Dissertations / Theses on the topic 'Substrate identification'
To see the other types of publications on this topic, follow the link: Substrate identification.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Substrate identification.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Thomas, Daniel Alexander. "Application of peptide and cDNA libraries to protease substrate identification." Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418926.
2
Redbird, Ruth Ann. "Identification of a protein kinase substrate in Sulfolobus solfataricus P2." Diss., Virginia Tech, 2010. http://hdl.handle.net/10919/26884.
Abstract:
Living organisms rely on many different mechanisms to adapt to changes within their environment. Protein phosphorylation and dephosphorylation events are one such way cells can communicate to generate a response to environmental changes. In the Kennelly laboratory we hope to gain insight on phosphorylation events in the domain Archaea through the study of the acidothermophilic organism Sulfolobus solfataricus. Such findings may provide answers into evolutionary relationships and facilitate an understanding of phosphate transfer via proteins in more elaborate systems where pathway disturbances can lead to disease processes.
A λ-phage expression library was generated from S. solfataricus genomic DNA. The immobilized expression products were probed with a purified protein kinase, SsoPK4, and radiolabeled ATP to identify potential native substrates. A protein fragment of the ORF sso0563, the catalytic A-type ATPase subunit A (AtpA), was phosphorylated by SsoPK4. Full length and truncated forms of AtpA were overexpressed in E. coli. Additional subunits of the ATPase were also overexpressed and ATPase activity reconstituted in vitro. Phosphoamino acid analysis and MS identified the phosphorylation sites on AtpA. Several variants of AtpA were derived via site-directed mutagenesis and assayed for ATPase activity. Chemical cross-linking was employed to determine possible ATPase subunit interactions; tryptic digests of AtpA and its mutant variants were performed to examine protein folding. The phosphorylated-mimic variant of AtpA, T98D, resulted in an inactive ATPase complex as determined by ATPase activity assays and native-PAGE indicating potential phosphoregulation by SsoPK4 on enzyme activity. Ultimately, any findings would need verification with in vivo studies.Ph. D.
3
Zhang, Peng. "Functional Characterization of Protein Tyrosine Phosphatases in Tumorigenesis through Substrate Identification." Case Western Reserve University School of Graduate Studies / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=case1365174835.
4
Campbell, Timothy. "Methods for Arrhythmogenic Substrate Identification and Procedural Improvements for Ventricular Arrhythmias." Thesis, The University of Sydney, 2022. https://hdl.handle.net/2123/29925.
Abstract:
Ventricular arrhythmias (VA) are a frequent precursor to sudden cardiac death (SCD) in patients with
structural heart disease (SHD). Patients with SHD are at risk of recurrent ventricular tachycardia
(VT), which generally occurs due to re-entry within and around the presence of an arrhythmogenic
scar. Therefore, scarred myocardium forms the necessary substrate for arrhythmogenesis to occur. A
scar may occur due to obstructive coronary artery disease, causing ischaemic cardiomyopathy (ICM),
or from cardiac injury due to several other causes, including inflammatory, infiltrative, toxin-mediated,
or genetic heart disease, termed non-ischaemic cardiomyopathy (NICM). An implantable
cardioverting defibrillator (ICD) can abort SCD from recurrent VAs. However, they do not stop VAs
from occurring in the first place. Anti-arrhythmic drugs (AADs) may reduce the frequency and burden
of VAs but have limited efficacy. Some have a narrow therapeutic window or the potential for multiorgan
toxicity and can be poorly tolerated. Catheter ablation (CA) is a class I indication for treating
sustained monomorphic VT refractory to AADs. CA reduces VT burden, the number of defibrillator
therapies, greater freedom from recurrent ventricular arrhythmia, and improves quality of life.
However, recurrences can be experienced in up to 50% of patients with SHD-related VT. Some
reasons for the failure of CA include reliable identification of critical components of substrate that can
harbour VAs both in sinus rhythm and during ongoing VT using electroanatomic mapping (EAM) and
imaging techniques, as well as limitations in assessing intraprocedural endpoints. Further refinement
of electroanatomic mapping techniques is required to improve the efficacy of CA. This thesis aims to
expand on current techniques for substrate identification and methods to improve the efficacy of VA
ablation procedures.
5
Chin, Wing Hong. "Identification of TrkB as a p35 interacting protein and a Cdk5 substrate /." View abstract or full-text, 2005. http://library.ust.hk/cgi/db/thesis.pl?BICH%202005%20CHIN.
6
Humphreys, D. "Identification of a novel substrate of the Salmonella protein tyrosine phosphatase SptP." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604780.
Abstract:
A novel cellular target was captured when purified SptP substrate-trapping derivatives and cell extracts were combined, which was isolated and identified by MALDI-TOF mass spectrometry as VCP (valosin-containing protein). VCP is a AAA family ATPase that regulates multiple ubiquitin-dependent cellular processes, including proteasome-dependant protein degradation, protein-membrane association and organelle biogenesis. Next, interaction between native SptP and VCP was reconstituted in vitro. Further biochemical analyses revealed that binding was blocked by phosphatase inhibitors and required both SptP domains (GAP and PTP). Furthermore, SptP exhibited potent phosphatase activity towards VCP in vitro, and TTSS-delivered SptP associated with and specifically dephosphorylated VCP during infection of cultured cells with S. typhimurium. Two additional entry effectors, the Salmonella GTP exchange factor mimics SopE/2 and inositol phosphatase SopB are ubiquitinated following their delivery into target cells. Immunoprecipitation of SopE2 and SopB from cells following infection with a S. typhimurium ΔsptP null mutant localisation occurred independently of SptP and therefore VCP dephosphorylation. Bacterial entry efficiency was unaffected by VCP gene silencing by RNA interference (RNAi), suggesting that SptP-dependent VCP dephosphorylation was relevant later during infection. Such a role was further supported by immunofluorescence and immunoprecipitation analyses demonstrating that SptP persists within infected cells. Immunofluorescence microscopy showed that both SptP and VCP transiently associate with internalised S. typhimurium and that this SptP targeting was dependent upon it s GAP activity. Infection with S. typhimurium engineered to delivery augmented levels of SptP resulted in a 2.1-fold increase in bacterial replication 8-hours post-infection and reciprocally, a 1.7-fold reduction was observed at equivalent time-points after cells were infected with the S. typhimurium ΔsptP mutant. VCP knockdown resulted in increased bacterial replication and parallel immunofluorescence microscopy revealed elevated numbers of Salmonella localised in the infected cell cytosol. Examination of wild-type SCV morphology after VCP knockdown demonstrated that Sif formation was abolished. This thesis reports that the Salmonella entry effector SptP persists after invasion and regulates S. typhimurium replication at late stages of infection through alteration of the phosphorylation status of VCP, a novel cellular target of the SptP PTP domain located on the SCV.
7
Kusevic, Denis [Verfasser], and Albert [Akademischer Betreuer] Jeltsch. "Biochemical investigation of the substrate specificity of protein methyltransferases and the identification of novel substrates / Denis Kusevic ; Betreuer: Albert Jeltsch." Stuttgart : Universitätsbibliothek der Universität Stuttgart, 2016. http://d-nb.info/1165574489/34.
8
Boswell, Nicholas William Bradford. "Biochemical characterization of the [FeFe]-hydrogenase maturation protein HydE and identification of the substrate." Thesis, Montana State University, 2011. http://etd.lib.montana.edu/etd/2011/boswell/BoswellN1211.pdf.
Abstract:
Hydrogenases catalyze the reversible reduction of protons using complex metal clusters with unusual ligands. The catalytic center of the [FeFe]-hydrogenases is called the H-cluster, and is characterized by a [4Fe-4S] cluster connected via a cysteine thiolate to a 2Fe subcluster coordinated by carbon monoxide and cyanide ligands as well as a bridging dithiolate. Assembly of the H-cluster is carried out by three hydrogenase maturation proteins: HydE, HydF, and HydG. HydF is a GTPase and has been implicated to serve as a scaffold for assembly of the 2Fe subcluster of the H-cluster. HydE and HydG are radical S-adenosylmethionine (SAM) enzymes and thus are thought to utilize reductive cleavage of SAM to initiate radical chemistry. HydG has been shown to catalyze the formation of the carbon monoxide and cyanide ligands of the H-cluster utilizing tyrosine as a substrate. HydE, therefore, has been proposed to be responsible for biosynthesis of the dithiolate ligand of the H-cluster. The aim of this study was to biochemically characterize active, Fe-S reconstituted HydE and to identify the substrate of this radical SAM enzyme. Questions to be studied also included studying the role of HydE in H-cluster maturation. This study used protein purified from recombinant E. coli. The purified protein was chemically reconstituted with iron and sulfide, and used for spectroscopic characterization and HPLC based activity assays. Colorimetric assays were also used for protein characterization and to test for the consumption of substrate. The results indicate that cysteine is likely the substrate of HydE. Activity assays show that HydE- catalyzed SAM cleavage is stimulated in the presence of cysteine, and HydF purified from different genetic backgrounds shows a spectroscopic shift in the lambda max when both HydE and cysteine are present during growth. Spectroscopic characterization confirms that HydE is an Fe-S containing radical SAM enzyme and that cysteine may be a substrate during [FeFe]-hydrogenase H-cluster maturation.
9
Farah, Sahar. "Identification of Rho-associated protein kinaseà as an insulin receptor substrate-1 binding protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ28422.pdf.
10
Cheng, Kai. "Identification of Pctaire1 as a p35-interacting protein and a novel substrate for Cdk5 /." View Abstract or Full-Text, 2003. http://library.ust.hk/cgi/db/thesis.pl?BICH%202003%20CHENG.
Abstract:
Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003.Includes bibliographical references (leaves 153-177). Also available in electronic version. Access restricted to campus users.
11
Farah, Sahar. "Identification of Rho-associated protein kinase-alpha as an insulin receptor substrate-1 binding protein." Thesis, University of Ottawa (Canada), 1998. http://hdl.handle.net/10393/4152.
Abstract:
Insulin receptor substrate-1 (IRS-1) is phosphorylated on multiple tyrosine residues by ligand-activated insulin receptor. These tyrosine phosphorylation sites serve to dock several SH2-containing signaling proteins. In addition, IRS-1 also contains several protein modules that have been implicated in protein-protein or protein-lipid interactions. In an attempt to identify novel proteins that may interact with these IRS-1 protein modules, yeast two-hybrid screening was employed. The bait, corresponding to the N-terminal 500 amino acids of the Xenopus IRS-1 (XIRS-1), was comprised of a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain and a SAIN (Shc and IRS-1 NPXY binding) domain. Screening of a Xenopus oocyte cDNA library with the bait resulted in the isolation of a partial Xenopus cDNA, XROK$\alpha$. The cloned cDNA contains an open reading frame of about 500 amino acids (in frame with the N-terminal GAL4 activating domain) which are 90% identical to the C-terminus of the recently identified RhoA-associated protein kinase $\alpha$ (ROK$\alpha$). The partial XROK$\alpha$ cDNA contains the putative Rho binding domain as well as the C-terminal pleckstrin homology/cysteine rich domain (PH/CRD) but lacks the N-terminal serine/threonine kinase domain. Using the yeast two-hybrid system, we showed that XROK$\alpha$ interacts strongly with a constitutively active form of RhoA (V14-RhoA). Further analysis indicated that the XIRS-1 PTB domain is specifically involved in binding to XROK$\alpha$. To further characterize the potential role of XROK$\alpha$ in insulin signalling, I have cloned the entire coding sequence of XROK$\alpha$ by a combination of hybridyzation screening and PCR amplification. I have also generated XROK$\alpha$ specific polyclonal antibodies. Using these antibodies I have detected endogenous XROK $\alpha$ protein by both Western-blotting and by immune kinase assay in vitro. Taken together, these studies have identified an IRS-1 binding serine/threonine kinase which may play an important role in modulating insulin signalling.
12
Mueller, Martin J. "Leukotriene A₄ hydrolase : identification of amino acid residues involved in catalyses and substrate-mediated inactivation /." Stockholm, 2001. http://diss.kib.ki.se/2001/91-628-4934-4/.
13
Berglund, Frederick M. "Identification of hnRNP-U as a DNA-PK substrate phosphorylated in response to DNA damage." Thesis, University of Dundee, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505651.
14
Cole, Kathryn O. "Printability and environmental testing using silver-based conductive flexographic ink printed on a polyamide substrate /." Online version of thesis, 2007. http://hdl.handle.net/1850/4490.
15
Kahl, Philipp. "Identification of long-range solid-like correlations in liquids and role of the interaction fluid-substrate." Thesis, Le Mans, 2016. http://www.theses.fr/2016LEMA1002/document.
Abstract:
Les liquides diffèrent des solides par une réponse retardée à la sollicitation en cisaillement; c’est-à-dire une absence d’élasticité de cisaillement et un comportement d'écoulement à basses fréquences (<1 Hz). Ce postulat pourrait ne pas être vrai à toutes échelles. A l’échelle submillimétrique, les mesures viscoélastiques (VE) réalisées en améliorant l'interaction entre le liquide et le substrat, montrent qu’une élasticité basses-fréquences existe dans des liquides aussi variés que les polymères, les surfondus, les liquides à liaison H, ioniques ou van der Waals. Ce résultat implique que les molécules à l'état liquide ne seraient pas dynamiquement libres, mais élastiquement corrélées.En utilisant les propriétés biréfringentes des fluctuations prétransitionnelles qui coexistent dans la phase isotrope des cristaux liquides, nous montrons qu'il est possible de visualiser ces corrélations « cachées ». Dans des conditionssimilaires aux mesures VE, une biréfringence optique synchrone à la déformation est observée dans la phase isotrope à des fréquences aussi basses que 0.01 Hz et des températures éloignées de toute transition. Le comportement dela biréfringence basses-fréquences a des similitudes avec l'élasticité; elle est en phase avec la déformation à faibles amplitudes de déformation, puis en phase avec le taux de déformation à plus grandes amplitudes. La biréfringence basses- fréquences est forte, sans défaut et réversible. Elle indique un ordre à longue portée. La synchronisation de la réponse à la sollicitation en fréquence et l’état ordonné qu’elle produit ne sont pas compatibles avec un état liquide isotrope mais montrent qu’il s’agit d’un état élastique soumis à déformation (entropie élastique)Liquids differ from solids by a delayed response to a shear mechanical solicitation; i.e. they have no shearelasticity and exhibit a flow behaviour at low frequency (<1 Hz). This postulate might be not verified at thesub-millimeter scale. By optimizing the measurement in particular by improving the liquid/substrate interactions (wetting), a low frequency shear elasticity has been found in liquids including molten polymers, glass-formers, H-bond polar, ionic or van der Waals liquids. This result implies that molecules in the liquid state may not be dynamically free but weaklyelastically correlated. Using the birefringent properties of the pretransitional fluctuations coexisting in the isotropic phase of liquid crystals, we show that it is possible to visualize these “hidden” shear-elastic correlations. We detect a synchronized birefringent optical response in the isotropic phase that is observable at frequencies as low as 0.01 Hz and at temperatures far away from anyphase transition. The low-frequency birefringence exhibits a strain dependence similar to the low frequency elasticity: An optical signal that is in-phase with the strain at low strain amplitudes and in-phase with the strain-rate at larger strain amplitudes. The birefringent response is strong, defect-free, reversible and points out a collective response. This long-range ordering rules out the condition of an isotropic liquid and its synchronized response supports the existenceof long-range elastic (solid-like) correlations. In the light of this, the strain dependence of the harmonic birefringent signal and the shear elasticity may correspond to an entropy-driven transition
16
Cowan, Jon Walter. "Proteolysis and the growth hormone receptor identification and characterization of GHR as a [gamma]-secretase substrate /." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2009r/cowan.pdf.
17
Myers, Alexandra. "Identification of CaMK-II Protein Targets in Tissue Culture and Zebrafish Embryos using Tandem Mass Spectrometry." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/10.
Abstract:
Calcium (Ca2+)/calmodulin-dependent kinase 2 (CaMK-II) is one member of a family of Ca2+/calmodulin-dependent protein kinases that responds to intracellular Ca2+ signals (Hudmon, A. and H. Schulman (2002)). CaMK-II is a multifunctional regulator of transcription, cell cycle progression, cell motility and neuronal development. (Wang, C., et al. (2008), Easley, C. A. IV, et al. (2008), Osterhoff, M., et al. (2003), Faison, M. O., et al. (2002)). Recently, CaMK-II has been shown to be important in the early development of vertebrates. In developing zebrafish, disruption of CaMK-II expression has been shown to induce phenotypes similar to those documented in several human diseases. The identification of the tissue-specific binding partners and substrates of CaMK-II which are responsible for specific developmental fates remains a key step in understanding this important protein kinase family. In this thesis research, specific “substrate-trapping” mutants of CaMK-II were designed, introduced into a variety of rodent and human cell lines in culture and used in conjunction with tandem mass spectrometry to identify binding partners, such as β-actin, tropomodulin-3 and Fli-I as well as novel, putative substrates, such as the tumor suppressor protein 53 (p53). This approach was subsequently applied to zebrafish embryos where an overlapping subset of CaMK-II binding proteins to those found in mammalian cell culture were identified. This project represents one of the first studies to identify binding proteins in zebrafish embryos using epitope tagging and mass spectrometry. This research has also established a technical framework for the use of mass spectrometry to characterize the developmental proteome of whole zebrafish embryos or specific zebrafish tissues at any developmental time point.
18
Kenny, Thomas Donald. "Identification of High-Velocity Pseudo-surface Acoustic Wave Substrate Orientations and Modeling of Surface Acoustic Wave Structures." Fogler Library, University of Maine, 2011. http://www.library.umaine.edu/theses/pdf/KennyT2011.pdf.
19
Nguyen, Hue Bach Thi [Verfasser], and Wolfgang [Akademischer Betreuer] Schumann. "Identification of substrate proteins of FtsH during sporulation of Bacillus subtilis / Hue Bach Thi Nguyen. Betreuer: Wolfgang Schumann." Bayreuth : Universitätsbibliothek Bayreuth, 2012. http://d-nb.info/1023305542/34.
20
Sathanantham, Preethi, Shiva K. Devaiah, and Cecelia A. McIntosh. "Structure-Function Analysis of Grapefruit Glucosyltransferase Protein – Identification of Key Amino Acid Residues for its Rigid Substrate Specificity." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/352.
Abstract:
Flavonoids are an important class of secondary metabolites widely distributed in plants. The majority of naturally occurring flavonoids are found in glucosylated form. Glucosyltransferases are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor flavonoid substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi (Cp3OGT) has strict substrate and regiospecificity. In this study, amino acid residues that could potentially alter the rigidity observed in this enzyme were mutated to position equivalent residues of a putative anthocyanin specific glucosyltransferase from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling followed by site directed mutagenesis to identify candidate regions, three double mutations were made. To test the basis of substrate specificity, biochemical analysis of the three recombinant mutant proteins was carried out. Recombinant protein with mutation S20G+T21S revealed that the enzyme retained activity similar to the wildtype (Cp3OGT) (WT- Km app-104.8 µM; Vmax = 24.6 pmol/min/µg, Mutant- Km app-136.42 µM; Vmax -25pmol/min/µg) but the mutant was more thermostable compared to the WT. The (S290C+S319A) mutant protein retained 40% activity relative to wildtype and has an optimum pH shifted towards the acidic side (pH 6) (Km app-8.27 µM; Vmax-90.9 pmol/min/µg). Mutation of Glutamine87 and Histine154 (H154Y+Q87I) have rendered this recombinant protein inactive with every class of flavonoid tested. Interestingly, the single point mutations H154Y and Q871I had significant activity, slightly greater than that of wildtype enzyme. The two active recombinant proteins will further be analyzed to determine whether the mutations have altered regiospecificity of the original enzyme. Product identification is being conducted using HPLC.
21
Herdendorf, Timothy J. Miziorko Henry M. "Phosphomevalonate kinase investigation of the recombinant human enzyme and identification of key residues involved in substrate binding and catalysis /." Diss., UMK access, 2007.
Abstract:
Thesis (Ph. D.)--School of Biological Sciences. University of Missouri--Kansas City, 2007."A dissertation in molecular biology and biochemistry and cell biology and biophysics." Advisor: Henry M. Miziorko. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Sept. 12, 2008. Includes bibliographical references (leaves 109-126). Online version of the print edition.
22
Yang, Li. "Design and development of novel radio frequency identification (RFID) tag structures." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/31824.
Abstract:
Thesis (Ph.D)--Electrical and Computer Engineering, Georgia Institute of Technology, 2010.Committee Chair: Tentzeris, Manos; Committee Member: DeJean, Gerald; Committee Member: Ingram, Mary; Committee Member: Kavadias, Stylianos; Committee Member: Laskar, Joy. Part of the SMARTech Electronic Thesis and Dissertation Collection.
23
Ferguson, Alexandra. "Synthesis and ring-opening of NH-aziridine-2-carboxylates, and preparation of novel pyrazolo[3,4-d]pyrimidines for kinase-substrate identification." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/11074.
Abstract:
1: The ring-opening of aziridine-2-carboxylates dominates their reactivity, providing diverse, biologically relevant compounds. Less is known about the chemistry of NH-aziridine-2-carboxylates, owing to multi-step preparation, and lack of N-functionality to aid ring-opening. Building on the recent disclosure of an NH-aziridination methodology applicable to enones, the substrate scope was expanded to include enoates. Aziridination provided access to NH-aziridine-2-carboxylates in a single step from readily available starting materials. Treatment of the resulting aziridine-2-carboxylates with nucleophiles proceeded with high regio- and stereoselectivity, providing access to natural and unnatural amino acid derivatives (Scheme 1).[Molecular structure diagrams appear here. To view, please open pdf attachment] Scheme 1: The synthesis and ring-opening of aziridine-2-carboxylates 2: Identification of kinase protein-substrate sets is crucial to further understand cellular processes, but is challenging with known methodology. Previous study had proposed a new methodology, incorporating two known strategies, bump-hole inhibition and photo-affinity labelling. Novel C3-aryl pyrazolo[3,4-d]pyrimidines were designed for the purpose (Fig. 1), although preparation of the compounds had proved challenging, with several key steps in the synthesis of analogues low-yielding and lengthy. A new synthetic route was developed, using hydrazone allylation to establish the α-tertamine functionality of the pyrazolo[3,4-d]pyrimidines. A late-stage oxidative cleavage was used to re-connect with the original synthetic strategy, to provide access to the C3-hydro analogues. Application of the revised route to the synthesis of the target compounds, using an alternative enol ether for C3-aromatic installation, provided access to two novel pyrazolo[3,4-d]pyrimidines for testing. [Molecular structure diagrams appear here. To view, please open pdf attachment] Fig. 1: C3-aryl pyrazolo[3,4-d]pyrimidines
24
Hammes, Clay. "Identification and analysis of upper extremity cumulative trauma disorders in the substrate disk polishing areas of ABC Company in central Minnesota." Online version, 1999. http://www.uwstout.edu/lib/thesis/1999/1999hammesc.pdf.
25
Wegner, Nina Vanessa [Verfasser], Jens C. [Akademischer Betreuer] Brüning, and Günter [Akademischer Betreuer] Schwarz. "Identification of in vivo interactions of insulin receptor substrate 1 in murine liver / Nina Vanessa Wegner. Gutachter: Jens C. Brüning ; Günter Schwarz." Köln : Universitäts- und Stadtbibliothek Köln, 2011. http://d-nb.info/1038168716/34.
26
Moheshwarnath, Issur. "Structural definition of substrate recognition by model RNA capping enzymes and the identification of a novel class of viral RNA capping enzymes." Thèse, Université de Sherbrooke, 2011. http://savoirs.usherbrooke.ca/handle/11143/4315.
Abstract:
The RNA cap structure is a fundamental feature of most known eukaryotic mRNAs and some viral RNAs, It is important for the stability, transport and translation of mRNAs. It is co-transcriptionally synthesized via the action of 3 consecutive enzymatic reactions: (1) A RNA triphosphatase which cleaves off the 5' terminal phosphate of nascent RNAs; (2) A RNA guanylyltransferase which transfers a GMP moiety onto the acceptor RNA; (3) A RNA (guanine-N7) methyltransferase which methylates the cap guanine at the N7 position. Through the end of the 1990's until now, the crystal structures of several capping enzymes have been solved. However, these structures, although very insightful in themselves, failed to provide any instructive information on several key issues regarding enzyme-substrate interactions. For instance, one of the first breakthrough crystallographic studies in RNA capping chemistry led to the elucidation of the yeast RNA triphosphatase structure (the Cet1 protein). However, in the crystal structure, the Cet1 protein was bound to a sulphate molecule, which was hypothesised to be mimicking the product of the RNA triphosphatase reaction- a phosphate molecule.The inability to capture the RNA triphosphatase in complex with its ligands is probably on account of the inherent thermodynamic instability of this protein when bound to RNA or a nucleotide. A structural definition of the active site of the yeast RNA triphosphatase in complex with an appropriate substrate is still lacking. In addition, the elucidation of the structure of the RNA guanylyltransferase of the Paramecium bursaria chlorella virus -1 (PBCV-1) in several different conformations has been a landmark study which greatly contributed towards the understanding of the catalytic pathway of this model enzyme. On the other hand, despite the presence of the natural substrate-GTP, within the active site of the enzyme, the rationale behind the GTP specificity of RNA guanylyltransferase remains poorly understood. Moreover, a molecular mechanism for the RNA guanylyltransferase reaction is still missing. Finally, the importance of the RNA cap for the process of eukaryotic translation is undisputable. However, the relationship between the RNA cap and translation has been mostly studied indirectly through proteins which bind to the cap structure. Most studies pertaining directly to the impact of the binding of the RNA cap structure have been restricted to investigating the inhibitory potential of various cap analogues on the translation process. Studies on the effects of modified RNA caps at the 5' ends of RNAs have only started in the last few years, and more importantly, the necessity of the N7-methyl group on RNA cap analogues had not been addressed. This thesis therefore aims to provide a structural insight into the structural dynamics of enzyme-ligand(s) interactions of the model S. cerevisiae's RNA triphosphatase and the PBCV-1 RNA guanylyltransferase. In addition, we show that purine analogues can be a useful tool for the study of several cellular processes, such as RNA translation. In the process we have uncovered a novel class of RNA capping enzyme in the flavivirus genus of the Flaviviridae family of RNA viruses, thus providing a more succinct insight into the flaviviral replication complex.
27
David, Marion. "Identification and functional characterization of an ABC transporter of Haemonchus contortus, the P-glycoprotein 13." Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30206/document.
Abstract:
Les lactones macrocycliques (LM) sont des anthelminthiques (AH) à effet paralysant très utilisés chez les animaux et les humains contre les nématodes parasites. Cependant, leur succès thérapeutique est compromis par la résistance croissante aux LM, qui pourrait être en partie dû aux ABC transporteurs P-glycoprotéines (Pgps) sélectionnés et surexprimés chez les nématodes résistants aux LM. Dans ce travail, nous avons étudié plus précisément la P-glycoprotéine 13 du parasite de petits ruminants, Haemonchus contortus. Son orthologue chez le modèle nématode C. elegans, Cel-Pgp-13, est exprimé dans les amphides, structures qui ont été associées à la sensibilité aux AH chez C. elegans et H. contortus. Pour prédire la capacité des Pgps de nematode à transporter des drogues, incluant des LM et autres AH, nous avons développé un modèle de docking in silico. Nous avons utilisé la structure cristallographique de C. elegans Pgp-1 (Cel-Pgp-1), et nous avons montré la liaison avec une forte affinité de plusieurs ligands décrits comme activateurs de sa fonction ATPasique. Nous avons aussi décrit une forte affinité des LM, et un site spécifique de liaison de ces composés à Cel-Pgp-1. Cette approche représente un outil important pour prédire les interactions entre AH, et pour concevoir rationnellement de nouveaux inhibiteurs compétitifs des Pgps de nématode, dans le but d'améliorer les stratégies thérapeutiques. Sur la base de cette approche, nous avons prédit la structure 3D de Hco-Pgp-13 à partir du cristal de Cel-Pgp-1 afin d'étudier son intéraction avec des substrats potentiels, en particulier les LM. Nous avons trouvé des affinités similaires pour différents composés précédemment testés sur Cel-Pgp-1. In vitro, la mesure de l'activité ATPasique montre que l'actinomycine D est un substrat de Hco-Pgp-13. Nos données démontrent la présence possible d'un domaine de reconnaissance multispécifique sur ce transporteur de parasite. La détermination par immunofluorescence de l'expression de Hco-Pgp-13 a montré une distribution tissulaire large indiquant que Hco-Pgp-13 pourrait jouer un role important dans le transport de substrats endogènes et/ou exogènes. En conclusion, ce travail permet de mieux comprendre le rôle des Pgps de nématodes dans le transport de médicaments AH, tant au niveau de l'organisme modèle C. elegans que du nématode parasite H. contortus. Cette étude suggère la conservation de la fonction de tranporteur ABC multidrogue dans ces espèces. La localisation de Hco-Pgp-13 sur les structures amphidiales, et son éventuelle implication dans la résistance aux médicaments et à la survie de H. contortus à l'exposition à des composés AH, restent à préciserMacrocyclic lactones (ML) are paralyzing anthelmintics used in animals and humans against parasite nematodes. However, their therapeutic success is compromised by the spread of ML resistance. This might be at least partly due to P-glycoproteins (Pgps) ABC transporters that are selected and overexpressed in ML-resistant nematodes. Deciphering the role of the 10 Pgps expressed in the parasite of small ruminants Haemonchus contortus is thus of major importance to guaranty anthelmintic (AH) efficacy of various drugs. Here we focused on Hco-Pgp-13 due to the expression in the amphids of its closest ortholog in the model nematode C. elegans. Indeed, the amphids represent a putative entry route of drugs to reach AH targets in the nervous system and have been linked to AH susceptibility in C. elegans and H. contortus. In order to predict the capacity of nematode Pgps to transport drugs, including ML and otherAH, we have developed an in silico drug docking model. We have used C. elegans Pgp-1 (Cel-Pgp-1) crystal structure and have showed a high affinity binding of several ligands that have been shown to be activators of its ATPase function. ML were also found to bind with high affinity to Cel-Pgp-1, on a specific binding site. This approach provides a valuable tool to predict drug-drug interactions and to rationally design new competitive inhibitors of nematode Pgps, in order to improve anthelmintic therapeutics. We then predicted a putative 3D structure of Hco-Pgp-13 based on the recently released crystal of Cel-Pgp-1, with which it presented a high homology. This allowed the study of the interaction of Hco-Pgp-13 with potential substrates, in particular ML. We found similar affinities for various drugs previously tested on Cel-Pgp-1, supporting the good homology of these two proteins. Together with in vitro ATPase assay experiments that confirmed the substrate status of actinomycin D, this indicates a possible multispecifc recognition capacity of this parasitic transporter. The determination of Hco-Pgp-13 localization using immunohistochemistry showed a wide tissue expression consistent with a critical role for Hco-Pgp-13 in endogenous and/or exogenous substrate transport. In conclusion, this work provides insights into the role of nematode Pgps in transporting AH drugs, both at the level of the model organism C. elegans and of the parasitic nematode H. contortus. This suggests a high homology of function conserved between ABC tranporters in these species. The localization of such protein on amphidial structures and its possible involvement in drug resistance and survival of H. contortus to exposure to AH compounds remain to be precised
28
Rehman, Saba [Verfasser], and Koepsell [Gutachter] Hermann. "Identification of accessible and closed substrate binding sites in the outward open cleft of rat Organic Cation Transporter 1 (rOCT1) / Saba Rehman ; Gutachter: Koepsell Hermann." Würzburg : Universität Würzburg, 2018. http://d-nb.info/1170061710/34.
29
Nimmagadda, Subbaiah Chary Verfasser], Thomas [Akademischer Betreuer] [Seufferlein, Sven-Erik [Akademischer Betreuer] Behrens, and Michael [Akademischer Betreuer] Seckl. "Identification and characterization of Abelson Interactor 1, as a novel substrate of Protein Kinase D2 / Subbaiah Chary Nimmagadda. Betreuer: Thomas Seufferlein ; Sven-Erik Behrens ; Michael Seckl." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2013. http://d-nb.info/1037725433/34.
30
Wasserman, Jacob Israel. "Progress toward the synthesis of structurally novel cytotoxic chlorinated diterpenes and identification of 3-isopropylmalate as an endogenous substrate of a methyl transferase in the yeast S. Cerevisiae." Diss., Restricted to subscribing institutions, 2004. http://proquest.umi.com/pqdweb?did=765405381&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
31
Eggert, Erik. "Development of a cellular mechanistic assay for the SET and MYND domain containing methyltransferase SMYD2, identification and validation of a novel substrate, and functional characterization of its inhibition." Doctoral thesis, Humboldt-Universität zu Berlin, 2017. http://dx.doi.org/10.18452/18145.
Abstract:
Protein Methyltransferasen sind oftmals fehlreguliert in Tumorzellen und stellen potenzielle Ziele in der Krebstherapie dar. Das SET und MYND Domain enthaltene Protein 2 (SMYD2) wurde als potenzielles Onkogen beschrieben und eine Überexpression korreliert mit einer schlechten Prognose. Für SMYD2 wurden verschiedene Substrate beschrieben u.a. Histon H3 und der Tumorsuppressor p53, allerdings ist die Biologie dieses Enzymes kaum verstanden. Durch die Entwicklung einer Testsubstanz zur spezifischen Hemmung von SMYD2 könnte ein möglicher therapeutischer Nutzen besser untersucht werden. Hierfür wurde ein zellulärer mechanistischer Test zur Messung der SMYD2 Aktivität mittels eines methylierungs-spezifischen Antikörpers etabliert. Mit Hilfe dieses Tests wurde BAY-598 als selektiver und potenter zellulärer Hemmer für SMYD2 identifiziert. Im weiteren Verlauf dieser Arbeit wurden mittels eines Proteomansatzes nach SMYD2 Überexpression hunderte neue zelluläre Lysinmethylierungen identifiziert. Hierbei wurde das AHNAK Protein als neues SMYD2-Substrat identifiziert und validiert. Die AHNAK Methylierung konnte in verschiedenen Zelllinien und im Muskelgewebe von Mäusen nachgewiesen werden. Im letzten Teil der Arbeit wurde die spezifische Testsubstanz BAY-598 genutzt, um verschiedene in der Literatur aufgekommene Hypothesen zur SMYD2 Funktion zu testen. Die vorliegende Arbeit hat dazu beigetragen die potente und selektive SMYD2 Testsubstanz BAY-598 zu entwickeln. Außerdem wurde mit AHNAK ein neues SMYD2 Substrat identifiziert und validiert. Die Relevanz des SMYD2 Enzymes und der AHNAK Methylierung erfordert weitere Forschungsarbeit, die durch die Bereitstellung der spezifischen Testsubstanz BAY-598 deutlich verbessert werden sollte.Protein methyltransferases are often misregulated in tumor cells and display a potential target for cancer therapy. The SET and MYND domain containing protein 2 (SMYD2) was described as a potential oncogene and overexpression correlated with a worse prognosis. Several substrates for SMYD2 had been described among them histone H3 and p53. However, the biology of SMYD2 is poorly understood. By developing a small molecule inhibitor of SMYD2 its therapeutic role could be better evaluated. Therefore, a cellular mechanistic assay was developed using a methylation specific antibody. With that assay BAY-598 was identified as a potent and selective cellular inhibitor of SMYD2. In the following a proteomic approach revealed hundreds of novel cellular lysine methylation sites in SMYD2 overexpression cells. Among these AHNAK protein was validated as a novel SMYD2 substrate, which was present in several cell lines as well as in muscle of mice. Finally, BAY-598 was used to test several hypothesized functions of SMYD2 in different cell line models. Taken together, the current work strongly supported the development of the probe inhibitor BAY-598 and the discovery of AHNAK as a novel SMYD2 methylation substrate. The relevance of SMYD2 and AHNAK methylation needs further investigation, which should be supported by BAY-598.
32
Alonso, Tarajano Manuel Alejandro. "Systematic identification and quantification of substrate specificity determinants in human protein kinases = Identificación y cuantificación sistemática de determinantes de la especificidad por sustrato en las proteínas quinasas de humano." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/125027.
Abstract:
In human there are 518 protein kinases reported and many of them are involved in several cellular processes and also in important pathologies. Kinases have a diverse specificity for the sequences they phosphorylate, and it has been shown that their in vivo specificity is guided by several elements such as the sequence surrounding the phosphorylated amino acid in the substrate, the co-localization with the substrate, interactions mediated by docking sites and the association of kinases to adaptor or scaffold proteins (AS).
The objective of the current thesis has been the identification and quantification of the contribution to the specificity of i) the phosphorylated site and its surrounding residues in sequence and ii) the association of kinases to AS proteins.
By integrating data from public resources we compiled a set of kinase-phosphorylation sites in human corresponding to 325 (62.7%) kinases, 1856 substrates and 5946 phosphorylation sites. We have used sequence logos for representing the phosphorylation motifs recognized by the kinases in our set and we have used these logos to guide the classification of kinases attending to the residue composition of the sequences surrounding the phosphorylation site.
We have used position-specific scoring matrices (PSSMs) as the probabilistic representation of the sequences phosphorylated by the kinases. Based on the score in the PSSMs relative to the phospho-acceptor amino acid, we classified several residues as specificity-determinant residues (SDR) for several kinase families. The identity, position in the sequence alignment and the frequency of the SDRs identified vary considerably among the kinase families analyzed. The statistical significance of the PSSMs was assessed taking into account their recall and their information content (IC). We found negative correlations between the the number of seed sequences and i) the recall of the PSSMs and ii) the IC of the PSSMs. Based on the IC value, and in the comparison to random distributions, we found that statistically significant PSSMs differ from the non-statistically significant ones regarding their IC, recall, number of seed phosphorylation sites and AUC-ROC.
We have developed a computational strategy for the identification of proteins with known function as adaptors or scaffolds of human kinases (kAS). In total, we have identified a set of 191 kAS proteins which is enriched in domains and functional terms that support their role as AS. These 191 kAS associate to 55% of human kinases, which suggest that the association to this type of AS molecules is common among human kinases. Our results suggest that, when compared to random proteins, kAS proteins are five times more likely to interact with a significantly large fraction of the substrates of a the kinases to which the kAS are associated.
Starting from a set of 156 human kinases for which we count with at least five substrates, we identified a set of 279 proteins with a potential function as adaptor or scaffold (pAS). This set is enriched in protein domains and functional terms related to the predicted function and that also suggest a relationship to signalling processes. Our analysis on cellular co-localization suggest that, for 74.6% of the kinase-pAS associations found, the pAS protein may play a role in the co-localization of the kinases and their corresponding sets of substrates.
Finally, we have analyzed the relationship between the association of different kinases to common AS proteins and the number of in vivo substrates shared by the kinases.
Our results suggest that kinases with AS proteins in common do not share more in vivo substrates than what would be expected only due to chance. To our opinion, this suggests that AS proteins may diminish the substrate crossed specificity of the kinases to which they associate.
33
Sundermann, Lena. "Identification and characterization of new Greatwall kinase substrates." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT016.
Abstract:
La Division mitotique est une phase essentielle du cycle cellulaire qui assure la répartition correcte du contenu génétique. La mitose implique une réorganisation cellulaire profonde qui est principalement induite par une phosphorylation massive de protéines. Cette phosphorylation a lieu grâce à un équilibre fin entre kinases et phosphatases. À l'entrée mitotique, la phosphorylation protéique est induite par l'activation de la kinase cycline B/CDK1 et par l'inhibition de la phosphatase PP2A-B55. Résultats de notre et d'autres laboratoires ont récemment découvert une nouvelle voie essentielle pour moduler la phosphatase PP2A-B55 pendant la transition G2-M. Cette voie inclut la kinase Greatwall (GW) et ses substrats Arpp19 et ENSA. À l'entrée mitotique GW est activé et phosphoryle Arpp19 et ENSA les convertissant en inhibiteurs puissants de PP2A-B55. Étonnamment, aucun autre substrat de GW n'a été identifié jusqu'ici. Cependant, plusieurs éléments suggèrent fortement de nouveaux rôles de GW indépendamment de Arpp19 et de ENSA. L'objectif principal de ce travail était l'identification de nouveaux substrats de GW. À cette fin, j'ai utilisé plusieurs approches, y compris: (1) fractionnement biochimique des lysats de cellules ou des extraits d'oeufs de Xenopus combiné suivi d’une phosphorylation in vitro avec une kinase GW recombinante, (2) SILAC/phosphoproteomique des lysats de cellules exprimant différents niveau de GW, (3) Co-Immunoprecipitation, (4) BioID, et (5) une approche dirigée candidat. Les résultats de la phosphorylation in vitro ont révélé la présence de deux bandes de phosphorylation intéressantes qui sont actuellement analysées. Les deux approches SILAC/phosphoprotéinique et interactome ont révélé l'enrichissement des protéines impliquées dans la régulation post-transcriptionnelle de l'expression génique et des processus liés à l'ARN, une fonction physiologique déjà décrite pour cette voie chez la levure. Enfin, nous avons directement étudié la phosphorylation présumée par GW de trois candidats connus pour être impliqués dans le contrôle du cycle cellulaire. Bien que phosphorylées in vitro par GW, nous n’avons pu identifier le site de phosphorylation que dans l'une de ces trois protéines. Cette protéine, qui correspond à un inhibiteur de phosphatase, semble contrôler la sortie mitotique par la modulation de la déphosphorylation protéique. Un mutant non phosphorylable de cet inhibiteur induit une sortie mitotique perturbée avec une déphosphorylation ralentie des substrats mitotiques et une altération de la dégradation de la cycline B. J’ai pu attribuer ce défaut à une association perturbée de l'inhibiteur avec la phosphatase et, par conséquent, à un timing aberrant de l'inhibition de la phosphatase. Enfin, j'ai identifié le site de phosphorylation par GW comme le facteur clé contrôlant cette association. En résumé, j'ai identifié dans cette étude un nouveau substrat de GW contrôlant l'activité de la phosphatase essentielle pour une division mitotique correcteMitotic division is an essential phase of the cell cycle that ensures the correct repartition of the genetic content. Mitosis involves profound cellular reorganization that is mostly induced by massive protein phosphorylation. This phosphorylation is achieved thanks to the fine-tuning of the balance between kinases and phosphatases. At mitotic entry, protein phosphorylation is induced by the activation of the master kinase Cdk1-cyclin B and the inhibition of the phosphatase PP2A B55. Previous results from our and other laboratories recently discovered a new pathway essential to modulate PP2A-B55 during G2-M transition. This pathway includes the kinase Greatwall (GW) and its substrates Arpp19 and Ensa. At mitotic entry GW is activated and promotes the phosphorylation of Arpp19/Ensa converting them into potent inhibitors of PP2A B55. Surprisingly, no other substrates of GW have been identified so far. However, several pieces of data strongly suggest new roles of GW independently of Arpp19 and Ensa. The main aim of this work was the identification of new substrates of GW. To this end, I used several approaches including: (1) Biochemical fractionation of cell lysates or Xenopus egg extracts combined with in vitro phosphorylation with recombinant GW kinase, (2) SILAC/phosphoproteomics from cell lysates expressing different GW amounts, (3) Co-Immunoprecipitation, (4) BioID and (5) a candidate directed approach. Results from in vitro phosphorylation revealed the presence of two interesting phosphorylated bands that are currently being analysed. Both SILAC/phosphoproteomic and interactome approaches yielded the enrichment of proteins involved post-transcriptional regulation of gene expression and RNA related processes, a physiological function already described for this pathway in yeast. Finally, we directly investigated the putative phosphorylation by GW of three candidates known to be involved in the control of cell cycle. Although phosphorylated in vitro by GW, we could only identify the phosphorylation site in one of these three proteins. This protein, corresponding to a phosphatase inhibitor, appears to control mitotic exit through the modulation of mitotic protein dephosphorylation. A non-phosporylable mutant of this inhibitor promotes a perturbed mitotic exit with delayed dephosphorylation of mitotic substrates and impaired cyclin B degradation. I could attribute this defect to a perturbed association of the inhibitor with the phosphatase and consequently to an aberrant timing of phosphatase inhibition. Finally, I identified the GW phosphorylation site as a key factor controlling this association. In summary, I identified in this study a new substrate of GW controlling phosphatase activity essential for correct mitotic division
34
Eggert, Erik [Verfasser], Ana [Gutachter] Pombo, Bernard [Gutachter] Haendler, and Ingo [Gutachter] Morano. "Development of a cellular mechanistic assay for the SET and MYND domain containing methyltransferase SMYD2, identification and validation of a novel substrate, and functional characterization of its inhibition / Erik Eggert ; Gutachter: Ana Pombo, Bernard Haendler, Ingo Morano." Berlin : Humboldt-Universität zu Berlin, 2017. http://d-nb.info/1189330334/34.
35
Jagdeo, Julienne. "Identification of novel picornavirus proteinase substrates using terminal amine isotopic labeling of substrates." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/61277.
Abstract:
Viruses have exploited strategies of proteolysis for the purposes of processing viral proteins and manipulating cellular processes to direct synthesis of new virions and subvert host antiviral responses. Many viruses encode proteases within their genome, of which many have been well studied among the family of positive-sense single-stranded RNA picornaviruses. A subset of host proteins have already been identified as targets of picornaviral proteinases; however, the full repertoire of targets is not known. In this thesis, a novel proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) was used to conduct a global analysis of protease-generated N-terminal peptides by mass spectrometry and identify novel substrates of the 3C (3Cpro) and 2A (2Apro) proteinases from poliovirus and coxsackievirus type B3 (CVB3). TAILS was performed on HeLa cell extracts subjected to purified poliovirus 3Cpro or CVB3 2Apro, and on mouse HL-1 cardiomyocyte extracts subjected to purified CVB3 3Cpro. A list of high confidence candidate substrates for all three proteinases was generated, which included a peptide corresponding to the known poliovirus 3Cpro substrate polypyrimide tract binding protein at a known cleavage site, thus validating this approach. Furthermore, three identical peptides in both the poliovirus and CVB3 3Cpro list of high confidence substrates were identified, suggesting that cleavage of these substrates may contribute to general strategy of picornaviral infection. A total of seven high confidence substrates were validated as novel targets of 3Cpro in vitro and during virus infection. Moreover, mutations in the TAILS-identified cleavage sites for these candidates blocked cleavage in vitro and during infection. Depletion of these proteins by siRNAs modulated virus infection, suggesting that cleavage of these substrates either promotes or inhibits virus infection. In summary, an in vitro TAILS assay can be utilized to identify novel substrates of viral proteinases that are cleave during infection. Moreover, TAILS can identify common substrates of viral proteinases between different viral species, revealing general strategies of infection utilized by related viruses. Finally, the identification of novel host substrates provides new insights the viral-host interactions mediated by viral proteinases that are required for successful infection.Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
36
Birkin, Ria H. "The identification of novel protein kinase B substrates." Thesis, University of Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435741.
37
Ehrhardt, Michael. "Identification of novel MARK3 substrates and upstream regulators." Thesis, Bangor University, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.516120.
38
Probst, Brandon Linn. "Identification of substrates and pathways regulated by PAS kinase." Access to abstract only; dissertation is embargoed until after 12/20/2006, 2005. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=137.
39
Kinstrie, Ross Stuart. "Identification of Drosophila DYRK family substrates and interacting proteins." Thesis, University of Glasgow, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433084.
40
Berwick, Daniel. "The identification of novel substrates of protein kinase B." Thesis, University of Bristol, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274824.
41
Sylvestre-Gonon, Elodie. "Caractérisation biochimique et structurale de quelques glutathion transférases de la classe Tau d'arabette (Arabidopsis thaliana) et de peuplier (Populus trichocarpa)." Electronic Thesis or Diss., Université de Lorraine, 2020. http://www.theses.fr/2020LORR0253.
Abstract:
Les glutathion transférases (GSTs) constituent une famille multigénique d’enzymes ubiquitaires impliquées notamment dans la détoxication des xénobiotiques et le métabolisme secondaire. Les GSTs canoniques sont constituées d’un domaine N-terminal de type thiorédoxine et d’un domaine C-terminal formé d’hélices α. Chez les plantes terrestres, les GSTs peuvent être regroupées en 14 classes et selon le résidu conservé au sein de leur motif catalytique en GSTs à cystéine (Cys-GSTs) ou à sérine (Ser-GSTs). Les Ser-GSTs présentent des activités de réduction des peroxydes et/ou de conjugaison de glutathion (GSH) alors que les Cys-GSTs portent des activités de déglutathionylation et déshydroascorbate réductase. Certaines d’entre elles présentent également des propriétés non-catalytiques de type ligandine à des fins de transport ou de stockage de molécules diverses. Les GSTs Tau (GSTUs) correspondent à la classe regroupant le plus d’isoformes chez les plantes et leur sont spécifiques. Les GSTUs sont souvent surexprimées lors de stress biotiques et abiotiques et participent notamment à la détoxication des herbicides. Toutefois, le rôle physiologique des GSTUs reste encore lacunaire in planta. En combinant des approches phylogénétiques, biochimiques et structurales, ces travaux ont conduit à la caractérisation de neuf GSTUs d’Arabidopsis thaliana (AtGSTUs) et de six GSTUs de Populus trichocarpa (PtGSTUs). L’analyse phylogénétique des Ser-GSTs d’organismes photosynthétiques a révélé que l’expansion des GSTUs est apparue de façon concomitante à l’apparition du réseau vasculaire chez les plantes bien que quelques mousses et bryophytes possèdent des GSTUs. Au sein d’un organisme, les GSTUs peuvent être classées en groupes distincts en fonction de leur motif catalytique. Les essais enzymatiques réalisés ont montré que quasiment toutes les GSTUs d’intérêt portent des activités de conjugaison du GSH et de réduction des peroxydes envers différents substrats modèles (CDNB, dérivés d’isothiocyanates, hydroperoxydes). Les structures tridimensionnelles de deux GSTUs ont été résolues et ces dernières présentent le repliement classique des GSTs canoniques avec des différences notables entre elles. Les analyses biochimiques et structurales réalisées sur les protéines AtGSTUs et PtGSTUs d’intérêt ont montré que certaines d’entre elles lient des porphyrines bactériennes et d’autres des composés polyphénoliques. Parmi les complexes enzyme-ligand identifiés, la structure d’un complexe baicaléine-GSTU a été résolue. L’utilisation d’échantillons enrichis en métabolites extraits de plantes représente la prochaine étape sur le chemin de l’analyse fonctionnelle des GSTUsGlutathione transferases (GSTs) constitute a ubiquitous multigene superfamily of enzymes involved in xenobiotic detoxification and secondary metabolism. Canonical GSTs consist of an N-terminal thioredoxin domain and a α-helical C-terminal domain. In terrestrial plants, GSTs can be grouped in 14 classes but also according to the conserved residue found in their catalytic site either cysteine (Cys-GSTs) or serine (Ser-GSTs) GSTs. Ser-GSTs exhibit reduction of peroxides and/or glutathione (GSH) conjugation activities while Cys-GSTs rather exhibit deglutathionylation and dehydroascorbate reductase activities. Some of them also appear to have non-catalytic ligandin properties for the transport or storage of various molecules. The plant-specific Tau GST (GSTU) class is usually the most expanded one. The GSTUs are often over-expressed during biotic and abiotic stresses contributing notably to herbicide detoxification. However, the physiological role of most GSTUs is still poorly documented in planta. By combining phylogenetic, biochemical and structural approaches, this work led to the characterisation of nine GSTUs from Arabidopsis thaliana (AtGSTUs) and six GSTUs from Populus trichocarpa (PtGSTUs). Phylogenetic analysis of the Ser-GSTs present in photosynthetic organisms revealed that the expansion of GSTUs occurred concomitantly with the appearance of vasculature in plants, although some mosses and bryophytes possess GSTUs. Within an organism, GSTUs can be classified into distinct groups according to their catalytic motif. Enzymatic tests using recombinant proteins showed that almost all studied GSTUs exhibit GSH conjugation and peroxide reduction activities against different model substrates (CDNB, isothiocyanate derivatives, hydroperoxides). The three-dimensional structures of two GSTUs have been resolved and these adopt the classical canonical GST fold with some notable difference between them. The biochemical and structural analyses of these AtGSTUs and PtGSTUs further showed that some of them bind bacterial porphyrins while others bind polyphenolic compounds. Among the enzyme-ligand complexes identified, the structure of a bacalein-GSTU has been solved. The use of metabolites enriched samples extracted from A. thaliana and P. trichocarpa is the next step to decipher the role of GSTUs in planta
42
Shuaib, Ahmad. "Optimisation thermique de lasers à cavité vertical à base d’InP et reportés sur substrat silicium, pour des applications télécom." Rennes, INSA, 2011. http://www.theses.fr/2011ISAR0014.
Abstract:
En tant que sources de lumière cohérente bas-coût, les VCSELS sont incontournables dans beaucoup de domaines dont celui des telecoms optiques. Cependant, des améliorations des propriétés des VCSELS sont indispensables notamment dans le domaine de la « fibre-vers-l’abonné » utilisant la technologie InP à 1,5 µm. En effet, ce domaine ne se développera qu’en remplaçant les lasers à émission par la tranche par des lasers VCSELs bas-coûts. Ceci sera possible à condition d’optimiser l’évacuation de la chaleur générée par la zone active et limitant la puissance de ces VCSELs. Ce travail de thèse est dédié à l’optimisation de l’évacuation thermique de VCSELs émettant à 1,5 µm pompés optiquement et fonctionnant en continu à température ambiante. En particulier, nous avons utilisé des miroirs de Bragg diélectriques (a-SiH/a-SiNx) reportés sur substrats de Si. De plus, nous montrons qu’une optimisation de l’épaisseur d’InP intra-cavité permet d’améliorer l’évacuation de la chaleur dans nos VCSELs. Finalement, nous proposons un nouveau design de VCSEL composé d’un miroir de fond de cavité enterré dans des couches métalliques. Les optimisations présentées dans le cadre du VCSEL pompé optiquement ont pour but d’être utilisés ultérieurement pour le VCSEL pompé électriquementFor the last years, Vertical cavity surface emitting lasers (VCSELs) have become of large commercial importance since they are the dominant low cost, low power coherent sources in many applications such as short distance data communication. However, despite their great success, there remain opportunities for improvement, in particular in the fiber-to-the-home (FTTH) segment which needs 1550 nm long-wavelength VCSELs, based on the InP technology. Indeed, the FTTH infrastructure would develop only if the optical network becomes less costly, that is only if the laser component becomes more cheaper by replacing the edge-emitting lasers by VCSEL lasers. In particular, to be cost-effective, these devices must avoid complicating factors like cooling systems. Indeed, the high temperature generated in the active region is one of the limiting factors in achieving high power operation. This thesis work is focused on investigating the effects of thermal management on improving properties of optically-pumped VCSELs in order to achieve higher thermal performance devices. In particular, improved thermal performance has been obtained by using (a-SiH/a-SiNx)-based efficient dielectric Bragg mirror bonded on silicon substrate. Moreover, we found that a broad heat spreading in the intra-cavity InP layer is essential for obtaining low thermal resistance, we report on an optimisation of the intra-cavity InP thickness. We finally introduce a new design for the VCSEL device by using a bottom Bragg mirror patterned and embedded inside a metallic layer. Such device has been studied and implemented. Continuous wave (CW) single mode laser operation is demonstrated at room temperature (RT) with a photopumping experiment. The optimisations on the optically pumped VCSELs shown in this thesis work can be used in electrically pumped VCSEL
43
Yap, Jessica. "Identification of Plasmodium falciparum protein kinase substrates and interacting proteins." Honors in the Major Thesis, University of Central Florida, 2012. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/644.
Abstract:
Characterization of PfPKA and PfPK5 substrates, as well as the proteins they interact with, will help us to develop innovative therapies targeting binding sites.; Malaria is a devastating disease that results in almost one million deaths annually. Most of the victims are children under the age of five in Sub-Saharan Africa. Malaria parasite strains throughout developing countries are continually building resistance to available drugs. Current therapies such as mefloquine, chloroquine, as well as artemisinin are becoming less effective, and this underscores the urgency for therapeutics directed against novel drug targets. In order to identify new drug targets, the molecular biology of the malaria parasite Plasmodium needs to be elucidated. Plasmodium exhibits a unique cell cycle in which it undergoes multiple rounds of DNA synthesis and mitosis without cytokinesis. Thus, cell cycle regulatory proteins are likely to be promising pathogen-specific drug targets. It is expected that fluctuating activity of key proteins, such as protein kinases, play an essential role in regulating the noncanonical life cycle of Plasmodium. Consequently, malarial kinases are a prime target for therapy. One way to better understand the role of malarial kinases in Plasmodium cell cycle regulation is to identify putative protein kinase substrates and interacting proteins. Two malarial kinases that have been implicated in regulating malaria parasite cell cycle stages were investigated in this study: P. falciparum CDK-like Protein Kinase 5 (PfPK5) and cAMP-Dependent Protein Kinase A (PfPKA). A transgenic P. falciparum line was created for the expression of epitope-tagged PfPK5 for pull-down analysis. Phospho-substrate antibodies were used to identify physiological substrates of both PfPK5 and PfPKA. Immunoblotting with these antibodies identified several potential substrates. Identities of the PfPKA physiological substrates were determined from the global P. falciparum phosphoproteome dataset that has recently been generated in our laboratory.B.S.
Bachelors
Burnett School of Biomedical Sciences
Molecular and Microbiology
44
Floen, Miranda J. "Thioredoxin-1| Identification of redox substrates and response to hyperoxia." Thesis, University of South Dakota, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10132866.
Abstract:
Bronchopulmonary dysplasia (BPD) is a serious respiratory complication for the preterm newborn characterized clinically by prolonged respiratory distress and histologically by alveolar simplification and decreased pulmonary vasculature. The development of BPD is well linked to oxidative stress suffered by the newborn as a result of a preterm fetal-neonatal transition, supplemental oxygen, infection, increased inflammation, and mechanical ventilation. Damage suffered by oxidative stress may be through direct mechanisms or through alteration of redox¬sensitive pathways involved in cell death, cell survival, differentiation, and proliferation. Redox¬sensitive modifications regulating protein function and redox-sensitive pathways have mainly been ascribed to oxidative modification of cysteine thiols. As their modification is critical for protein function, maintenance of the thiol redox status is crucial. Thioredoxin-1 (Trx1) functions in maintenance of thiol redox homeostasis, and its redox activity is intimately linked to antioxidant, cytoprotection, proliferation responses, and cytoprotection. While Trx1 targets of redox regulation have been identified, we hypothesize that additional protein may be redox regulated and that Trx1 target profiles may change during oxidative stress. Therefore a novel immunoprecipitation approach, identified as the substrate trap approach, was developed to identify Trx1 targets. The following demonstrates the use of the substrate trap approach for identification of Trx1 redox targets and further application of the approach to identify alterations in target profiles in response to oxidative stress. Use of nuclear targeted substrate trap was successfully employed to enrich from nuclear Trx1 targets. As a final component the characterization of the Trx1 system in mouse from late embryonic development through the first week of life animals were exposed to room air or hyperoxia (model of BPD). Characterization indicates impairment of the Trx1 system in response to hyperoxic injury. As Trx1 is known to regulate proliferation, cell death, survival, differentiation pathways, impairment of the Trx1 system during early neonatal development may potentiate hyperoxic injury and alterations in lung development. Better understanding of Trx1 interactions occur through the substrate trap in a physiological model of BPD will help elucidate redox-signaling pathways involved in BPD pathogenesis.
45
Loflin, Paul T. (Paul Tracey). "Identification of Endogenous Substrates for ADP-Ribosylation in Rat Liver." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc277847/.
Abstract:
Bacterial toxins have been shown to modify animal cell proteins in vivo with ADPR. Animal cells also contain endogenous enzymes that can modify proteins. Indirect evidence for the existence in vivo of rat liver proteins modified by ADPR on arginine residues has been reported previously. Presented here is direct evidence for the existence of ADP-ribosylarginine in rat liver proteins. Proteins were subjected to exhaustive protease digestion and ADP-ribosyl amino acids were isolated by boronate chromatography.
46
Agban, Amégninou. "Synthèse de substrats chromogéniques et fluorogéniques pour l'identification des bactéries." Université Joseph Fourier (Grenoble), 1989. http://www.theses.fr/1989GRE19003.
47
Shao, Wei 1970. "Identification of caspase-1 and caspase-3 substrates and study on caspase-1 substrates in glycolytic pathway." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=100248.
Abstract:
Apoptosis is executed by caspase-mediated cleavage of various proteins. Elucidating the consequence of substrate cleavage provides us with insight into cell death and other biological processes. In this study, we applied the diagonal gel approach, a proteomic strategy, to identify substrates of the inflammatory caspase, caspase-1 and the cell death executioner caspase, caspase-3. Our results showed significant overlap between the substrates cleaved by both caspase-1 and -3. Such substrates are implicated in common cellular functions, including maintenance of the cytoskeleton, folding of proteins, translation, glycolysis, bioenergetics, signaling and trafficking. An important finding is that many glycolysis enzymes were targeted specifically by caspase-1. Processing of these glycolysis enzymes by caspase-1 was confirmed by cleaving in vitro transcribed and translated substrates with recombinant caspase-1. We have focused our further analysis on certain glycolysis enzymes. We have characterized the caspase-1 cleavage site in GAPDH. Point mutation of the Aspartic acid at position 189 to Alanine (D189A) in GAPDH blocked its cleavage by caspase-1. In vivo, in a mice model of septic shock, characterized by hyperactivation of caspase-1, we observed depletion of the full-length forms of these glycolysis enzymes in the diaphragm muscle. Further studies in caspase-1 deficient mice will confirm whether this depletion, in caspase-1 proficient mice, was due to caspase-1 processing of the glycolysis enzymes. This provides a direct link between caspase-1 activation and inhibition of glycolysis, which might have important implications on loss of muscle contractility in septic shock.
48
Grimaldi, Giovanna. "Identification and roles of cell substrates of intracellular mono-ADP-ribosylation." Thesis, Open University, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.551612.
Abstract:
Mono-ADP-ribosylation is a reversible post-translational modification that is catalysed by either bacterial pathogenic toxins or endogenous cellular ADP- ribosyltransferases (ARTs), and that can modulate the functions of target proteins. This latter aspect has only recently emerged as an important mechanism in mammalian cells for the control of a vast array of cellular processes. Some of the members of the poly-ADP-ribosyl-polymerase (PARP) family are among the mammalian enzymes that can catalyse mono-ADP-ribosylation. Here, based on biochemical data, I provide evidence that the P ARP family member P ARP 12 is a mono-ART (mART) that can modify acidic amino acid residues. Using an affinity purification method based on a protein module that recognises ADP-ribosylated proteins (the macro domain), together with liquid chromatography coupled to tandem mass spectrometry, I have identified 25 putative PARP12 substrates that are involved in different cellular processes. The identification of this range of substrates suggests that P ARP 12 is involved in multiple cellular functions, ranging from regulation of small-GTPases to the control of newly synthesized RNA. To determine its biological role, I analysed its intracellular localisation. Data reported here show that P ARPl2 is a mART that is localised tothe Golgi complex, where it can use the NAD+ pool present on this organelle to modify Golgi-localised proteins. In line with these findings, among the PARP12 substrates, I identified Rab1A, a small GTPase that controls both . transport from the endoplasmic reticulum to the Golgi- complex, and the structure of the Golgi itself My data suggest a role for PARP12 in maintenance of Golgi structure, potentially through ADP-ribosylation of RablA. A further aspect I have analysed is the ADP-ribosylation of the dual function protein C-terrninal binding protein 11 BFA- dependent ADP-ribosylated substrate (CtBP1IBARS) as a mechanism of action of the toxin brefeldin A (BFA). BFA-induced ADP-ribosylation of CtBP1IBARS occurs through a novel mechanism that comprises two steps: (i) synthesis of a BF A-ADP- ribose conjugate (BAC); and (ii) covalent binding of the BAC into the NAD+ -binding pocket of CtBPIIBARS. Here, I demonstrate that this reaction requires the involvement of ADP-ribosyl-cyc1ases, such as CD38. Importantly, this modification abolishes CtBPIIBARS fissioning activity, while it increases CtBPIIBARS co- repressor activity, in a promoter-dependent context. As with sumoylation and phosphorylation, ADP-ribosylation thus represents a new mechanism for the regulation of the functions ofCtBPlIBARS.
49
Kondacs, Laszlo. "Novel substrates for the improved detection and identification of pathogenic bacteria." Thesis, University of Sunderland, 2018. http://sure.sunderland.ac.uk/10222/.
Abstract:
Many diseases are caused by pathogenic bacteria. A key example of this is sepsis, which is mostly caused by staphylococci and Gram-negative bacteria. In addition, the highly resistant ESKAPE pathogens are responsible for the majority of hospital acquired infections. In order to treat bacterial infections effectively, and to avoid promoting bacterial resistance against antibacterial drugs, the correct agents must be used, for which in turn the detection and identification of pathogenic strains is essential. This research aims to develop selective chromogenic culture media, by introducing new antibacterial agents for the improved selectivity and new chromogenic substrates for selective visualisation of certain bacterial strains. The intention of the major part of this work was to inhibit the growth of commensal bacteria in clinical samples, as they mask the growth of the infection-causing bacteria. New and known compounds were prepared for 3 evaluation as alanine racemase inhibitors. The compounds were tested on a range of clinical pathogenic and non-pathogenic bacterial strains. The molecules developed were based on the amino acid alanine and utilised bioisosteres and other replacements for the carboxylic acid moiety. Key compounds targeted included alanylmethanesulfonamide 27-L, 1-aminoethyl5-oxo-1,2,4-oxadiazole 33-L and 1-aminoethyltetrazole 32a-L. Each compound was tested initially as the alanyl-X dipeptide form. While most of the alanine bioisosteres were known structures, their novel peptide derivatives required synthetic development using both solution and solid phase techniques. The solid phase synthesis of several C-terminal 1aminoethyltetrazole peptides was successfully established by using 2-chlorotrityl chloride resin. The investigation of the antimicrobial activity of the synthesised compounds identified several clinically applicable selective inhibitors. These compounds were shown to provide differentiation between Salmonella and Escherichia coli, or enterococci and streptococci. This work also gave a useful comparison between the different alanine bioisosteres, and showed the importance of di- and oligopeptide permease systems in order to reach sufficient bacterial activity. The microbiological activity of 1- aminoethyltetrazole peptide derivatives was studied in more detail, due to their potential in clinical applications for the diagnosis of food poisoning. In other work, also directed towards the rapid and selective detection and identification of pathogenic bacteria in a clinical environment, new chromogenic substrates were prepared. Each of these compounds contained a chromogen with a phenoxazin-3-one scaffold linked to an amino acid residue. The purpose of the amino acid is to act as a unit recognised and cleaved by specific hydrolytic bacterial enzymes. Upon liberation, electronic differences between the conjugated and free forms of the chromogen resulted in the development of distinct colour changes, which provide the basis of 4 bacterial detection and identification. Synthetic methods have been developed for the efficient and economical production of this series of substrates. After preparation, these compounds were tested against a panel of clinically relevant bacteria. The aim of these substrates was to present an alternative substrate for (N-β-alanyl)-7-amino-1-pentylphenoxazine-3-one 86a, which is applied commercially in chromID® Pseudomonas aeruginosa chromogenic medium designed for the clinical detection of P.aeruginosa. The new substrates are designed to fully explore the chemical space of phenoxazinonebased chromogenic substrates, and to decrease the colour, as substrate 86a causes significant background colour in culture media. The future application of these substrates in chromogenic media resides in their potential to advance the identification of specific pathogenic bacteria and to thus facilitate the treatment of bacterial infections.
50
Lee, Caroline H. "IDENTIFICATION OF NOVEL SUBSTRATES OF LRRK2, A PARKINSON’S DISEASE ASSOCIATED KINASE." Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1390561222.