Dissertations / Theses on the topic 'Binding component'
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1
Troup, Timothy J. "A component system architecture to enable user-directed component binding at run-time." Thesis, University of Glasgow, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414045.
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Watson, Keith. "Characterisation of the dextrin 2-sulphate cell surface binding component." Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265875.
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Whyte, J. "Studies on the alpha-bungarotoxin binding component in human brain." Thesis, University of Bath, 1985. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.352843.
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Maximum levels of alpha-bungarotoxin binding to human foetal brain membranes remained constant between gestational ages of 10-24 weeks at 30-50 gmol/mg protein (1.1-1.5 p mol/g wet weight in whole brain). Subcellular fractionation of whole brain homogenate showed an enrichment of alpha-bungarotoxin binding in the crude mitochondrial fraction and the subsequent nerve ending sub-fraction. The high proportion of occluded LDH in the nerve ending fraction was indicative of the presence of synaptosomes. Analysis of the fraction at the electron microscope level revealed the existence of synaptosome-like profiles containing mitochondria and very few synaptic vesicles. The specific binding of alpha-bungarotoxin to membranes and crude detergent extracts was shown to be saturable with KD (app) of 3.5 x 10-9 and 2.4 x 10-9M respectively. Scatchard and Hill analysis revealed the presence of a single class of sites exhibiting no cooperativity. Association rate constants, determined by measuring the binding of alpha-bungarotoxin with time to membranes and crude detergent extracts were 2.3 x 105M-1 sec-1 and 2.6 x 105M-1 sec-1 respectively. Dissociation of alpha-bungarotoxin from membranes and crude detergent extract was rapid with a T1/2 in the order of 12 min. and off rate constants of 9.2 x 10-4 sec-1 and 9.8 x 10-4 sec -1 respectively. The intrinsic dissociation constant (KD) was calculated from the on and off rate constants to be 4.0 x 10-9M and 3.3 x 10-9M for membranes and crude detergent extracts respectively. Competition studies with several cholinergic ligands indicated that the alpha-bungarotoxin binding sites displayed a predominantly nicotinic pharmacology. Unlabelled a-BgTx, nicotine and d-tubocurarine were found to be the most effective inhibitors of [125I]-alpha-bungarotoxin binding. A histological study employing autoradiographic techniques showed that [125I] -alpha-bungarotoxin binding was concentrated primarily in the dorsal horn of the spinal cord of a 14 weekold foetus. Clusters of silver grains above the background density were also observed in the ventral horn. Silver grain densities above background were abolished by pre-treatment of the sections with unlabelled alpha-bungarotoxin. A sample of myasthenic plasma and the subsequently prepared immunoglobulin fraction containing antibodies directed against the nAChR from human skeletal muscle were able to precipitate a low but measurable amount of the alpha-bungarotoxin binding sites in human foetal brain. Samples of serum in which an anti-(muscle nAChR) titre was absent failed to precipitate the brain component.
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Marafie, Sulaiman. "TRIM7, a novel binding protein of the mTORC2 component Sin1." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/trim7-a-novel-binding-protein-of-the-mtorc2-component-sin1(c344b542-0706-4ec0-be06-ce6683cee52e).html.
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TRIM7 is a member of the TRIM (tripartite motif-containing) protein superfamily. This family has been implicated in many disorders such as genetic diseases, neurological diseases, and cancers. Little is known about the function of TRIM7 except that it interacts with glycogenin and may regulate glycogen biosynthesis. Recently, a yeast two-hybrid protein-protein interaction screen revealed the binding of TRIM7 to Sin1, a protein found in a complex with the mammalian target of rapamycin (mTOR) protein kinase. mTOR can form two complexes, mTORC1 and mTORC2, which are important for cell growth, differentiation, and survival. Sin1 is a core component of mTORC2 and is critical for mTORC2 stability and activity. It was confirmed by co-immunoprecipitation that TRIM7 associates with Sin1 and mTOR in cultured mammalian cells. Furthermore, it was demonstrated that TRIM7 is a phosphoprotein, although it was not directly targeted by mTOR in vitro. Similar to some other TRIM family proteins, it was demonstrated that TRIM7 has a ubiquitin E3 ligase function allowing it to autoubiquitinate both in vitro and in cells. The autoubiquitination of TRIM7 was dependent on its RING domain. Further characterization of TRIM7 indicated that it can both homo-oligomerise as well as hetero-oligomerise with other members of its sub-class of TRIM proteins and that it co-localises with them into discrete cytoplasmic loci. To determine the cellular function of TRIM7, a stable cell line expressing an shRNA directed against TRIM7 was generated. Successful knock down of TRIM7 was achieved and this led to an increase in the protein levels of components of the mTORC2 complex, including Sin1. This coincided with an increase in cell proliferation. In conclusion, this research identifies a novel role for TRIM7 as a ubiquitin ligase involved in regulating cell proliferation and provides a potential link between TRIM7 and the mTOR pathway, a major transducer of proliferative and cell survival signals.
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Ray, Nandita. "Characterisation of an α-bungarotoxin binding component of chick optic lobe." Thesis, Imperial College London, 1990. http://hdl.handle.net/10044/1/46521.
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Christner, Robert B. "Studies on the binding specifications of human serum amyloid P- component /." The Ohio State University, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487847761305544.
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Vaughan, P. J. "Studies on a component of the herpes simplex virus DNA polymerase." Thesis, University of Leeds, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379648.
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Rodrigues, Daniel Joseph. "Structure-function relationships in the NADP (H) binding component of proton-translocating transhydrogenase from Rhodospirillum rubrum." Thesis, Oxford Brookes University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.289256.
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Grancell, Adam Scott 1969. "Biochemical characterization of CBF3, an essential DNA-binding component of the yeast kinetochore." Thesis, Massachusetts Institute of Technology, 1998. http://hdl.handle.net/1721.1/49652.
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Vinger, Gift. "THE STATUS OF THE PROJECTION PRINCIPLE IN GOVERNMENT-BINDING THEORY." Journal for New Generation Sciences, Vol 6, Issue 2: Central University of Technology, Free State, Bloemfontein, 2008. http://hdl.handle.net/11462/509.
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Published ArticleThe role of the Projection Principle within Chomsky's Government-Binding (GB) Theory is to preserve the subcategorisation properties of lexical items at all levels of syntactic representation, viz. D-structure, S-structure, and Lexical Form. Arguments have been made that the Projection Principle is a new concept that is simply an extension of theTransformational Component (XFM) and Emonds' Structure-Preserving Constraint (SPC), and that it does not deserve the high status it has been accorded in GB theory. This paper provides evidence, based on sentences involving movement operations, that the Projection Principle is innovative and that it convincingly addresses what theXFMandSPChave failed to address.
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Peake, Sarah Jayne. "Structure and function of the NADP(H)-binding component (dIII) of human heart transhydrogenase." Thesis, University of Birmingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367626.
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Huxley, Lucinda. "Kinetics and specificity of nicotinamide nucleotide binding to the dIII component of transhydrogenase from Rhodospirillum Rubrum." Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/1441/.
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Transhydrogenase is an enzyme located in the cytoplasmic membrane of bacteria or the inner membrane of animal mitochondria. Using the energy of the proton electrochemical gradient (Δp), transhydrogenase translocates protons across the membrane whilst undergoing its redox reaction, in which hydride ion equivalents are transferred from NADH to NADP+ producing NAD+ and NADPH. Transhydrogenase comprises three components; dI binds NA(H), dIII binds NADP(H) and dII spans the membrane. Transhydrogenase is thought to function by way of a binding-change mechanism, which involves “open” and “occluded” conformations of the enzyme. In the open conformation, nucleotides can readily bind and dissociate from the enzyme but the hydride transfer reaction is blocked. In the occluded conformation, hydride transfer is permitted but the binding and release of nucleotides is blocked. Hydride transfer and proton translocation are coupled. The coupling is not well understood due to the lack of structural information about the membrane-spanning dII component. However, it is believed to involve conformational changes of the enzyme, particularly the dII and dIII components, resulting in the switch between the open and occluded conformations. Enzyme assays and tryptophan fluorescence experiments using apo-dIII in complex with dI revealed two features: Firstly, the binding of NADP(H) to dIII is very slow and is probably limited by the conversion from the occluded to the open conformation. Since the switch between the occluded and open conformations is thought to be central in the coupling of hydride transfer and proton translocation, the results presented here give an insight into the binding-change mechanism of transhydrogenase. Secondly, NAD(H) is able to slowly bind into the NADP(H)-binding site of dIII (the “wrong” site). This brought into question the specificity of the dIII component of transhydrogenase for NADP(H). The significance and likelihood of NAD(H) binding to dIII in the intact enzyme in the living cell are discussed.
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Harris, Claire Louise. "Analysis of covalent binding reactions between human complement component C3 and other proteins of the alternative pathway." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309802.
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Martin, Daniel Dalton. "Purification of Anthrax Toxin Protective Antigen Component and Characterization of its Binding Interaction with Bovine Kidney Cells." DigitalCommons@USU, 1986. https://digitalcommons.usu.edu/etd/4641.
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Protective antigen component of B. anthracis toxin was produced and purified to the >99% level. Toxin was purified from culture supernatant utilizing concentration and liquid chromatography techniques. Purity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The purified protective antigen retained biological and antigenic activity as evidenced respectively by lethality in Fischer 344 rats when injected in combination with lethal factor, and by positive results on the Ouchterlony double diffussion assay.
Radioiodinated protective antigen was used both in the in vivo and the in vitro experiments. In vivo distribution of labelled protective antigen was determined in Fischer 344 rats. Assay of organ tissues for labelled protective antigen aided in the decision to use Maden-Darby bovine kidney cells for the cell cultures in the protective antigen binding studies.
Protective antigen binding studies, all performed at 37°C, evaluated criteria for receptor existence. Labelled protective antigen was found to bind specifically and reversibly to Maden-Darby bovine kidney cells. Receptors proved to be saturable. Scatchard analysis showed a relatively high dissociation constant (KD= 17 X 10-9M) compared to other toxins in similar studies. This indicated moderately low affinity for protective antigen.
The receptor was also partially characterized. It was shown that cholera toxin subunit B blocked the binding of labelled protective antigen to Maden-Darby bovine kidney cells and that the protective antigen receptor was insensitive to trypsin treatment. Both of these observations suggest a ganglioside as the receptor for protective antigen.
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Pereira, M. F. V. T., J. M. Benson, M. Williams, and H. Chikwanda. "Component and die design principles and process parameters for the metal injection moulding of a Ti alloy." Journal for New Generation Sciences, Vol 8, Issue 2: Central University of Technology, Free State, Bloemfontein, 2010. http://hdl.handle.net/11462/562.
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Published ArticleMetal injection moulding (MIM) offers advantages for mass production of components over conventional production methods for parts with complex shapes and large production runs. The MIM process includes mixing a fine metallic powder with a polymeric binder to produce a homogeneous feedstock. This enables the production of metallic components in a similar manner to plastic injection moulding. After undergoing a process of binder removal the components undergo a conventional sintering cycle. As significant shrinkage occurs (as much as 30%) this must be considered when designing the die cavity. This paper describes the design and manufacture of a die to produce tensile specimens. Extensive injection moulding trials to produce acceptable tensile components were undertaken. The complexities and possible implications of the design of a mould on the process are discussed. The outcomes of this research will be used by the CSIR for further development and application of the MIM technology for manufacture of high value components, such as dental implants.
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Zamora, Garcia Rafael E. "Characterisation of the Doxorubicin pump of Streptomyces peucetius : the DrrA component of the DrrAB ATP-binding cassette transporter." Thesis, University of Glasgow, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.438971.
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McHugh, Anna. "T cell responses to the E3 binding protein component of the pyruvate dehydrogenase complex in primary biliary cirrhosis." Thesis, University of Newcastle Upon Tyne, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424090.
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Smith, Adam Campbell. "Functional deficiency in the 67-kD elastin binding protein is a crucial component of the pathomechanism of Costello syndrome." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0020/MQ54081.pdf.
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Hopkins, Adam P. "Molecular and biochemical characterisation of SiaP as a sialic acid binding protein component of a TRAP transporter of sialic acid." Thesis, University of York, 2010. http://etheses.whiterose.ac.uk/1030/.
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Sialic acid utilisation plays an important role in the growth and persistence of the obligate human mucosal pathogen Haemophilus influenzae, which causes respiratory tract infections, septicaemia and meningitis. Like many other bacteria, H. influenzae can use host-derived sialic acids as carbon, nitrogen and energy sources, but also as a terminal modification on the LPS to better evade the human immune system. H. influenzae takes up exogenous sialic acid via a tripartite ATP-independent periplasmic (TRAP) transporter, SiaPQM. This possesses an extracytoplasmic substrate binding protein (SBP), SiaP, which binds the substrate in the periplasm and delivers it to the specific membrane permease, SiaQM. SiaP contains two globular domains, which close around the substrate upon binding. Here, the mechanism of sialic acid binding by SiaP is investigated using site-directed mutagenesis of residues in the ligand binding site and analogues of sialic acid. These, and several mutations on the surface of SiaP, were investigated for their effect on transport by SiaPQM in vitro, using SiaQM reconstituted into proteoliposomes, and in vivo, using expression of siaPQM in an E. coli strain lacking its native sialic acid transporter, NanT. It is demonstrated that stabilisation of the carboxylate group of sialic acid by the totally conserved Arginine-147 is important for high-affinity ligand binding, but is not essential for transport. Mutation of Aparagine-150 to Aspartate abolishes the function of the transporter without affecting ligand binding, suggesting the existence of a critical interaction between the components of the transporter. The catabolism of the sialic acid analogues was also examined in E. coli expressing different sialic acid transporters. This indicates that a wide variety of sialic acid analogues are potential carbon sources in many pathogenic bacteria.
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Gardner, Stewart G. "Studies of PhoU in Escherichia coli: Metal Binding, Dimerization,Protein/Protein Interactions, and a Signaling Complex Model." BYU ScholarsArchive, 2014. https://scholarsarchive.byu.edu/etd/5685.
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Phosphate is an essential nutrient for all forms of life. Escherichia coli has a PhoR/PhoB two component regulatory system that controls the expression of various genes whose products allow the cell to thrive in low phosphate environments. The signaling mechanism of the PhoR/PhoB system has been studied and the phosphorylation cascade that controls gene expression is well understood. What is still unknown is how PhoR senses the phosphate level of the environment. The PstS, PstC, PstA, PstB, and PhoU proteins play a role in this signal sensing. This work confirms the hypothesis that the PstSCAB complex senses the environmental phosphate and that phosphate signal is passed through PhoU to PhoR. Further, this work characterizes residues important for interaction on PhoU and PhoR and identifies a structural model for interaction. This model points to a potential mechanism for PhoU mediated signaling to PhoR. We tested this model with direct coupling analysis and obtained further confirmation. Further use of these techniques may elucidate more of the interactions necessary for proper phosphate signaling.
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Moore, Jocelyn. "Post-transcriptional control of Drosophila pole plasm component, germ cell-less." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115700.
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Mechanisms of post-transcriptional control are critical to deploy RNAs and proteins asymmetrically to a discrete region of cytoplasm at the posterior of the Drosophila oocyte and embryo, called the pole plasm and thus allow differentiation of the germline. Research presented in this thesis investigates the post-transcriptional control of Drosophila pole plasm component germ cell-less (gcl ). Maternal gcl activity is required for germ cell specification and gcl RNA and protein accumulate asymmetrically in the pole plasm. gcl RNA, but not Gcl protein, is also detected in somatic regions of the embryo, and ectopic expression of Gcl in the soma causes repression of somatic patterning genes suggesting that gcl RNA is subject to translational control. I find that Gcl is expressed during oogenesis, where its expression is regulated by translational repressor Bruno (Bru). Increased levels of Gcl are observed in the oocyte when Bru is reduced (i.e., in an arrest heterozygote) and Bru overexpression reduces the amount of Gcl. Consistent with this, reduction of the maternal dosage of Bru leads to ectopic Gcl expression in the embryo, which, in turn, causes repression of anterior huckebein RNA expression. Bruno binds directly to the gcl3'UTR in vitro, but surprisingly, this binding is largely independent of a Bruno Response Element (BRE) in the gcl 3'UTR and depends upon a novel site. Furthermore, the gcl BRE-like region is not required to repress Gcl expression during oogenesis or embryogenesis. I concluded that Bru regulates gcl translation in a BRE-independent manner. In addition, I established the role of the gcl 3'UTR in gcl RNA localization and translation using transgenes that replace the endogenous 3'UTR with the alpha-tubulin 3'UTR or place it in tandem to the bicoid 3'UTR. I find that accumulation of gcl RNA in the embryonic pole plasm requires the gcl 3'UTR. Moreover, Gel is restricted to the pole plasm by translational repression mediated by the gcl 3'UTR and a limiting pool of trans-acting translational repressors. The phenotypic consequences of loss of this translational control are relatively mild, suggesting that gcl translation does not require stringent repression in the soma.
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Jayasinghe, Wedhigangoda Arachchilage Pradeep Aruna. "Synthesis of charged receptors with a bis phenolic ether scaffold, and studies of their binding to Phosphatidylglycerol, a bacterial membrane component." Diss., Wichita State University, 2013. http://hdl.handle.net/10057/10611.
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An ongoing project of our research group is to develop synthetic receptors for the head group of Phosphatidylglycerol, a bacterial membrane component. Previous studies with bis phenolic oxygen linked scaffoldings with neutral binding sites showed relatively weak binding to the Phosphatidylglycerol (PG) anion. This study reports a fourteen step synthesis of a receptor molecule with a bis-phenolic oxygen ether linked scaffold, leading to an expansion of the binding pocket. The receptor is multifunctional with ammonia binding units for the phosphate anion portion of PG and two bis hydroxyl groups to bind to the glycerol hydroxyls of PG head group. The receptor's initial characterization by means of 1H NMR binding studies with Phosphatidylglycerol anion has also been reported. It also describes the synthesis of a control receptor molecule, and its binding stoichiometry with Phosphatidylglycerol and phosphate anions.Thesis (Ph.D.)--Wichita State University, Fairmount College of Liberal Arts and Sciences, Dept. of Chemistry
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Pan, Qun. "Two clusters of acidic amino acids at the NH¦2-terminus of complement component C4 Ã'-chain are important for C2 binding." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0028/MQ50359.pdf.
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Guidi, Cynthia J. "The Role of the SWI/SNF Component INI1 in Mammalian Development and Tumorigenesis: a Dissertation." eScholarship@UMMS, 2003. https://escholarship.umassmed.edu/gsbs_diss/69.
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In vivo DNA is compacted tightly, via its association with histones and non-histone proteins, into higher-order chromatin structure. In this state, the DNA is refractory to the cellular factors that require access to DNA. The repressive nature of chromatin is alleviated in part by the action enzymes that modify chromatin structure. There are two major groups of chromatin modifying enzymes: those that post-translationally modify histones by the addition of small chemical moieties and those that utilize the energy derived from ATP hydrolysis to physically disrupt chromatin structure. The SWI/SNF enzyme belongs to this latter group.
The SWI/SNF complex was identified originally in yeast. Several of its subunits are required for the expression of a subset of inducible genes. The ATPase activity is provided by the SWI2/SNF2 protein. In mammals, there are two biochemically separable SWI/SNF complexes that contain either BRG1 or BRM, both homologs of yeast SWI2/SNF2. The yeast and mammalian SWI/SNF complexes are able to disrupt the Dnase I digestion pattern of in vitro assembled mononucleosomes and arrays, as well as facilitate the accessibility of restriction nucleases and transcription factors. The mechanism by which SWI/SNF functions has yet to be elucidated.
SNF5 is a component of the yeast SWI/SNF complex. It is required for sucrose fermentation and mating type switching. The mammalian homolog of Snf5 is SNF5/INI1. SNF5/INI1 was identified simultaneously by two groups as a protein that shares homology with Snf5 and via a yeast two hybrid assay as a protein that interacts with HIV integrase (INtegrase Interactor). INI1 is a component of all mammalian SWI/SNF complexes purified to date.
In humans, mutations and/or deletions in INI1 are associated with a variety of cancers, including malignant rhabdoid tumors, choroid plexus carcinomas, medullablastomas, primitive neuralectodermal tumors, and some cases of leukemia. Furthermore, constitutional mutations within INI1in individuals presenting with these tumors support the role of INI1 as a tumor suppressor.
In this thesis, we show that Ini1 also functions as a tumor suppressor in mice. Approximately 20% of mice heterozygous for Ini1 present with tumors. Most of these tumors are undifferentiated or poorly differentiated sarcomas with variable rhabdoid features. All tumors examined to date show loss of heterozygosity at the Ini1 locus. We also show that Ini1 is essential for embryonic development. Mice homozygous-null for Ini1die between days 4 and 5.5 post-fertilization due to an inability to adhere to their substratum, form trophectoderm, and expand their inner cell mass.
We further characterize the function of Ini1 in tumor suppression by generating mice heterozygous for both Ini1 and either Rb or p53. While heterozygosity at the Ini1 locus appears to have no effect on the rate of tumorigenesis in Rb-heterozygous mice, many of the tumors arising in compound heterozygous mice present with an altered morphology. This finding suggests that Ini1 may contribute to tumor progression due to loss of Rb. In contrast, mice compound heterozygous for Ini1 and p53 show a marked reduction in the rate of tumorigenesis compared to p53-heterozygous mice. Furthermore, the tumor spectrum is altered in these compound heterozygous mice. These findings suggest that Ini1 may function normally to repress p53 activity.
Lastly, we show that expression of the Ini1 tumor suppressor itself is regulated tightly. Tissues and cells heterozygous for Ini1 express roughly equivalent levels of Ini1 protein and mRNA as their wild-type counterparts. We further show that this compensation is mediated by an increase in the rate of transcription from the wild-type Ini1 allele. Moreover, when exogenous Ini1 is introduced into Ini1-heterozygous cells, expression from the Ini1 promoter is reduced. These data indicate that a compensatory mechanism exists to ensure that the steady-state levels of Ini1 are constant.
In summary, research detailed in this thesis has contributed to our understanding of the regulation of Ini1 as well as the role this protein plays in mammalian development and tumor suppression.
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Dian, Cyril. "Adaptive Responses by Transcriptional Regulators to small molecules in Prokaryotes : Structural studies of two bacterial one-component signal transduction systems DntR and HpNikR." Doctoral thesis, Stockholm : Department of Biochemistry and Biophysics, Stockholm University, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-7052.
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Dayibas, Orcun. "Feature Oriented Domain Specific Language For Dependency Injection In Dynamic Software Product Lines." Master's thesis, METU, 2009. http://etd.lib.metu.edu.tr/upload/3/12611071/index.pdf.
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Base commonality of the Software Product Line (SPL) Engineering processes is to analyze commonality and variability of the product family though, SPLE defines many various processes in different abstraction levels. In this thesis, a new approach to configure (according to requirements) components as building blocks of the architecture is proposed. The main objective of this approach is to support domain design and application design processes in SPL context. Configuring the products is made into a semi-automatic operation by defining a Domain Specific Language (DSL) which is built on top of domain and feature-component binding model notions. In order to accomplish this goal, dependencies of the components are extracted from the software by using the dependency injection method and these
dependencies are made definable in CASE tools which are developed in this work.
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Згоранець, Олег Васильович. "Робочі властивості стрижневих сумішей з комплексними зв’язувальними компонентами на основі металофосфатів." Master's thesis, КПІ ім. Ігоря Сікорського, 2020. https://ela.kpi.ua/handle/123456789/42987.
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Магістерська дисертація: 98 с., 49 рис., 30 табл., 32 посилань.
Об’єкти дослідження: стрижні, виготовлені із металофосфатних сумішей експериментального складу; виливки із залізовуглецевих сплавів (технологічні проби на пригар та на вибиваємість).
Предмет дослідження: пригар на поверхні виливків; шорсткість литих поверхонь; робота вибивання та гігроскопічність стрижневих сумішей.
Мета роботи: визначення комплексу робочих властивостей: стійкості до пригару, вибиваємості, а також технологічної властивості – гігроскопічності стрижневих сумішей, які містять зв’язувальні компоненти металофосфатного типу.
Методи дослідження: виготовлення спеціального модельного оснащення; виготовлення ливарних форм і стрижнів, плавлення і заливання металу; контроль шорсткості литих поверхонь за еталонами, визначення роботи вибивання та гігроскопічності традиційними методами.
Результати дослідження: установлено закономірності утворення пригару залежно від складу стрижневих сумішей та товщини стінок виливка, визначено роботу вибивання та гігроскопічність усіх сумішей. Показано, що стрижні із ряду сумішей можуть бути застосовані без протипригарних покриттів.
Значущість роботи: в роботі вперше підтверджено з технологічної точки зору реальну можливість використання стрижневих сумішей з фосфатами кремнію, алюмінію, натрію, калію, марганцю для виготовлення виливків із залізовуглецевих сплавів.
Галузі застосування: дрібні ливарні стрижні (масою до декількох кг), які зміцнюються у печі або в нагрітому оснащенні (150…300 ºС, залежно від складу суміші), для виготовлення виливків із залізовуглецевих сплавів.
Економічна ефективність – умовний економічний ефект 44 777 грн.
Прогнозовані припущення: поступове впровадження стрижневих сумішей, починаючи з цехів із індивідуальним і дрібносерійним виробництвом. Зниження собівартості литва, покращення якості литих поверхонь, санітарно-гігієнічних умов праці, екологічної ситуації.Master's dissertation: 98 pp., 49 figs., 30 tables, 32 references. Objects of research: rods made of metal-phosphate mixtures of experimental composition; castings from ferrocarbon alloys (technological tests for burn and knockout). Subject of research: burns on the surface of castings; roughness of cast surfaces; beating work and hygroscopicity of rod mixtures. Purpose: to determine the set of working properties: resistance to scorching, knockout, as well as technological properties - hygroscopicity of the core mixtures, which contain binders of metal-phosphate type. Research methods: production of special model equipment; production of molds and rods, melting and pouring of metal; control of roughness of cast surfaces according to standards, definition of work of knocking out and hygroscopicity by traditional methods. The results of the research: the regularities of burn formation depending on the composition of the core mixtures and the thickness of the casting walls are established, the work of knocking out and hygroscopicity of all mixtures are determined. It is shown that rods from a number of mixtures can be used without non-stick coatings. Significance of the work: the work for the first time confirmed from a technological point of view the real possibility of using core mixtures with phosphates of silicon, aluminum, sodium, potassium, manganese for the manufacture of castings from ferrocarbon alloys. Areas of application: small casting rods (weighing up to several kg), which are hardened in a furnace or in heated equipment (150… 300 ºC, depending on the composition of the mixture), for the manufacture of castings from iron-carbon alloys. Economic efficiency - conditional economic effect UAH 44,777. Projected assumptions: gradual introduction of core mixtures, starting with shops with individual and small-scale production. Reducing the cost of casting, improving the quality of cast surfaces, sanitary and hygienic working conditions, environmental situation.
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Stone, Howard C. "Isolation and characterisation of cadmium binding components of the scallop, Pecten maximus." Thesis, University of Aberdeen, 1985. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU356206.
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1. The digestive gland of the scallop Pecten maximus naturally contains a very high concentration of cadmium (100 ppm wet weight), which does not show individual or seasonal variations. 2. About 60% of the tissue cadmium is soluble (i.e. is found in the supernatant when homogenate is centrifuged for 1 hour at 100,000g) and is bound to three main types of macromolecule. Most (about 60%) of this metal is associated with a component of molecular weight 55,000, the rest being bound to a low molecular weight species and, probably non-specifically, to components of very high molecular weight (greater than 100,000 molecular weight). The latter components were not further characterised. 3 The major binding component complexes the Cd2+ via sulphydryl groups, and so has a high cysteine content, but the binding is weaker than that of cadmium to metallothionein. The component has a high content of glutamate and aspartate (or their amides) and contains aromatic amino acids. It may also have a small carbohydrate content.4. The major cadmium binding component was susceptible to degradation by endogenous proteolytic enzymes. A major digestive enzyme was identified as a chymotrypsin which could be inactivated with phenylmethanesulphonylfluoride. Addition of PMSF to the homogenate reduced the total proteolytic activity of the digestive gland cytosol by up to 75%. Attempts to further inhibit, or remove, the remaining protease activity were largely unsuccessful. Probably as a result of the action of the proteolytic enzymes on the major cadmium component efforts to isolate the latter were characterised by irreproducibility, and satisfactory purification was not achieved. 5. The low molecular weight cadmium binding component binds 10-15% of the total soluble cadmium and exhibits many of the characteristics of a metallothionein. It has an apparent molecular weight of 10,000 on gel exclusion chromatography, high cadmium and cysteine contents and a high A250/A280 ratio. It is also heat stable and contains copper and zinc as well as cadmium. It can be detected by the metallothionein assay of Eaton & Toal (1982). 6. Preparations of both the major Cd binding component and metallo-thionein-like component contained relatively large amounts of carbohydrate, but the latter was probably not associated with these proteins. Its origin is unknown.
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Nordesjö, Olle. "Searching for novel protein-protein specificities using a combined approach of sequence co-evolution and local structural equilibration." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-275040.
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Greater understanding of how we can use protein simulations and statistical characteristics of biomolecular interfaces as proxies for biological function will make manifest major advances in protein engineering. Here we show how to use calculated change in binding affinity and coevolutionary scores to predict the functional effect of mutations in the interface between a Histidine Kinase and a Response Regulator. These proteins participate in the Two-Component Regulatory system, a system for intracellular signalling found in bacteria. We find that both scores work as proxies for functional mutants and demonstrate a ~30 fold improvement in initial positive predictive value compared with choosing randomly from a sequence space of 160 000 variants in the top 20 mutants. We also demonstrate qualitative differences in the predictions of the two scores, primarily a tendency for the coevolutionary score to miss out on one class of functional mutants with enriched frequency of the amino acid threonine in one position.
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Whalley, Caroline. "Estimating binding strength and chemical phases of metals adsorbed to sediment components." Thesis, University of East Anglia, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259990.
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Campbell, Veronica Ann. "Components of neuronal calcium channels and their interaction with GTP-binding proteins." Thesis, University College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363058.
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Paris, Guillaume. "Effets dynamiques et conformationnels sur le rôle de transport des albumines sériques." Thesis, Besançon, 2014. http://www.theses.fr/2014BESA2024/document.
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L’albumine sérique humaine (HSA) est une protéine connue pour ses propriétés de transport exceptionnelles et son contenu élevé en ponts disulfure. L’étude de sa dynamique conformationnelle représente un défi important dans la compréhension de ses fonctions physiologiques. Le but de notre travail a été d’étudier cette dynamique conformationnelle et de comprendre le rôle des ponts disulfure dans le maintien de la structure native de la protéine. Notre analyse est basée sur des simulations de dynamique moléculaire couplées à des analyses par composantes principales. Outre la validation de la méthode de simulation les résultats fournissent de nouveaux éclairages sur les principaux effets de la réduction des ponts disulfure dans les albumines sériques. Les processus de dépliement/repliement protéique ont été détaillés. La prédiction de la structure réduite d’équilibre a également fait l’objet d’une attention particulière. Une étude détaillée de la dynamique conformationnelle globale de la protéine ainsi que celle des deux sites principaux de complexation a été effectuée. D’éventuels effets allostériques entre ces deux sites ont été recherchés. Les résultats théoriques obtenus ont été discutés avec les données expérimentales disponiblesHuman serum albumin (HSA) is a protein known for its exceptional transport properties and its high content of disulfide bridges. The study of the conformational dynamics represents a major challenge in the comprehension of its physiological functions. The aim of our work was to study the conformational dynamics and to understand the roleof disulfide bonds in the stability of the native protein structure. Our analysis is based on simulations of molecular dynamics coupled with principal component analysis. Beyond the validation of the simulation method, the results provide new insights on the main effects of the disulfide bonds reduction in serum albumins. Protein unfolding/refolding processes were detailed. A special attention is paid to the prediction of the reduced structure at the equilibrium. A detailed study of the global protein conformational dynamics as well as the two main binding sites were performed. Possible allosteric effects between these two sites were researched. The theoretical results have been discussed with the available experimental data
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Whittington, Christi Leigh. "Molecular Dynamics of the RNA Binding Cavity of Influenza A Non-structural Protein 1 (NS1) RNA Binding Domain." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4256.
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Molecular dynamics simulations were performed on the influenza A non-structural protein 1 (NS1) RNA binding domain (RBD), a homodimer. Fourteen simulations were performed at 298K, nine ionized with 0.1M KCl and five with no ions. Several analysis techniques were employed to study RBD residue flexibility. The focus of the study was the RNA binding cavity formed by side chains of helix 2 (chain A) and helix 2’ (chain B) and cavity intermonomeric salt bridges. Opening of the salt bridges D29–R46’ and D29’–R46 was observed in several of the trajectories. The RNA binding cavity has large flexibility, where the dimension and shape change during the dynamics. One pair of residues surrounding the cavity and necessary for RNA binding, residues R38 and R38’, have motions during the simulations which cover the top of the cavity. There is correlation between the salt bridge breaking, flexibility of R38 and R38’, and the cavity size and shape changes. Possible RBD small molecule drug targets are these two salt bridges and the pair R38 and R38’. Disrupting the events that occur around these areas could possibly inactivate RNA binding function of the domain. These results could have implications in searching for potential molecules that effectively treat influenza A.
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Andersson, David. "Multivariate design of molecular docking experiments : An investigation of protein-ligand interactions." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-35736.
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To be able to make informed descicions regarding the research of new drug molecules (ligands), it is crucial to have access to information regarding the chemical interaction between the drug and its biological target (protein). Computer-based methods have a given role in drug research today and, by using methods such as molecular docking, it is possible to investigate the way in which ligands and proteins interact. Despite the acceleration in computer power experienced in the last decades many problems persist in modelling these complicated interactions. The main objective of this thesis was to investigate and improve molecular modelling methods aimed to estimate protein-ligand binding. In order to do so, we have utilised chemometric tools, e.g. design of experiments (DoE) and principal component analysis (PCA), in the field of molecular modelling. More specifically, molecular docking was investigated as a tool for reproduction of ligand poses in protein 3D structures and for virtual screening. Adjustable parameters in two docking software were varied using DoE and parameter settings were identified which lead to improved results. In an additional study, we explored the nature of ligand-binding cavities in proteins since they are important factors in protein-ligand interactions, especially in the prediction of the function of newly found proteins. We developed a strategy, comprising a new set of descriptors and PCA, to map proteins based on their cavity physicochemical properties. Finally, we applied our developed strategies to design a set of glycopeptides which were used to study autoimmune arthritis. A combination of docking and statistical molecular design, synthesis and biological evaluation led to new binders for two different class II MHC proteins and recognition by a panel of T-cell hybridomas. New and interesting SAR conclusions could be drawn and the results will serve as a basis for selection of peptides to include in in vivo studies.
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Fajardo, Mark A. "Defining cis- and trans-acting components for Prm-1 temporal translational control during murine spermatogenesis /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/10251.
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Rodríguez, Solovey Leisa Natacha. "IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs)." Doctoral thesis, Universitat Politècnica de València, 2015. http://hdl.handle.net/10251/58862.
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[EN] ABSTRACT
Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYR/PYL/RCAR receptors (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS) for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches.
Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calciumdependent interactions of PYR/PYL/RCAR ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL/RCAR receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL/RCAR function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL/RCAR-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL/RCAR subcellular localization and positively regulates ABA signaling.[ES] RESUMEN La señalización por la hormona vegetal ácido abscísico (ABA) desempeña un papel crítico en la regulación del crecimiento de la raíz y en la arquitectura del sistema radical. La promoción de crecimiento de la raíz en condiciones de estrés hídrico mediada por ABA es clave para la supervivencia de las plantas bajo condiciones limitantes de agua. En este trabajo, hemos explorado el papel de los receptores PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) de Arabidopsis (Arabidopsis thaliana) en la ruta de señalización de ABA en raíz. Así, hemos descubierto que el receptor de ABA PYL8 juega un papel no redundante en la regulación de la percepción de ABA en raíz. Inesperadamente, dada la naturaleza multigénica y la redundancia funcional parcial observada en la familia PYR/PYL/RCAR, el mutante pyl8 fue el único mutante sencillo de pérdida de función de los receptores PYR/PYL/RCAR que mostraba una sensibilidad reducida a la inhibición del crecimiento mediada por ABA en raíz. Este efecto se debe a la falta de inhibición mediada por PYL8 de varias fosfatasas del grupo A tipo 2C (PP2Cs), ya que PYL8 es capaz de interactuar in vivo con al menos cinco PP2Cs, denominadas HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 según lo han revelado la purificación por afinidad en tándem (TAP por sus siglas en inglés) y estudios proteómicos de espectrometría de masas. La transducción de la señal del ABA localizada en la membrana plasmática celular juega un papel crucial en los pasos iniciales de la señalización de la fitohormona, pero los mecanismos moleculares que unen los componentes básicos de la señalización y la membrana plasmática no están claros. Estudiando las interacciones de los receptores del ABA PYR/PYL/RCAR con la membrana plasmática hemos encontrado que éstos pueden interaccionar transitoriamente con ella de forma dependiente de calcio gracias a una familia de proteínas con dominios C2 relacionadas con la ruta de señalización de ABA (denominadas C2-domain ABA-related (CAR) proteins). Específicamente, se encontró que PYL4 interacciona de manera independiente de ABA con CAR1 tanto en la membrana plasmática como en el núcleo de las células vegetales. La proteína CAR1 pertenece a una familia multigénica constituida por 10 miembros en Arabidopsis thaliana, desde CAR1 hasta CAR10, y que solo se encuentra en plantas. Los ensayos de complementación bi-molecular de fluorescencia y de co-immunoprecipitación confirmaron la interacción en células vegetales tanto de PYL4-CAR1 como de otras parejas de PYR/PYL-CAR. La cristalización de la proteína CAR4 reveló que, además de un dominio C2 clásico de unión a lípidos dependiente de calcio, las proteínas de la familia CAR presentan un dominio específico que probablemente es responsable de la interacción con los receptores PYR/PYL/RCAR y de su posterior reclutamiento a las vesículas de fosfolípidos. Esta interacción es relevante para la función de los receptores PYR/PYL/RCAR en la señalización del ABA, ya que diferentes mutantes triples car de pérdida de función, que tienen afectados los genes CAR1, CAR4, CAR5, y CAR9, demostraron una reducción de la sensibilidad al ABA en ensayos de establecimiento de plántula y crecimiento de la raíz. En resumen, hemos identificado nueva familia de proteínas que son capaces mediar las interacciones transitorias dependientes de Ca2+ con vesículas de fosfolípidos, lo que a su vez afecta localización de PYR/PYL/RCAR y regula positivamente la señalización de ABA.
[CAT] RESUM La senyalització per l'hormona vegetal àcid abcíssic (ABA) exerceix un paper crític en la regulació del creixement de l'arrel i també en l'arquitectura del sistema radical. La promoció del creixement de l'arrel en condicions d'estrés hídric, regulada per ABA és clau per la supervivència de les plantes sota condicions limitants d'aigua. Amb aquest treball, hem investigat el paper dels receptors PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) d'Arabidopsis (Arabidopsis thaliana) en el camí de senyalització d'ABA en arrel. Així, hem descobert que el receptor d'ABA PYL8 exerceix un paper no redundant en la regulació de la percepció d'ABA en arrel. Inesperadament, donada la naturalesa multigènica i la redundància funcional parcial que s'observa en la família PYR/PYL/RCAR, el mutant pyl8 va ser l'únic mutant senzill de pèrdua de funció dels receptors PYR/PYL/RCAR que mostrava una sensibilitat reduïda a la inhibició del creixement mitjançada per l'ABA en l'arrel. Doncs aquest efecte es deu a la falta d'inhibició regulada per PYL8 de diverses fosfatases del grup A tipus 2C (PP2Cs), ja que PYL8 té la capacitat d'interactuar in vivo almenys amb cinc PP2Cs, anomenades HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABAHYPERSENSITIVE GERMINATION3 segons ho han revelat per una banda la purificació per afinitat en tàndem (TAP són les seues sigles en anglés) i per altra banda, estudis proteòmics d'espectrometria de masses. Pel que fa a la transducció del senyal del l'ABA, la qual es localitza en la membrana plasmàtica cel¿lular, juga un paper molt important en els primers instants de la senyalització de la fitohormona, no obstant això els mecanismes moleculars que uneixen els components bàsics d'aquesta senyalització amb la membrana plasmàtica, no es troben del tot clars. Per tant, s'han estudiat les interaccions que tenen els receptors del ABA PYR/PYL/RCAR amb la membrana plasmàtica, i hem trobat que aquests tenen la capacitat d'interaccionar transitòriament amb la membrana de forma dependent al calci, gràcies a una família de proteïnes amb domini C2, les quals es troben relacionades amb la ruta de senyalització d'ABA(anomenades C2domain ABArelated (CAR) proteins).Específicament, es va trobar que PYL4 interacciona d'una manera independent al ABA amb CAR1, tant en la membrana plasmàtica, com en el nucli de les cèl¿lules vegetals. La proteïna CAR1 pertany a la família multigènica constituïda per 10 components en Arabidopsis thaliana, des de CAR1 fins CAR10, que tan sols es troba en plantes. Els assajos de complementació bimolecular de fluorescència i de co-immunoprecipitació, van confirmar la interacció en cèl¿lules vegetals, tant de PYL4CAR1 com d'altres parelles de PYR/PYL-CAR. La cristal¿lització de la proteïna CAR4 va revelar que, a més d'un domini C2 clàssic de unió a lípids dependent del calci, les proteïnes de la família CAR presenten un domini PYR/PYL/RCAR, i del seu posterior reclutament a les vesícules fosfolipídiques. Doncs, aquesta interacció és rellevant en la funció dels receptors PYR/PYL/RCAR, ja que participa en la senyalització del l'ABA. Aquesta interacció es clau per a la funció dels receptors, ja que diferents mutants triples car de pèrdua de funció, els quals posseïxen afectats els gens CAR1, CAR4, CAR5 i CAR9, van mostrar una reducció de la sensibilitat a l'ABA en assajos d'establiment de plàntula i creixement de l'arrel. En conclusió, hem identificat una nova família de proteïnes amb la capacitat d'organitzar les interaccions transitòries dependents del calci amb vesícules de fosfolípids, fet que al seu torn afecta la localització de PYR/PYL/RCAR i regula positivament la senyalització d'ABA.
Rodríguez Solovey, LN. (2015). IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58862
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Flory, Mark Randall. "Isolation and characterization of calmodulin-binding centrosome components related to Saccharomyces cerevisiae Spc110p from the fission yeast Schizosaccharomyces pombe and humans /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/5061.
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Dunaiski, Vera. "Effects of IGF-1 or LR3IGF-1 infusion on components of the GH/IGF-1 axis in pigs /." Title page, contents and abstract only, 1997. http://web4.library.adelaide.edu.au/theses/09PH/09phd897.pdf.
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Kaczmarczyk, Aneta. "Proteins of the Inter-α-inhibitor Family : Biosynthesis, Plasma Clearance and Interaction with Extracellular Matrix Components." Doctoral thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3495.
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Bikunin, a chondroitin sulfate-containing protein of 25 kDa, has protease inhibitory activity and occurs in the plasma in free and complexed form. In inter-α-inhibitor (IαI) and pre-a-inhibitor (PαI) it is covalently linked through its chondroitin sulfate (CS) chain to two or one other polypeptide of about 80 kDa – heavy chains 1 and 2 (H1, H2) and heavy chain 3 (H3) – respectively. Bikunin and the heavy chains are synthesized as precursors, which are proteolytically cleaved and assembled into IαI and PαI in the secretory pathway. The C-terminal extension (CTX) of the heavy chains seems to mediate its own cleavage and theassembly of the complexes. The heavy chains of the IαI family become transferred to hyaluronan during ovulation and inflammation.
In this thesis, the biosynthesis of PαI, the plasma clearance of bikunin and the binding of IαI to collagen were studied. We found that in H3, a short segment on the N-terminal side of the CTX cleavage site is required for cleavage. Furthermore, the H3 could become linked to free CS chains primed by a xyloside, showing that the bikunin protein core is not needed for coupling. We also identified His649 as a residue essential for coupling, but not for cleavage.
Bikunin labelled with a residualizing agent, 125I-tyramine cellobiose, was injected into mice to identify tissues involved in its uptake. Half of the radioactivity was recovered in the kidneys, 10% in the liver, and the rest distributed in other tissues. We determined the half-life of bikunin in rat plasma using two independent methods: injection of 125I-bikunin, or hepatectomy followed by assessing the rate of disappearance of endogenous bikunin. Both methods yielded half-time values of 5-7 minutes. Removal of the CS chain did not affect the clearance rate of bikunin.
IαI and its heavy chains were found to bind to collagen with dissociation constants greater than 2 μM and 0.4-0.6 μM, respectively and this binding was independent of divalent metal ions. We suggest that the interaction of IαI with collagen may play a modulatory role in cell migration or in remodelling of the extracellular matrix.
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Molento, Carla Forte Maiolino. "Effects of insulin and the interaction between insulin and recombinant bovine somatotropin on the production of milk and its components and on IGF-I plasma levels." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38085.
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The effects of insulin on milk production were tested employing two different approaches. Firstly, 12 Holstein cows were used to determine the effects of feeding calcium propionate (Ca prop) on dry matter intake (DMI) and production traits. The experimental design was a switchback with 2 treatments (Ca prop at 0 or 300 g/d). The DMI was lower when animals received Ca prop. Ca prop did not affect the yield of milk and its components; however, Ca prop increased protein content. The (acetate+butyrate)/propionate ratio in rumen fluid 2 h after feeding was lower when cows received Ca prop. Plasma insulin concentration was not different between treatments and the putative effect of propionate as an insulin secretagogue was probably related to the maintenance of insulin levels when DMI was lower. In conclusion, Ca prop is a potential feed ingredient to increase protein content in milk. The second approach consisted of intravenous infusion of insulin. A trial was designed to test the effects of insulin, recombinant bovine somatotropin (rbST) and their interaction in lactating dairy cows. Eight Holstein cows were used in a Latin Square design with 4 treatments: (1) intravenous infusion of saline, (2) infusion of saline and administration of 40 mg of rbST per day, (3) intravenous infusion of 12 mg of insulin per day coupled with glucose infusion and (4) rbST administration combined with insulin and glucose infusion. The theory that rbST causes a peripheral resistance to insulin was confirmed. Insulin infusion increased percent protein, percent casein and decreased milk urea content regardless of rbST administration. For milk yield, protein yield, casein yield, lactose percent and lactose yield, there was an interaction between insulin and rbST administration. Similarly, there was an interaction between insulin and rbST on plasma IGF-I levels. Fat yield was higher, with a higher content of long chain fatty acids, during rbST administration, regardless of insulin infusion. I
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Tejedor, Vaquero Juan Ramón 1984. "Systematic functional analyses of spliceosomal components reveal novel mechanisme of alternative splicing regulation." Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/385718.
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Alternative splicing is an essential regulatory layer of gene expression that expands the coding potential of the genome in multicellular organisms. The spliceosome -the sophisticated machinery involved in intron removal- allows versatile regulation of gene expression programs. The splicing process relies on the dynamic interplay between hundreds of components of the spliceosome, and the steps at which the complex process of the splicing reaction can be regulated remain largely unknown.
The main objective of this thesis has been to develop high- throughput approaches to systematically identify novel regulators of alternative splicing, as well as to study the mechanisms by which they modulate splice site choice. We have identified a variety of regulators of Fas/CD95 alternative splicing within and outside of the splicing machinery and provide novel insights into connections between iron homeostasis and alternative splicing regulation. Using computational networks, we carried out a systematic functional analysis of the spliceosome components and their regulatory potential. Our results reveal the extensive regulatory plasticity of core spliceosome components throughout its assembly process. They also identified links between alternative splicing and iron homeostasis, providing a mechanism by which iron modulates alternative splicing through regulation of the RNA binding properties of a Zinc knuckle domain in the SR regulatory protein SRSF7.
The results of this thesis highlight the value of high throughput technologies and network analyses to study complex molecular mechanisms, and unveils novel functional connections between the splicing machinery and other cellular processes.El procesamiento alternativo del pre-ARNm constituye uno de los pilares esenciales en la regulación de la expresión génica y expande la capacidad codificadora del genoma en organismos multicelulares. El Espliceosoma – la maquinaria encargada de la eliminación alternativa de los intrones- permite una regulación multifacética de los programas genéticos en el interior de la célula. El proceso de corte y empalme se sustenta en la interacción dinámica de cientos de componentes del Espliceosoma, y los distintos niveles de regulación de la compleja reacción de splicing permanecen aun sin descubrir. El objetivo principal de esta tesis se ha centrado en el desarrollo de tecnologías sistematicas de alto cribado para identificar reguladores potenciales del procesamiento alternativo del pre- ARNm, así como los mecanismos implicados en su regulación. Hemos identificado una gran variedad de reguladores del procesamiento alternativo de Fas/CD95, tanto componentes esenciales del espliceosoma como factores implicados en otros procesos biologicos, y hemos observado una conexión inédita entre la regulación del splicing alternativo y el proceso de homeostasis modulado por hierro. Mediante el uso de redes computacionales, hemos llevado a cabo un análisis sistemático y funcional de los componentes del Espliceosoma y hemos identificado el potencial regulador de los mismos en la reacción de corte y empalme. Nuestros resultados reflejan una inmensa plasticidad de los factores esenciales del Espliceosoma a lo largo de toda a reacción de ensamblaje. Además, hemos conseguido identificar el mecanismo potencial por el cual la homeostasis del hierro ejerce su función en splicing alternativo a través de la modulación de la actividad de unión a RNA -mediada por un dominio de unión a cinc- en la proteína reguladora de splicing SRSF7. Los resultados de esta tesis enfatizan la relevancia de las tecnologías emergentes de alto cribado y el análisis de redes computacionales en el estudio de complejos mecanismos moleculares, y desvelan nuevas conexiones funcionales entre la maquinaria de splicing y otros procesos celulares.
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Bertacchini, Eleonora. "Molecular study of stress system in the European sea bass (Dicentrarchus labrax): cloning of different components and effects of essential oil of Lippia alba during stress situation." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2018.
Find full textAbstract:
European sea bass (Dicentrarchus labrax) is one of the most important species in the Mediterranean aquaculture. In this context, fish are subjected to practices that activate the stress system and can adversely affect their health and welfare. To minimise the effects of stress on fish, investigators have begun to examine the use of natural products with anaesthetic properties that are more effective, safer and less expensive than the currently available synthetic drugs. The aim of the present study was to assess the effectiveness of the essential oil of Lippia alba (EOLA) to mitigate the stress response in D. labrax individuals disturbed by persecution. For this purpose, sea basses were subjected to 3 and 6 hours of stress procedure and sedated with two different concentrations of the EOLA, 25 and 50 μL L-1. Partial cDNA sequences of crhbp and trh genes were cloned and their mRNA expression, together with that of crh, pomc, star, nr3c1 and nr3c2, all stress-related genes of the hypothalamus-pituitary-interrenal axis, were analysed in different tissues. Results elucidated that the most conspicuous variations in gene expression were found out for crh at 6 hours, which pattern was inversely proportional to cortisol levels. This may indicate the existence of a negative feedback mechanism exerted by cortisol. Expression of mineralocorticoid and glucocorticoid receptors (nr3c1 and nr3c2) showed a trend that diverged in relation to the belonging tissue, but in hypothalamus it mirrored the variation of crh, highlighting again a role of cortisol in regulating gene expression during the stress response. Furthermore, an appreciable increment in trh mRNA occurred in every treatment after 3 hours, suggesting that this hormone can be involved in the stress response. The best concentration of EOLA in order to reduce stress is represented by 25 μL L-1. On the contrary, at 50 μL L-1, the oil can be itself a stressor and after 6 hours it loses its effectiveness and it degrades.
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Labusch, Corinna [Verfasser]. "Patatin-related phospholipases (pPLA) and other cytosolic components in auxin binding protein1 (ABP1)-mediated auxin signaling are revealed by using early auxin-induced gene expression as a biotest in Arabidopsis thaliana / Corinna Labusch." Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover (TIB), 2013. http://d-nb.info/1041238843/34.
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Lindström, Anton. "A multivariate approach to characterization of drug-like molecules, proteins and the interactions between them." Doctoral thesis, Umeå universitet, Kemi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1924.
Full textAbstract:
En sjukdom kan många gånger härledas till en kaskadereaktion mellan proteiner, co-faktorer och substrat. Denna kaskadreaktion blir många gånger målet för att behandla sjukdomen med läkemedel. För att designa nya läkemedelsmoleyler används vanligen datorbaserade verktyg. Denna design av läkemedelsmolekyler drar stor nytta av att målproteinet är känt och då framförallt dess tredimensionella (3D) struktur. Är 3D-strukturen känd kan man utföra så kallad struktur- och datorbaserad molekyldesign, 3D-geometrin (f.f.a. för inbindningsplatsen) blir en vägledning för designen av en ny molekyl. Många faktorer avgör interaktionen mellan en molekyl och bindningsplatsen, till exempel fysikalisk-kemiska egenskaper hos molekylen och bindningsplatsen, flexibiliteten i molekylen och målproteinet, och det omgivande lösningsmedlet. För att strukturbaserad molekyldesign ska fungera väl måste två viktiga steg utföras: i) 3D anpassning av molekyler till bindningsplatsen i ett målprotein (s.k. dockning) och ii) prediktion av molekylers affinitet för bindningsplatsen. Huvudsyftena med arbetet i denna avhandling var som följer: i) skapa modeler för att prediktera affiniteten mellan en molekyl och bindningsplatsen i ett målprotein; ii) förfina molekyl-protein-geometrin som skapas vid 3D-anpassning mellan en molekyl och bindningsplatsen i ett målprotein (s.k. dockning); iii) karaktärisera proteiner och framför allt deras sekundärstruktur; iv) bedöma effekten av olika matematiska beskrivningar av lösningsmedlet för förfining av 3D molekyl-protein-geometrin skapad vid dockning och prediktion av molekylers affinitet för proteiners bindningsfickor. Ett övergripande syfte var att använda kemometriska metoder för modellering och dataanalys på de ovan nämnda punkterna. För att sammanfatta så presenterar denna avhandling metoder och resultat som är användbara för strukturbaserad molekyldesign. De rapporterade resultaten visar att det är möjligt att skapa kemometriska modeler för prediktion av molekylers affinitet för bindningsplatsen i ett protein och att dessa presterade lika bra som andra vanliga metoder. Dessutom kunde kemometriska modeller skapas för att beskriva effekten av hur inställningarna för olika parametrar i dockningsprogram påverkade den 3D molekyl-protein-geometrin som dockingsprogram skapade. Vidare kunde kemometriska modeller andvändas för att öka förståelsen för deskriptorer som beskrev sekundärstrukturen i proteiner. Förfining av molekyl-protein-geometrin skapad genom dockning gav liknande och ickesignifikanta resultat oberoende av vilken matematisk modell för lösningsmedlet som användes, förutom för ett fåtal (sex av 30) fall. Däremot visade det sig att användandet av en förfinad geometri var värdefullt för prediktion av molekylers affinitet för bindningsplatsen i ett protein. Förbättringen av prediktion av affintitet var markant då en Poisson-Boltzmann beskrivning av lösningsmedlet användes; jämfört med prediktionerna gjorda med ett dockningsprogram förbättrades korrelationen mellan beräknad affintiet och uppmätt affinitet med 0,7 (R2).A disease is often associated with a cascade reaction pathway involving proteins, co-factors and substrates. Hence to treat the disease, elements of this pathway are often targeted using a therapeutic agent, a drug. Designing new drug molecules for use as therapeutic agents involves the application of methods collectively known as computer-aided molecular design, CAMD. When the three dimensional (3D) geometry of a macromolecular target (usually a protein) is known, structure-based CAMD is undertaken and structural information of the target guides the design of new molecules and their interactions with the binding sites in targeted proteins. Many factors influence the interactions between the designed molecules and the binding sites of the target proteins, such as the physico-chemical properties of the molecule and the binding site, the flexibility of the protein and the ligand, and the surrounding solvent. In order for structure-based CAMD to be successful, two important aspects must be considered that take the abovementioned factors into account. These are; i) 3D fitting of molecules to the binding site of the target protein (like fitting pieces of a jigsaw puzzle), and ii) predicting the affinity of molecules to the protein binding site. The main objectives of the work underlying this thesis were: to create models for predicting the affinity between a molecule and a protein binding site; to refine the geometry of the molecule-protein complex derived by or in 3D fitting (also known as docking); to characterize the proteins and their secondary structure; and to evaluate the effects of different generalized-Born (GB) and Poisson-Boltzmann (PB) implicit solvent models on the refinement of the molecule-protein complex geometry created in the docking and the prediction of the molecule-to-protein binding site affinity. A further objective was to apply chemometric methodologies for modeling and data analysis to all of the above. To summarize, this thesis presents methodologies and results applicable to structure-based CAMD. Results show that predictive chemometric models for molecule-to-protein binding site affinity could be created that yield comparable results to similar, commonly used methods. In addition, chemometric models could be created to model the effects of software settings on the molecule-protein complex geometry using software for molecule-to-binding site docking. Furthermore, the use of chemometric models provided a more profound understanding of protein secondary structure descriptors. Refining the geometry of molecule-protein complexes created through molecule-to-binding site docking gave similar results for all investigated implicit solvent models, but the geometry was significantly improved in only a few examined cases (six of 30). However, using the geometry-refined molecule-protein complexes was highly valuable for the prediction of molecule-to-binding site affinity. Indeed, using the PB solvent model it yielded improvements of 0.7 in correlation coefficients (R2) for binding affinity parameters of a set of Factor Xa protein drug molecules, relative to those obtained using the fitting software.
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López, Muñoz Laura. "Homology modeling and structural analysis of the antipsychotic drugs receptorome." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7228.
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Classically it was assumed that the compounds with therapeutic effect exert their action interacting with a single receptor. Nowadays it is widely recognized that the pharmacological effect of most drugs is more complex and involves a set of receptors, some associated to their positive effects and some others to the side effects and toxicity. Antipsychotic drugs are an example of effective compounds characterized by a complex pharmacological profile binding to several receptors (mainly G protein-coupled-receptors, GPCR). In this work we will present a detailed study of known antipsychotic drugs and the receptors potentially involved in their binding profile, in order to understand the molecular mechanisms of the antipsychotic pharmacologic effects.The study started with obtaining homology models for all the receptors putatively involved in the antipsychotic drugs receptorome, suitable for building consistent drug-receptor complexes. These complexes were structurally analyzed and compared using multivariate statistical methods, which in turn allowed the identification of the relationship between the pharmacological properties of the antipsychotic drugs and the structural differences in the receptor targets. The results can be exploited for the design of safer and more effective antipsychotic drugs with an optimum binding profile.
Tradicionalmente se asumía que los fármacos terapéuticamente efectivos actuaban interaccionando con un único receptor. Actualmente está ampliamente reconocido que el efecto farmacológico de la mayoría de los fármacos es más complejo y abarca a un conjunto de receptores, algunos asociados a los efectos terapéuticos y otros a los secundarios y toxicidad. Los fármacos antipsicóticos son un ejemplo de compuestos eficaces que se caracterizan por unirse a varios receptores simultáneamente (principalmente a receptores unidos a proteína G, GPCR). El trabajo de la presente tesis se ha centrado en el estudio de los mecanismos moleculares que determinan el perfil de afinidad de unión por múltiples receptores de los fármacos antipsicóticos.
En primer lugar se construyeron modelos de homología para todos los receptores potencialmente implicados en la actividad farmacológica de dichos fármacos, usando una metodología adecuada para construir complejos fármaco-receptor consistentes. La estructura de estos complejos fue analizada y se llevó a cabo una comparación mediante métodos estadísticos multivariantes, que permitió la identificación de asociaciones entre la actividad farmacológica de los fármacos antipsicóticos y diferencias estructurales de los receptores diana. Los resultados obtenidos tienen interés para ser explotados en el diseño de fármacos antipsicóticos con un perfil farmacológico óptimo, más seguros y eficaces.
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Marcon, Edyta. "RNA binding protein, MIWI, a component of the synaptonemal complex /." 2004. http://wwwlib.umi.com/cr/yorku/fullcit?pMQ99355.
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Thesis (M.Sc.)--York University, 2004. Graduate Programme in Biology.Typescript. Includes bibliographical references (leaves 48-64). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ99355
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Chen, Yen-Chi, and 陳彥錡. "Biochemical Binding Characterization of Human Progesterone Receptor Membrane Component 1." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/46377342883670419342.
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碩士國立屏東教育大學
化學生物系碩士班
101
Human progesterone receptor membrane protein (Progesterone Receptor Membrane Component 1 , PGRCM 1) the study was to investigate the protein; Recent studies have pointed out, PGRMC1 Council and heme binding, but its specific physiological role has not yet been identified in the organism. In addition, although the name PGRCM 1 literature that progesterone receptor membrane protein for the human, but in fact do not directly bind progesterone, but with an unknown protein steroid / drug binding protein (S/D-BP) for Progesterone binding message passing and metabolism; expressed concern for the results of this literature. Therefore, in order to further understand the PGRCM 1 and progesterone combined with the actual situation, this study lab colleagues previously applied transgenic technology in Escherichia coli (E.coli) in a large number of performance PGRMC1; and were added to the culture of Heme precursor ALA (5-Aminolevul Acid Hydrochloride) in the culture medium; and in developing purification after adding hemin (Hemin Chloride) reconstruction, obtained by these two methods PGRMC1 iron containing metal center and comparison. We further use of UV - visible spectroscopy (UV-Vis) confirm PGRMC1 with metal centers of iron oxidation state (Fe3 +), and the use of quartz crystal microbalance QCM (Quartz Crystal Microbala) analysis of the binding constant between progesterone; also Discussion between PGRMC1 and binding of Heme. The results of this thesis show, PGRMC1 does not directly bind with Progesterone, but PGRMC1 active center Heme reconstruction may impact combined with Progesterone situation; while PGRMC1 and Heme Kd value can be achieved between 10-8.
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Wang, Li Jie, and 王立傑. "Interactome-wide Analysis Identifies End-Binding Protein 1 as a Crucial Component For The Speck-like Particle Formation of Activated Inflammasomes." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/37074499986072960485.
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博士長庚大學
生物醫學研究所
100
Inflammasomes are cytoplasmic receptors that can recognize intracellular pathogens or danger signals and are critical for IL-1β production. Although several key components of inflammasome activation have been identified, there has not been a systematic analysis of the protein components found in the stimulated complex. In this study, we used the isobaric tags for relative and absolute quantification (iTRAQ) approach to systemically analyze the interactomes of the NLRP3, AIM2 and RIG-I inflammasomes in NPC cells treated with specific stimuli of these interactomes (H2O2, poly (dA:dT) and EBER, respectively). We identified a number of proteins that appeared to be involved in the interactomes and also could be precipitated with anti-ASC antibodies after stimulation. Among them, EB1 was an interacting component in all three interactomes. Silencing of EB1 expression by small interfering RNA inhibited the activation of the three inflammasomes, as indicated by reduced levels of IL-1β secretion. We confirmed that EB1 directly interacted with AIM2 and ASC in vitro and in vivo. Most importantly, fluorescence confocal microscopy showed that EB1 was required for formation of the speck-like particles that represent activation of the AIM2 inflammasome. In NPC tissues, IHC staining showed that EB1 expression was elevated and significantly correlated with AIM2 and ASC expression in NPC tumor cells. In sum, we profiled the interactome components of three inflammasomes and show for the first time that EB1 is crucial for the speck-like particle formation that represents activated inflammasomes.
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Li, Pai-Chi, and 李珮琦. "Theoretical Study of The “Charge Center-Linker-Hydrogen Binding Site” Three-Component Signal Transducers: The Effect of Charge-Bearing Substituents and Counter Ions." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/11521827295153390809.
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碩士國立臺灣大學
化學研究所
89
We consider a charge-bearing molecule as a three-component system consisting of a charge center, a linker, and a hydrogen bond binding site. In the three-component system, the binding center is controlled by a reaction center (charge center). Basically, we want to maximize the long-range through-bond effect and turn the three-component system into an effective signal transducer. In real experiment, one can change the formal charge of the reaction center through protonation, oxidation, reduction, or chemical transformation of a functional group. In our theoretical study, protonation was calculated for the reaction site. For the linker part, fully conjugated alkenes, alkynes and -N=N- are good candidates for maximizing through-bond effects. For a binding site, pyrrole was chosen for its ability to donate electrons through conjugation with the linker. Our aim is to investigate how different reaction centers and counter ions affect the hydrogen bonding sites, so as to provide guidelines for the design of effective signal transducers. Calculation results at the HF/6-31+G* level showed that 1) for neutral species, the longer the linker unit, the larger its binding energy (5~6 kcal/mol); for charged species, the binding energy of the protonated cationic system was roughly double that of the neutral system (9~14 kcal/mol). 2) If the linker is -(CH=CH)n-, the longer the linker unit is, the less binding energy it has. With the azo linkers, we have demonstrated that it is even possible to amplify the signals when the charge center is an iminium group (-CH=NH2+). 3) Counter ions could decrease the binding energy of the binding site because of long-range electrostatic interactions and because some electron is transferred from the counter ion to the reaction center. The transducers containing iminium are greatly influenced by the counter ions, while the charge delocalized guanidinium group (-NHC(NH2)2+), whose electrons can not conjugate with the linker, seem to have better ability to preserve signal transduction in the presence of a counter ion (40~50%). The calculation results showed that the efficiency of changing counter ions to improve signal transduction is quite small. Experimentally, one could consider reducing the effect of counter ions by using anion-binding hosts.
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Wani, Saima Masood. "Elucidation of the Role of Nse1, a RING Domain Containing Component of Smc5/6 complex, in Maintenance of Chromosome Stability in Saccharomyces cerevisiae." Thesis, 2017. http://etd.iisc.ernet.in/2005/3570.
Full textAbstract:
Structural Maintenance of Chromosomes (SMC) proteins are a highly conserved class of proteins required for the maintenance of genome stability and regulate nearly all aspects of chromosome biology. Eukaryotes, such as the budding yeast Saccharomyces cerevisiae, have six Smc proteins that form three SMC complexes in association with non-SMC proteins, i.e., the cohesin complex, the condensin complex and the Smc5/6 complex. The yeast Smc5/6 complex consists of Smc5, Smc6 and six non-Smc elements (Nse1-6) that are all essential for the survival of cells.
Nse1 is the first non-smcelement that was identified associated with the Smc5/6 complex. Nse1 has a C-terminal RING-domain, which is a characteristic feature of some E3 ubiquitin ligases. A RING domain consists of eight conserved Zn-coordinating residues arranged in a cross-brace conformation. To understand the importance of this domain, we created site directed mutations in conserved residues identified by sequence alignment of the budding yeast Nse1 RING domain with that of other species. We found a new RING domain mutant nse1-103that was temperature sensitive at 37°C and showed an increased sensitivity towards genotoxic agents such as hydroxyurea (HU), methyl methane sulfonate (MMS) and ultraviolet (UV) radiation. Thense1-103 mutant cells are slow growing and show delayed chromosomal replication at the restrictive temperature. Genetic interactions with replication factors such as RRM3, TOF1 etc. revealed thatnse1-103shows a synthetic sick growth defect in combination with rrm3∆ that is partially suppressed by deletion of TOF1. We found an enhancement in chromosome loss in nse1-103 compared to wild type cells. This was accompanied by a slight reduction in cohesion between the sister chromatids in nse1-103,suggesting a plausible mechanism for the chromosome destabilization observed in the mutant.
Since Nse1 forms part of a trimeric sub-complex with Nse3 and Nse4 in the Smc5/6 complex, we performed a yeast two hybrid assay to test the interaction of nse1-103 with Nse3 or Nse4, and found a defect in interaction of nse1-103 with Nse3 and Nse4. In addition, a defect in association of nse1-103 with Smc5 or Smc6 could be observed by performing co-immunoprecipitation from yeast cell lysates, suggesting that the integrity of the RING-domain is critical for the interaction of Nse1 with other subunits of the Smc5/6 complex. However, there was no defect in the interaction between Nse3 and Smc5 in nse1-103, indicating that the interaction of these components within the complex isindependent of Nse1.
We also identified a novel sequence motif near the RING domain of Nse1, deletion of which leads to an increased sensitivity towards genotoxic stressors and higher temperature. Biochemical characterization of this mutant also suggests a defect ininteraction with Nse3 or Nse4, and also with Smc5. The nse1 mutants also showed defects in post translational modification of Smc5 and other proteins.
Since the Smc5/6 complex also has a SUMO E3 ligase, Mms21/Nse2, we also investigated genetic interactions between the RING domain mutant,nse1-103 and the SUMO ligase RING domain defective mutant,mms21∆sl, and found an exacerbation of the drug sensitive phenotypes in thense1-103 mms21∆sl double mutant relative to either of the single mutants nse1-103 or mms21∆sl, indicating that the two proteins contribute independently to the function of Smc5/6 complex in resisting genotoxic stress.
In conclusion, the present study emphasizes the role of the RING domain of budding yeast Nse1 in resisting genotoxic stress and maintaining chromosome stability and reveals that the integrity of the RING-domain is critical for interactions of Nse1 with Nse3 and other Smc5/6 complex components. In addition, we report identification of another novel sequence motif in Nse1 that is also crucial for its interaction with other subunits of the Smc5/6 complex and for maintenance of post-translational modifications of some cellular proteins.