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Arbuckle, Janeen Lynnae. "Identification and characterization of domains in non-core RAG1". Oklahoma City : [s.n.], 2007.
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Winter, Sherry Lynn. "Identification of BRCA1 interacting proteins". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0026/MQ50432.pdf.
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Baakdah, Fadi. "Identification of PfCRT interacting proteins". Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=121534.
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The lethal form of human malaria is caused by the most prominent human protozoan parasite, Plasmodium falciparum, responsible for an estimated ~1 million deaths annually. Malaria treatment and, prevention heavily rely on antimalarial drugs. The synthetic substitute of quinine, chloroquine, was the most effective treatment for this disease. The rise of chloroquine resistant malaria in endemic areas has suppressed control efforts. Chloroquine resistance has been associated with mutations in a transmembrane protein on the digestive vacuole of the parasite, designated PfCRT. Moreover, the normal function(s) and natural substrate(s) of PfCRT remain a matter of speculation as direct evidence for the normal functions or substrates are yet to be determined. The objectives of this thesis are to develop and characterize two antisera to the N- and C termini of PfCRT as tools for the identification of PfCRT interacting proteins in P. falciparum. Although the molecular mass of PfCRT was estimated as 48.67 kDa, immunobiochemical characterizations using different antiserums raised against the N- and C-cytosolic domains of this protein throughout the published literature yielded varying molecular masses of PfCRT on SDS PAGE. In this thesis, we report the generation and biochemical characterization of two antisera raised against the N- and C-cytosolic domains of PfCRT. Our results show PfCRT-C antiserum to detect non-phosphorylated epitopes or sequences in PfCRT. Moreover, our high-resolution epitope mapping results confirm the specificity of PfCRT-C antiserum to a non-phosphorylated form of PfCRT and show that phosphorylation at two sites in PfCRT sequence, Ser411 and Thr416, prevent its binding to the full-length and phosphorylated protein. In addition, we have identified a novel truncated form of PfCRT that is both unphosphorylated, as it is recognized by PfCRT-C antiserum, and likely to represent a differentially spliced product of PfCRT, that is expressed in vivo on the parasite digestive vacuole. Furthermore, our findings confirm the molecular mass of the full-length and phosphorylated PfCRT as a 52 kDa protein band on SDS PAGE. Using PfCRT antisera together with three different methods of protein interactions, we provide the first evidence of proteins interacting with PfCRT. Identification of these proteins which ranges from ~20 kDa to 200 kDa is under active investigation.La forme létale du paludisme humain est causée par le plus important parasite protozoaire humain, Plasmodium falciparum, responsable d'environ ~1 million de mort annuellement. Le traitement et la prévention du paludisme dépend des médicaments antipaludiques. Le substitut synthétique de la quinine, la chloroquine, était le traitement le plus efficace pour cette maladie. La montée du paludisme résistant à la chloroquine dans les pays endémiques a supprimé les efforts de contrôle. La résistance à la chloroquine a été associée aux mutations dans la protéine transmembranaire présente sur la vacuole digestive du parasite, désigné PfCRT. De plus, la ou les fonctions normales et substrats naturels de la protéine PfCRT restent une affaire de spéculation étant donné qu'une évidence directe des fonctions normales et des substrats de cette protéine sont encore à déterminer. Les objectives de cette thèse sont de développer et de caractériser deux antisérums de l'extrémité N- et C-terminale de PfCRT comme outils d'identification des protéines interagissant avec la protéine PfCRT de P. falciparum. Bien que la masse moléculaire de la protéine PfCRT était estimée à 48.67 kDa, des caractérisations immunobiochimiques utilisant différents antisérums dirigés contre les domaines cytosoliques N- et C-terminal de cette protéine et publiés à travers la littérature ont abouti à des masses moléculaires variable de la protéine PfCRT sur le SDS PAGE. Dans cette thèse, nous reportons la génération et la caractérisation biochimique de deux antisérums dirigés contre les domaines cytosoliques N- et C de PfCRT. Nos résultats montrent que l'antisérum C de PfCRT détecte les épitopes non-phosphorylés ou des séquences dans la protéine PfCRT. En outre, nos résultats de la cartographie à haute résolution de l'épitope confirment la spécificité de l'antisérum C de PfCRT à la forme non-phosphorylé de PfCRT et montrent que la phosphorylation à deux sites au niveau de la séquence de la protéine, Ser411 et Thr416, qui empêche sa fixation à la protéine complète et phosphorylée. De plus, nous avons identifié une nouvelle forme tronquée de PfCRT qui est à la fois phosphorylée puisqu'elle est reconnue par l'antisérum C de PfCRT, et susceptible de représenter un produit différemment épissé de PfCRT, qui est exprimé in vivo sur la vacuole digestive du parasite. En outre, nos résultats confirment la masse moléculaire de la protéine complète et phosphorylée de PfCRT à 52 kDa sur un SDS PAGE. En utilisant les antisérums de PfCRT combiné à trois méthodes différentes d'interactions protéiques, nous fournissons la première preuve de protéines interagissant avec la protéine PfCRT. L'identification de ces protéines dont la masse varie entre ~20 kDa et 200 kDa est sous enquête active.
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Wei, Heng. "Split PH domain identification & redundancy analyses in the classification of PDZ domains /". View abstract or full-text, 2006. http://library.ust.hk/cgi/db/thesis.pl?BICH%202006%20WEI.
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Hellborg, Fredrik. "Identification, cloning and characterization of the p53 induced gene human wig-1 /". Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-190-3/.
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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.
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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
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Wadahama, Hiroyuki. "Identification and Characterization of Soybean Protein Disulfide Isomerase Family Proteins as Functional Proteins for Folding of Seed-storage Proteins". Kyoto University, 2010. http://hdl.handle.net/2433/120458.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第15414号
農博第1799号
新制||農||978(附属図書館)
学位論文||H22||N4513(農学部図書室)
27892
京都大学大学院農学研究科食品生物科学専攻
(主査)教授 河田 照雄, 教授 村田 幸作, 教授 井上 國世
学位規則第4条第1項該当
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McLoughlin, D. M. "Identification of proteins interacting with the Alzheimer's disease amyloid precursor protein". Thesis, King's College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343724.
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Mac, Partlin Mary. "Identification of proteins interacting with the human mismatch repair protein MLH1". Thesis, University of Glasgow, 2000. http://theses.gla.ac.uk/1111/.
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Loss of expression of the human DNA mismatch repair (MMR) gene, hMLH1, is seen in a number of tumour cell lines resistant to a variety of cytotoxic drugs. The aim of this study was to identify other proteins that interact with hMLH1 to attempt to further elucidate its role in MMR and the engagement of downstream damage response pathways. A yeast two-hybrid system, an in vivo system for detecting protein-protein interactions was utilised for this purpose. Fifteen known and five unknown genes were identified as encoding proteins interacting with hMLH1. These included three known hMLH1 binding proteins, hMLH3, hPMS1 and MED1. Amongst the other genes identified was the proto-oncogene c-MYC, a gene previously implicated in genetic instability and apoptosis. Using in vitro derived mutants of c-MYC, it has been shown that hMLH1 interacts with the leucine-zipper domain of c-MYC. The effect of elevated c-MYC expression on functional MMR was examined. An inducible c-MYC expression system, Rat-1 fibroblasts expressing c-MYCERTM, a fusion of c-MYC to the hormone binding domain of the oestrogen receptor was utilised. Elevated expression of c-MYC did not effect the mismatch specific binding complex activity in these cells as measured in EMSA experiments. However c-MYC overexpression utilising the Rat-1 cMYCERTM system was shown to result in a mutator phenotype in these cells. The results suggest there may be a link between the mutator phenotype, induced through overexpression of c-MYC, and loss of MMR. Overexpression of c-MYC, which is associated with many cancers, may result in the sequestration of hMLH1 preventing functional MMR. The interaction between hMLH1 and c-MYC is proposed to act in a DNA damage response pathway which is disrupted upon aberrant c-MYC expression.
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Höglund, Pär J. "Identification, Characterization and Evolution of Membrane-bound Proteins /". Uppsala : Acta Universitatis Upsaliensis Acta Universitatis Upsaliensis, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9329.
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Almeida, T. B. "Identification and optimisation of ligands to target protein-protein interactions : EB1-SxIP proteins". Thesis, University of Liverpool, 2016. http://livrepository.liverpool.ac.uk/3004877/.
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End binding protein 1 (EB1) is a key element in the complex network of protein-protein interactions at microtubule growing ends which has a fundamental role in microtubule polymerisation. EB1 regulates the microtubule dynamic behaviour, through protein recruitment, and has been associated with several disease states, such as cancer and neuronal diseases. Diverse EB1 binding partners are recognised through a conserved SxIP motif within an intrinsically disordered region enriched with basic, serine and proline residues. Crystal structure of EB1 in complex with a peptide containing the SxIP motif demonstrated that the isoleucine-proline dipeptide is bound into a well‐defined cavity of EB1 that may be suitable for small molecule targeting. The research described herein reports the use of a multidisciplinary approach for the discovery of the first small molecule scaffold to target the EB1 recruiting domain. This approach included virtual screening (structure and ligand based design) and multiparameter compound selection. Solution NMR structures of the C-terminal domain of EB1 in the free form and in complex with the small molecule are also reported. A key finding from these structures is that the hydrophobic binding pocket reported to be essential for recruiting SxIP proteins is not pre-formed but highly dynamic in solution. This brings new insights to the protein recruitment mechanism regulated by EB1 and for the identification of new small molecule inhibitors for the EB1-SxIP protein interactions. The interaction of short length peptides containing the SxIP motif with EB1 was characterised through the use of solution NMR and ITC methods. The contributions for the binding of the SxIP motif and neighbouring residues to EB1 were quantified in terms of binding energy. A structural model shows that the binding pocket of EB1 is largely extended when in complex. This research describes not only the first chemical scaffold that targets EB1, it details important structural features of the interaction of this protein with SxIP containing peptides. This structural information provides fundamental understanding of this interaction that can be exploited in the future to discover higher affinity ligands.
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Pacheco, Sophia A. "Identification of Campylobacter jejuni secreted proteins". Pullman, Wash. : Washington State University, 2010. http://www.dissertations.wsu.edu/Thesis/Spring2010/s_pacheco_021610.pdf.
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Arianmanesh, Mitra. "Identification of embryo implantation-related proteins". Thesis, University of Aberdeen, 2010. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=165845.
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Identification of embryo implantation-related proteins Mitra Arianmanesh Embryo implantation is a complex process involving an active dialogue between the endometrium and embryo. Tightly controlled communication between the hypothalamus, pituitary, corpus luteum (CL), endometrium and embryo is essential for implantation. Unravelling molecules involved in embryo implantation is essential since implantation failure is one of the main causes of female infertility. Therefore, identification of molecular events during embryo implantation may result in enhancing implantation rates in both natural and assisted reproductive cycles, improving contraceptive design and reducing the rate of multiple pregnancies following embryo transfer in IVF cycles. Thus, in this study, sheep was used as an animal model in order to study endometrial, corpus luteal and plasma proteome changes during embryo implantation and early pregnancy. Endometrium, CLs and plasma were harvested from cyclic ewes on days 12 and 16 of the oestrous cycle (n=4 ewes/group) and from pregnant ewes on days 12, 16 and 20 of pregnancy (n=4 ewes/group). Furthermore, ovine endometrium were collected from pregnant and non-pregnant horns on days 16 (n=4) and 20 (n=4) of pregnancy to compare endometrial protein profiles of the gravid horn (in the presence of the conceptus) with the non-gravid horn (in the absence of the conceptus) in response to the conceptus to elucidate local embryo-endometrial signalling. 2DE gel, LC-MS/MS, Western blot, IHC and qRT-PCR were employed to quantify implantation processes. This study has identified proteins in the CL and endometrium with involvement in biological pathways that are fundamental for embryo implantation and gestation. In addition, it was found that the implanting embryo is capable of regulating the expression of endometrial proteins to establish an ideal environment for its implantation and establishment of pregnancy. These findings provide an addition to the field and a solid base for targeted studies to improve our understanding of implantation and its regulation.
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Tattersall, Daniel. "The identification of retromer partner proteins". Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615241.
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Prigge, Justin Robert. "Identification and characterization of novel protein-protein interactions with the basal transcription factor, TATA-binding protein". Diss., Montana State University, 2006. http://etd.lib.montana.edu/etd/2006/prigge/PriggeJ0506.pdf.
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Jing, Yonghua. "Identification of the Na,K-ATP interacting proteins". University of Toledo Health Science Campus / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=mco1139260222.
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Ler, Lian Wee. "Identification and characterization of novel mammalian eIF4E-Homologous Protein (4EHP) interacting proteins". Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66760.
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Translation of an mRNA begins with the recruitment of the eIF4F complex to the 5' cap of the mRNA and is completed upon start codon recognition by the preinitiation complex. Regulation of translation initiation is a major mechanism for the control of gene expression. The focus of this thesis is human 4EHP (h4EHP), a homolog of the cap-binding translation initiation factor eIF4E. 4EHP can bind to the cap structure but cannot initiate cap-dependent translation. Drosophila 4EHP functions as a translational repressor of specific mRNAs by forming a "closed loop", in which the binding partners of 4EHP determine the mRNA specificity. This study sought to investigate the function of h4EHP by identifying its interacting proteins. In total, three novel binding proteins of h4EHP have been identified: PERQ1, PERQ2, and eIF4E-Transporter (4E-T). All these proteins utilize a similar motif for h4EHP-binding. Experiments demonstrate that h4EHP:PERQ1/2 complexes are involved in translational repression. In addition, this study shows that the protein levels of PERQ1, PERQ2 and h4EHP are co-regulated. Among them, PERQ2 undergoes CUL7-mediated degradation upon h4EHP depletion. 4E-T has been shown to be involved in translational repression, eIF4E nuclear import, P-body formation and mRNA turnover. This study demonstrates that h4EHP can be localized to the nucleus. However, its nuclear localization is not affected by 4E-T depletion. The depletion of h4EHP results in a six-fold increase in the number of P-bodies but has no effect on the rate of turnover of a reporter mRNA. In conclusion, this study identifies PERQ1 and PERQ2 as two translational repressors; their function and stability are dependent on h4EHP. The function of h4EHP:4E-T interaction, however, remains elusive.La traduction d'un ARNm commence par le recrutement du complexe eIF4F au niveau de la coiffe (ou cap) en 5' de l'ARNm et se complète lors de la reconnaissance du codon d'initiation par le complexe de pré-initiation. La régulation de l'initiation de la traduction est une étape majeure dans le contrôle de l'expression génique. Cette thèse s'intéresse à la protéine humaine 4EHP (h4EHP), un homologue du facteur d'initiation de la traduction se liant à la coiffe, eIF4E. 4EHP peut d'ailleurs s'associer à la coiffe de l'ARNm mais ne peut initier la traduction dépendante de la coiffe. L'homologue chez la drosophile, d4EHP, est un inhibiteur de la traduction de certain ARNm et agit en formant une boucle 5'-3' avec l'ARNm. Les partenaires protéiques de 4EHP définissent alors la spécificité pour l'ARNm. Cette thèse s'intéresse à la fonction de h4EHP en recherchant ses partenaires protéiques. Les trois nouveaux partenaires identifiés (PERQ1, PERQ2 et 4E-T) utilisent un même motif de liaison à h4EHP. Mes expériences démontrent non seulement, que les complexes h4EHP:PERQ1/2 sont impliqués dans la répression de la traduction, mais aussi qu'il existe une co-régulation des niveaux relatifs des protéines h4EHP, PERQ1 et PERQ2. Ce dernier est d'ailleurs dégradé par CUL7 après la déplétion de h4EHP. La protéine 4E-T est connu pour son rôle dans l'inhibition de la traduction, l'import nucléaire d'eIF4E, la formation des « P-Bodies » et le renouvellement des ARNm. Cette étude démontre que h4EHP a une localisation nucléaire et indépendante de 4E-T. La suppression de 4E-T augmente cependant de six fois le nombre de « P-Bodies », sans pour autant affecter le taux de renouvellement d'un ARNm reporteur. En conclusion, cette étude identifie PERQ1 et PERQ2 comme deux inhibiteurs de la traduction dont la fonction et la stabilité sont dépendantes de h4EHP. Cependant, le rôle de l'inter
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Ritchie, Sian. "Identification of cytoskeletal proteins as substrates for Ca'2'+ dependent protein kinase". Thesis, University of Reading, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240317.
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Alzahrani, Ashwag. "Identification of Human Proteins Interacting with the Protein IcsB of Shigella flexneri". Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/38333.
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Sleeman, Katrina. "Identification of proteins interacting with the polymerase (L) protein of rinderpest virus". Thesis, University of Warwick, 2003. http://wrap.warwick.ac.uk/79583/.
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Rinderpest virus (RPV) is a morbillivirus which causes a highly contagious disease affecting members of the order Artiodactyla. The viral L protein is the catalytic subunit of the RNA-dependent RNA polymerase, but requires the P protein for activity. In previous studies it was found that, in addition to a direct L-P interaction, both the C and V non-structural proteins bind to L. The L proteins of morbilliviruses consist of three long highly conserved domains separated by short unconserved sequences. The interaction of P, C and V with these three domains was studied. Using co-immunoprecipitation, it was shown that P interacts with the first domain, whilst C and V were each shown to interact with the central domain. Further mutational analysis using the yeast two-hybrid system (Y2HS), showed that the P binding site lies in the amino-proximal domain of L, between amino acids 1 and 233, which fits with the co-immunoprecipitation data. However, the Y2HS suggested that the binding site for C and V includes a region between amino acids 1 and 363 of L, i.e. within the first domain. These data indicate (i) that the P binding site is distinct from that ofC and V, and (ii) that the C and V binding site(s) may be complex. To search for host cell proteins with which L interacts, a library screen was performed using the Y2HS and a porcine macrophage cDNA library. Three host cell proteins were recovered from the library screen as putative L interactors. The interaction with one of these, striatin, was confirmed by co-immunoprecipitation, and co-localisation of the two proteins was observed by confocal microscopy. The L sequence with which striatin interacts was investigated. Like the C and V proteins, striatin was shown to interact with the second conserved domain of L by co-immunoprecipitation and Y2HS data indicated that a possible second binding site for striatin includes a region of L sequence between amino acids 1 and 363.
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Gallia, Jason. "Protein identification by dynamic programming". Diss., Online access via UMI:, 2009.
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Fang, Neng. "Good riddance to bad proteins : identification of novel protein quality control pathways targeting cytosolic misfolded proteins for degradation". Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/46513.
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Chun, Stella Soyoung. "Identification and validation of CDK13 interacting proteins". Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43130.
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Cyclin dependent kinases (CDKs) are components of signal transduction pathways that regulate cellular functions by phosphorylation of substrate proteins in response to upstream signals. The kinase domains of CDK12 and CDK13 are most similar to CDK9; CDK9 phosphorylates the C terminal domain (CTD) of RNA Polymerase II in order to stimulate processive transcription elongation. However, while most human CDKs consist of little more than a kinase domain, CDK12 and CDK13 are much larger and have several protein-protein interaction domains suggesting that they could participate within regulatory cascades. They also have a RS domain found in the SR protein family of splicing factors. Consistent with these features CDK12 and CDK13 co-localize with splicing factors and RNA Polymerase II in nuclear speckles. Based on these features CDK12 and CDK13 have been proposed to coordinately regulate splicing and transcription. Consistent with this hypothesis, both kinases phosphorylate the CTD of RNA polymerase II and regulate the alternative splicing of the Adenovirus E1a mini-gene model substrate. CDK12 has been found to interact with the splicing factors PRP19, CDC5L, RBM25, FBP11 and SRP55. Due to the similarity of CDK13 to CDK12, I investigated the interacting partners of CDK13 by immunoprecipitation and mass spectrometry and determined that CDK13 interacts with same splicing factors as CDK12. These interactions were validated by immunoprecipitation – western blot analysis. My results also indicated that PRP19 and CDC5L interact as a complex with CDK13. Therefore, the protein interaction partners of CDK13 and CDK12 suggest functional mechanisms for their ability to regulate splicing. In parallel projects, to begin investigating the functional roles of the kinase domain of CDK12 I constructed and expressed different CDK12 mutants in insect cells and in mammalian cells. Also to investigate the role of the CDK12 mutants and the protein-protein interactions of CDK13 in alternative splicing, I also developed a PCR based E1A mini-gene splicing assay.
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Evans, R. Jane. "Identification and characterisation of RP2 interacting proteins". Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1444192/.
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Retinitis pigmentosa (RP) is a genetically and clinically heterogenous retinal degenerative disease, which is characterised by night blindness and constriction of visual fields in the early stages which progress to blindness. X-linked RP (XLRP) is the most severe form of the disease and 2 causative genes have been identified, RP2 and RPGR. RP2 encodes a ubiquitously expressed 350 amino acid protein with a tubulin folding cofactor C (TBCC) homology domain and a C-terminal NDK homology domain. Despite the ubiquitous expression of the RP2 protein, the disease pathogenesis in patients carrying RP2 gene mutations appears to be restricted to retina. The function of RP2 in retina, or other tissues, has yet to be determined. This study aims to increase the understanding of the pathobiology of RP2 by unravelling novel molecular pathways of RP2 function. The approach taken to elucidate the cellular roles of RP2 in retina was to identify potential interacting partner proteins from a retina library using a yeast-two-hybrid approach. The Sos Recruitment System (SRS) was exploited, as RP2 had previously been shown to be predominantly localised on the plasma membrane. Several candidate protein partners for RP2 were identified. ADP-ribosylation like factor 3 (Arl3) was the most common cDNA identified in the yeast-two-hybrid screen. The interaction between RP2 and Arl3 was further defined using site directed mutagenesis to model pathogenic mutations in RP2 and a series of deletion mutants. RP2 preferentially interacted with the active GTP-bound form of Arl3 via the RP2 TBCC-homology domain. An altered cellular localisation and behaviour of RP2 was observed when co-transfected with GTP-bound Ari3. Both Arl3 and RP2 exhibit partial relocalisation to the Golgi and increased intracellular transport of RP2-GFP. Arl2 is structurally very similar to Arl3, and was also identified as a potential interactor of RP2, however the physiological significance of the RP2-Ari2 interaction is unclear. Interestingly, TBCC did not bind Arl2 or Arl3 confirming that RP2 and TBCC have different cellular functions. Four other novel potential interactors were also identified from the yeast two-hybrid screen including another small GTPase, and their interaction mapped to the C-terminus of RP2. The cellular localisation and function of these novel interactors suggest RP2 may have multiple cellular functions.
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Wong, Yee-man Elaine, i 王怡雯. "Identification and characterization of VCY2 interacting proteins". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B31228008.
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Roux, Milena. "Identification and characterization of EFR-interacting proteins". Thesis, University of East Anglia, 2010. https://ueaeprints.uea.ac.uk/26674/.
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Renner, Sonja. "Identification of ADAM10 5`UTR binding proteins". Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-170738.
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Hanley, Jonathan Gordon. "Identification of GABAâ†C receptor interacting proteins". Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392909.
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Melillo, Amanda Adeline. "Identification of Francisella tularensis Outer Membrane Proteins". University of Toledo Health Science Campus / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=mco1121867713.
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Ryan, Niamh Marie. "Identification and charactisation of brcai interacting proteins". Thesis, Queen's University Belfast, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492495.
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BRCA1 is a tumour suppressor gene (TSG), mutations ofwhich predispose to early-onset breast and ovarian cancer. Involvement ofBRCAl in DNA damage repair maintains chromosome stability. BRCA1 also participates in a variety of other pathways including: transcriptional regulation, ubiquitination, cell-cycle checkpoint control and apoptosis. BRCA1 mediates these functions by acting as a scaffold protein, allowing the assembly of multiprotein complexes. Yeast-2-Hybrid analysis is an in vivo technique for identifying protein:protein interactions. A yeast-2-hybrid screen was undertaken using an ovarian eDNA library and an N-terminal BRCA1 'Bait', composed ofthe amino acid residues 1-1142. The aim ofthe yeast-2-hybrid screen was to identify novel protein binding partners ofBRCAl. We identified two novel BRCA1 interacting proteins, which were confirmed by endogenous co-immunoprecipitation with BRCA1 in mammalian cells. These were the bromodomain containing protein 7 (BRD7) and the DExDIH box RNA helicase 36 (DHX36). Due to time constraints, the association between BRCA1 and DHX36 was not investigated further. BRD7 is a bromodomain containing protein whose main role appears to be in transcriptional regulation. We have demonstrated that both BRCA1 and BRD7 affect the transcription ofknown transcripts associated with the basallike subtype ofbreast cancer. BRCA1 transcriptionally represses expression ofKRT5, KRT17 and p-cadherin. Specifically, a BRCA1:c-Myc co-repressor complex is present at the promoter ofthese basal genes. We have shown that inhibition ofendogenous BRD7. expression, in the presence of functional BRCA1, abrogates expression ofthe BRCA1regulated KRT5 and p-cadherin basal marker genes. Further work is necessary to fully Supplied by The British Library - 'The world's knowledge' elucidate the mechanism and biological significance of this mode oftranscriptional regulation. Co-immunoprecipitation experiments also demonstrated an unexpected association between BRD7 and DHX36. Therefore, as well as the ability ofBRD7 and DHX36 to interact separately with BRCAl, it is possible that all three ofthe above proteins form a novel multimeric protein complex. Proving the existence ofthis BRCAl:BRD7:DHX36 multimeric complex and determination ofits function has still to be investigated.
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Powell, Karen L. "Identification of type 1 diabetes-related proteins". Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/27408.
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Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease, whereby the insulin-secreting beta-cells of the pancreas are targeted and destroyed by the patient's immune system. Although genetic susceptibility is required for the onset of this autoimmune disorder, environmental factors, including infectious agents and diet, also play an important but poorly understood role. Recently, WP5212 was identified as the first candidate diabetes-related wheat protein. To identify candidate molecular mimics of this wheat protein in target tissues, a novel approach was developed. Antibodies raised against two short peptides of this dietary antigen cross-react with self proteins: an exocrine pancreatic 33 kDa protein and a 28 kDa protein in the islets, spleen and mesenteric lymph nodes. In order to identify additional putative targets, a broad spectrum polyclonal serum was generated. In addition, a second antigenic wheat protein was identified by screening a wheat cDNA expression library with antibodies from a highly wheat-sensitive patient with both T1D and celiac disease. Further characterization of candidate molecular targets and antigenic wheat proteins may reveal novel pathways related to environmentally-induced T1D.
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Hou, Yuqing. "Identification and Characterization of Components of the Intraflagellar transport (IFT) Machinery: a Dissertation". eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/323.
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Intraflagellar transport (IFT), the bi-directional movement of particles along the length of flagella, is required for flagellar assembly. The IFT particles are moved by kinesin II from the base to the tip of the flagellum, where flagellar assembly occurs. The IFT particles are then moved in the retrograde direction by cytoplasmic dynein 1b/2 to the base of the flagellum. The IFT particles of Chlamydomonas are composed of ~16 proteins, organized into complexes A and B. Alhough IFT is believed to transport cargoes into flagella, few cargoes have been identified and little is known about how the cargos are transported. To study the mechanism of IFT and how IFT is involved in flagellar assembly, this thesis focuses on two questions. 1) In addition to a heavy chain, DHC1b, and a light chain, LC8, what other proteins are responsible for the retrograde movement of IFT particles? 2) What is the specific function of an individual IFT-particle protein? To address these two questions, I screened for Chlamydomonas mutants either defective in retrograde IFT by immunofluorescence microscopy, or defective in IFT-particle proteins and D1bLIC, a dynein light intermediate chain possibly involved in retrograde IFT, by Southern blotting. I identified several mutants defective in retrograde IFT and one of them is defective in the D1bLIC gene. I also identified several mutants defective in several IFT-particle protein genes. I then focused on the mutant defective in D1bLIC and the one defective in IFT46, which was briefly reported as an IFT complex B protein. My results show that as a subunit of the retrograde IFT motor, D1bLIC is required for the stability of DHC1b and is involved in the attachment of IFT particles to the retrograde motor. The P-loop in D1bLIC is not necessary for the function of D1bLIC in retrograde IFT. My results also show that as a complex B protein, IFT46 is necessary for complex B stability and is required for the transport of outer dynein arms into flagella. IFT46 is phosphorylated in vivo and the phosphorylation is not critical for IFT46’s function in flagellar assembly.
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Zhou, Yiqing, i 周怡青. "Identification of a cellular target of triptonide and its functional study". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46923561.
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Elston, Robert Conrad. "Identification of cellular proteins which interact with the human papillomavirus E6 proteins". Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627096.
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Dagvadorj, Bayantes. "Identification Of Proteins Interacting With Tagged-pathogen Effector Protein In Agro-delivered Planta". Master's thesis, METU, 2012. http://etd.lib.metu.edu.tr/upload/12614553/index.pdf.
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Wheat is one of the most essential food sources in the world. However, there has been serious yield loss of wheat production due to stripe rust disease caused by the fungal pathogen Puccinia striiformis f. sp. tritici. The cost-effective and long-lasting defense to the disease can be achieved by generating genetically resistant crops against the disease forming pathogens. To accomplish this, first step is to acquire knowledge in the plant pathogen interactions of the crop and the pathogen of interests at the cellular and the molecular level.
In this thesis research, PstHa2a5 candidate effector gene from Puccinia striiformis f. sp. tritici is investigated to identify its role and interaction between host factors in yellow rust infected Triticum aestivum L. The gene construct was engineered with FLAG-tag fusion at its N-terminus, and synthesized. This construct was cloned into pJL48-TRBO vector for an expression in Nicotiana benthamiana via agrobacterium-mediated gene transformation. The expressed protein structure with FLAG-tag was purified, and immunoprecipitated with one putative N. benthamiana interactor by immunoprecipitation experiments. This candidate interactor protein will be identified with Mass Spectroscopy. In addition to this, subcellular localization of the effector candidate was examined in N. benthamiana plant. This was achieved by cloning PstHa2a5 gene construct in pK7WGF2 gateway destination vector and localization is determined by GFP expression in N. benthamiana after agrobacterium-mediated gene transformation.
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Zhang, Xiao Xiao. "Identification of membrane-interacting proteins and membrane protein interactomes using Nanodiscs and proteomics". Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/39413.
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The insoluble nature of membrane proteins has complicated the identification of their interactomes. The Nanodisc has allowed the membrane and membrane proteins to exist in a soluble state. In this thesis, we combined Nanodisc and proteomics and applied the technique to discover the interactome of membrane proteins. Using the SecYEG and MalFGK membrane complex incorporated into Nanodisc, we identified, Syd, SecA, and MalE. These interactions were identified with high specificity and confidence from total soluble protein extracts. The protein YidC was also tested but no interactors were detected. Overall, these results showed that the technique can identify periplasmic and cytosolic interacting partners with high degree of specificity. In a second approach, the method was applied to detect proteins with high affinity for lipid using S. cerevisiae as a model organism. Using Nanodiscs containing different types of phospholipids, many known lipid interactors were identified, including: Ypt1, Sec4, Vps21, Osh6, and Faa1. Interestingly, Caj1 was identified as a PA specific interactor and this interaction was found to be pH dependent. Liposome sedimentation assay showed that Caj1 has affinity for acidic phospholipids. In vivo analysis confirmed the plasma membrane localization of N’-GFP-Caj1 and specifically to the yeast buds. However, pH dependent localization was not observed. Together, with the in vivo and in vitro results suggests that Caj1 is an acidic phospholipid interacting protein.
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Ginter, Joy M. "Protein identification and characterization by mass spectrometry". Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 133 p, 2008. http://proquest.umi.com/pqdweb?did=1597617911&sid=8&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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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.
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Long, Kimberly Renee. "Identification and Characterization of Agv1, a Pre-Metazoan Arf GAP: A Dissertation". eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/339.
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Human immunodeficiency virus type 1 (HIV-1) is a member of the lentivirus subfamily of retroviruses. HIV-1 expresses multiple genes from a single provirus by alternative splicing. Early in viral expression, fully spliced 2-kb viral RNA is exported from the nucleus and encodes the viral regulatory protein, Rev, which is essential for nuclear transport of partially spliced and unspliced genomic-length RNA. Rev binds to an RNA structural element called the Rev response element (RRE) and mediates nuclear export through the leucine-rich nuclear export signal (NES) pathway. The human Rev Interacting Protein (hRIP) interacts specifically with the Rev NES. Rev NES mutants that are unable to export Rev-dependent RNAs are also unable to bind to hRIP. The hRIP cDNA encodes a 562 amino acid protein containing an N-terminal zinc finger with homology to Arf GAP domains, a central serine and threonine rich region, and C-terminal phenylalanine-glycine (FG) repeats characteristic of nucleoporins.
To identify an hRIP ortholog in a genetically tractable organism, we performed database searches using the N-terminal zinc finger of hRIP. Using this approach, we identified a novel gene in Schizosaccharomyces pombe. Alignment of the entire reading frame of the putative ortholog with hRIP indicates similarity with the serine/threonine rich region and with the FG repeats, suggesting that S. pombecould be a good model system to study the cellular function of hRIP.
We find that the S. pombe ORF is an essential gene, which encodes a 483 amino acid protein that is also able to interact with the NES of HIV-1 Rev. Based on being an essential gene, and the presence of a putative Arf GAP domain, the ORF was named an Arf GAP essential for viability, agv1+. We show that Agv1 is not directly involved in the nuclear export of poly(A+) RNA or 5S rRNA, nuclear export of leucine-rich NES-containing proteins, or nuclear import of nuclear localization signal (NLS)-containing proteins. However, Agv1 does appear to play a role in the cytoplasmic localization of 5S rRNA.
We demonstrate that loss of Agv1 alters the localization of endoplasmic reticulum (ER) membrane and Golgi membrane resident proteins, accumulates intracellular membrane, and blocks processing of carboxypeptidase Y. Furthermore, the S. cerevisiae ADP-ribosylation factor (Arf) GTPase activating protein (GAP) Glo3, but not a catalytically inactive Glo3 mutant [R59K], is able to partially compensate for the loss of Agv1 function in temperature sensitive strains, indicating that Agv1 is an S. pombe Arf GAP with some functional features similar to S. cerevisiae Glo3.
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Höglund, Pär J. "Identification, Characterization and Evolution of Membrane-bound Proteins". Doctoral thesis, Uppsala universitet, Institutionen för neurovetenskap, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9329.
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Membrane proteins constitute approximately 30% of all genes in the human genome and two large families of membrane proteins are G protein-coupled receptors (GPCRs) and Solute Carriers (SLCs) with about 800 and 380 human genes, respectively. In Papers I, II and IV, we report 16 novel human Adhesion GPCRs found by searches in NCBI and Celera databases. In Paper I, we report eight novel human GPCRs, and six in Paper II. We identified two new human Adhesion GPCRs and 17 mouse orthologs in Paper IV. Phylogenetic analysis demonstrates that the 16 novel human genes are additional members of the Adhesion GPCR family and can be divided into eight phylogenetic groups. EST expression charts for the entire repertoire of Adhesions in human and mouse were established, showing widespread distribution in both central and peripheral tissues. Different domains were found in their N-terminus, some, such as pentraxin in GPR112, indicates that they take part in immunological processes. In Paper III, we discovered seven new human Rhodopsin GPCRs. In Paper V, we present the identification of two new human genes, termed SLC6A17 and SLC6A18 from the Solute Carriers family 6 (SLC6). We also identified the corresponding orthologs and additional genes from the mouse and rat genomes. We analysed, in total, 430 unique SLC6 proteins from 10 animal, one plant, two fungi and 196 bacterial genomes. In Paper VI, we provide the first systematic analysis of the evolutionary history of the different SLC families in Eukaryotes. In all, we analysed 2403 sequences in eight species and we delineate the evolutionary history of each of the 46 SLC families.
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Satijn, David Pierre Elisabeth. "Identification and characterization of human polycomb-group proteins". [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2000. http://dare.uva.nl/document/82593.
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Ferguson, Alison. "Identification and characterisation of Arabidopsis ER accessory proteins". Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12472/.
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ER accessory proteins are a novel class of endoplasmic reticulum (ER) proteins that facilitate the exit of polytopic membrane proteins from the ER. They are important for the correct targeting of their cognate polytopic membrane proteins to the plasma membrane (PM) and their absence leads to abnormal accumulation of their target in the ER. Until recently, it was not clear if such proteins exist in plants. However, work by Dharmasiri et al (2006) and Gonzales et al (2005) suggest that such proteins exists in plants too. Polytopic membrane proteins such as nutrient transporters, hormone transporters and sugar transporters are a very important class of proteins as they regulate many important physiological and biochemical processes. Better understanding of the targeting of these proteins to the PM is of considerable agronomic interest due to the importance of efficient use of resources in sustainable agriculture. One of the projects aims is to identify novel ER accessory proteins in Arabidopsis. Using a bioinformatics approach, 40 novel ER resident proteins were identified from a protein localisation database (LOPIT) generated by Dunkley et al (2006) as potential candidates for ER accessory proteins. Genetic, phenotypic and molecular approaches have been used to assess their role as potential ER accessory proteins. A few promising candidates have been identified, one of which AtBPL1 and related family. The AtBPL1 family has similarity to mammalian BAP31 which has been shown to function as an ER accessory protein (Ladasky et al, 2006). To determine if AtBPL1 family plays a similar role in plants a detailed molecular characterisation was carried out, this involved detailed expression analysis using reporter genes and in situ immunolocalisation and characterisation of miRNA lines. Smart screens suggest that BPL1 family members may be involved in the targeting of a nitrate transporter, however its precise target is currently unknown. A key focus of this present investigation have been on further characterisation of AXR4, which is required for the correct targeting of AUX1 to the plasma membrane (Dharmasiri et al, 2006). AUX1 belongs to a multi-gene family, involving three other members, LAX1, LAX2 and LAX3. Using genetic and cell biology approaches, AXR4 has been shown to be necessary for the correct localisation of at least two other members of this family LAX2 and LAX3. AXR4 mutants show defects in targeting of LAX2 and LAX3 to the plasmamembrane and show weak lax2 and lax3 phenotypes. Co-Immunoprecipitation studies revealed that AXR4 and AUX1 interact directly when co-expressed in insect cells. Finally molecular, bioinformatics and protein modelling approachs were used to probe the function of alpha beta hydrolase domain in AXR4 function. AXR4 appears to be tolerant to amino acid subsitition even at highly conserved amino acids, suggesting that the alpha beta hydrolase domain may not be important for its function.
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Shah, Bindiya. "Identification of genes encoding secreted proteins of schistosomes". Thesis, University of York, 2000. http://etheses.whiterose.ac.uk/9801/.
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Kimura, Atsuko. "Isolation and identification of imidazoline-2 binding proteins". Thesis, University of Bristol, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399960.
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Macer, Darryl Raymund Johnson. "Identification and analysis of proteins of the reticuloplasm". Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.330298.
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Lassen, Matthew Gordon. "Identification of proteins involved in chloroplast DNA replication /". Diss., CLICK HERE for online access, 2004. http://contentdm.lib.byu.edu/ETD/image/etd633.pdf.
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Lassen, Matthew G. "Identification of Proteins Involved in Chloroplast DNA Replication". BYU ScholarsArchive, 2004. https://scholarsarchive.byu.edu/etd/221.
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Chapter 1
Chloroplast nucleoids (ct-nucleoids) are DNA/protein complexes involved in compacting the chloroplast genome, and may play a role in regulating DNA replication. Ct-nucleoids were isolated from young soybean plants and separated by 2-D gel electrophoresis. Gel spots were excised and analyzed by MALDI-ToF mass spectrometry, resulting in several protein identifications. The proteins identified all have functions unrelated to DNA replication. While some of these proteins may be due to contamination, it is possible that some of these proteins are dual-functional, playing direct roles in the regulation of DNA replication.
Chapter 2
A 28 kDa soybean protein was isolated by sequence specific DNA affinity chromatography from total chloroplast protein isolations. Mass spectrometry analysis revealed that the 28 kDa protein contains some homology within an ssb domain of an Arabidopsis mitochondrial-targeted SSB (mtSSB) of approximately 21 kDa. N-terminal sequencing revealed that the 28 kDa soy protein is identical to a 36 amino acid region at the N-terminus of the Arabidopsis mtSSB. Protein fractions containing the 28 kDa protein shift oriA in electrophoretic mobility shift assays (EMSAs). Arabidopsis mtSSB fails to shift oriA in EMSAs run under identical conditions. Arabidopsis mtSSB causes a shift of ssDNA in EMSAs, while the ability of the 28 kDa soy protein to bind ssDNA is still unclear. Importantly, the 28 kDa soy protein was identified from total protein extracts obtained from intact chloroplasts, while in-vitro targeting experiments suggest that the Arabidopsis mtSSB localizes only to mitochondria and not to chloroplasts. BLAST searches of the available soybean genomic and EST databases do not produce any significant homologies to the 36 amino acid N-terminal sequence.
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Li, Zhaobo. "Identification of degradation pathways for HSP90 client proteins". Thesis, University of Sussex, 2017. http://sro.sussex.ac.uk/id/eprint/67006/.
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Heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone that plays critical roles in regulating the folding, stabilization, post-translational modification, activation and maturation of its various client proteins, of which many are oncoproteins. Impairing the function of HSP90 by the inhibition of its ATPase cycle with inhibitors such as AUY922 promotes the ubiquitylation and proteasomal degradation of its client proteins. However, we currently do not fully understand the mechanism for ATPase-inhibited triggered degradation of client proteins, and which E3 ligase systems are involved. Although previous studies revealed a number of E3 ligases including CHIP and CUL5 as potentially E3 ligases involved in the degradation of HSP90-dependent client protein, these have often used cancer cells that may have dysregulated systems. Additionally, other components of such E3 ligase systems have not been well characterised. Using a Reverse Transfection Format (RTF) siRNA screen system we identified two E3 ligases that are involved in two independent pathways for mediating proteasomal degradation of the HSP90-dependent protein kinase CRAF in HEK293 cells. The elongin BC-CUL5-SOCS-box protein (ECS) complex operates one pathway for the degradation of CRAF, while a novel but poorly described HECTD3 from the HECT-family was identified as the main E3 ligase for degrading CRAF following the pharmaceutical inhibition of HSP90. We revealed a potential complexes consisting of CRAF, HSP90 and HECTD3, which may contribute towards identifying the pathway for the degrading of such HSP90-dependent client protein kinases. We were also able to show that depriving access of CRAF to CDC37 and therefore HSP90 resulted in an HECTD3 and CUL5 independent degradation pathway. These studies form the basis of establishing the complex network of pathways that help to regulate CRAF protein levels.
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Cotrim, Cândida Zita dos Santos. "Identification of neuronal proteins that interact with PP1y2". Master's thesis, Universidade de Aveiro, 2009. http://hdl.handle.net/10773/4508.
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Mestrado em Métodos Biomoleculares AvançadosA proteína fosfatase 1 (PP1) está envolvida em múltiplos processos de grande relevância fisiológica (por ex. aprendizagem, memória e neurotransmissão) e patológica (envelhecimento, doenças neurodegenerativas). No entanto, restam ainda por estabelecer importantes interacções de relevância fisiológica, bem como a localização intracelular onde estas interacções ocorrem. Esta complexidade é originada pela existência de três isoformas da PP1, organizadas tanto espacialmente como temporalmente e que podem alterar a sua localização intracelular de forma dinâmica. Este projecto focalizou a identificação de proteínas expressas em cérebro humano que interagem com PP1γ2, através da técnica Dois Híbrido em leveduras. Através desta técnica foram obtidos 317 clones positivos, permitindo a identificação de 298 proteínas que ligam à PP1γ2 e 19 proteínas que ligam PP1γ2end, entre as quais algumas proteínas já conhecidas por interagirem com a PP1, outras nunca antes associadas com a PP1 e várias proteínas não caracterizadas. Foi feito um estudo mais detalhado para três proteínas das mais abundantes, PINK1, BRI2 e BRI3. A ligação destas proteínas com PP1 foi analisada através de várias técnicas e a sua localização subcelular e co-localização com a PP1γ e APP foi estudada por imunocitoquímica. Os resultados aqui apresentados corroboram a localização subcelular destas proteínas e a ligação já descrita entre a APP e BRI2. Permitem acrescentar que PINK1, BRI2 e BRI3 interagem com PP1γ e que não só a proteína BRI2 mas também a BRI3 interagem com APP. Estudos futuros serão necessários para compreender o papel destas interacções no sistema neuronal. Estes resultados também validam o uso do sistema YTH como um meio de estudo para melhor compreender as funções da PP1 e sua regulação em diferentes eventos celulares.
The ubiquitous protein phosphatase 1 (PP1) is involved in multiple processes of great physiological (e.g. learning, memory and neurotransmitter signaling) and pathological (aging, Alzheimer’s disease and other neurodegenerative conditions) relevance. However, many physiologically important interactions remain to be established, as well as the exact intracellular locations where these interactions take place. This complexity is provided by the existence of three PP1 isoforms that are organized both spatially and temporally, and can change their intracellular localization dynamically. This project focused on the identification of the PP1γ2 interacting proteins expressed in human brain using the Yeast Two-Hybrid system. With this technique 317 positive clones were recovered allowing the identification of 298 proteins that bind PP1γ2 and 17 proteins that bind PP1γ2end, among those are some previously known PP1 interacting proteins, other proteins never associated with PP1 before and several uncharacterized proteins. A more detailed study was carried out for three of the most abundant clones in the screen, PINK1, BRI2 e BRI3. The binding to PP1 was analyzed by several techniques and its subcellular localization and co-localization with PP1γ and APP studied by immunocytochemistry. These results corroborate the subcellular localization of this proteins and the interaction of BRI2 with APP already described. These results allow concluding that PINK1, BRI2 and BRI3 interact with PP1 and that BRI2 and BRI3 interact with APP. Further studies are needed to understand the function of the interaction of PP1 and PINK1, BRI2 and BRI3 in the neuronal system. Moreover, our results support the use of the YTH system as a means to study and understand PP1 function and regulation in different cellular events.
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Das, Lala Meenakshi. "Identification of Endogenously Biotinylated Proteins in Mammalian Spermatozoa". Kent State University Honors College / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1303846044.
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