradients form and in turn pattern the early embryo generating different cell types and spatial order. In the nematode Caenorhabditis elegans, the early embryo relies upon several RNA binding proteins that control mRNA stability, translation efficiency, and/or mRNA localization of cell fate determinants essential for proper development.
MEX-5 and MEX-3 are two conserved RNA-binding proteins required to pattern the anterior/posterior axis and early embryo. Mutation of either gene results in a maternal effect lethal phenotype with proliferating posterior muscle into the anterior blastomeres (Muscle EXcess). Several cell-fate determinants are aberrantly expressed in mex-5 and mex-3 embryos. Both proteins are thought to interact with cis-regulatory elements present in 3’-UTRs of target RNAs controlling their metabolism. However, previous studies failed to demonstrate that these proteins regulate maternal transcripts directly.
This dissertation presents a thorough assessment of the RNA binding properties of MEX-5 and MEX-3. Quantitative biochemical approaches were used to determine the RNA binding specificity of both proteins. MEX-5 has a relaxed specificity, binding with high affinity to linear RNA containing a tract of six or more uridines within an eight-nucleotide window. This is very different from its mammalian homologs Tristetraprolin (TTP) and ERF-2. I was able to identify two amino acids present within the MEX-5 RNA binding domain that are required for the differential RNA recognition observed between MEX-5 and TTP. MEX-3 on the other hand is a specific RNA binding protein, recognizing a bipartite element with flexible spacing between two four-nucleotide half-sites. I demonstrate that this element is required for MEX-3 dependent regulation in vivo. Previous studies only identify a small number of candidate regulatory targets of MEX-5 and MEX-3. The defined sequence specificity of both proteins is used to predict new putative targets that may be regulated by either protein. Collectively, this study examines the RNA binding properties of MEX-5 and MEX-3 to clarify their role as post-transcriptional regulators in nematode development.