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1
Gilker, Eva Adeline Gilker. "INTERACTIONS AND LOCALIZATION OF PROTEIN PHOSPHATASES, YWHA PROTEINS AND CELL CYCLE CONTROL PROTEINS IN MEIOSIS". Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1532699317257539.
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Yarbrough, Daniel Kenneth. "Structural and mutational analysis of chromophore maturation in long wavelength fluorescent proteins /". view abstract or download file of text, 2004. http://wwwlib.umi.com/cr/uoregon/fullcit?p3120630.
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Thesis (Ph. D.)--University of Oregon, 2004.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 142-152). Also available for download via the World Wide Web; free to University of Oregon users.
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Meissner, Christina Sylvia. "Influence of HCMV proteins pUL71 and pUL77 on viral maturation". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16414.
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Die Bildung infektiöser Viruspartikel des humanen Zytomegalievirus (HCMV) ist ein mehr-stufiger Prozess. Sie beginnt mit der Verpackung der DNA in die Kapside im Kern, gefolgt von weiterer Reifung während des Transports durch das Zytoplasma und der abschließenden Freisetzung aus der Zelle. Im Zuge dieser Arbeit wurden zwei Proteine, die Einfluss auf die ebengenannten Prozesse haben, analysiert. Der erste Teil der Arbeit befasst sich mit der funktionellen Charakterisierung des HCMV Pro-teins pUL77. Es ist bekannt, dass das homologe Protein pUL25 in alpha-Herpesvirinae essentiell für die DNA-Verpackung ist. Zunächst konnte das Protein als Kapsid-assoziiertes strukturelles Protein identifiziert werden. Es wurden Interaktionen von pUL77 mit DNA-Verpackungs- und Kapsidproteinen gezeigt. Weiterhin wurde die DNA-Bindungsfähigkeit von pUL77 in verschiedenen „in vitro“-Experimenten untersucht. Zusammengefasst weisen unsere Ergebnisse auf eine Funktion von HCMV pUL77 bei der DNA-Verpackung hin. Im zweiten Teil der Arbeit wurde das HCMV Protein pUL71 charakterisiert, das in allen Herpesviren konserviert vorkommt, dessen Funktion jedoch nicht charakterisiert ist. Zunächst wurde das Protein als strukturelles Tegumentprotein mit “earlylate“ Expressionskinetik klassifiziert. Weiterhin wurden die subzelluläre Lokalisation sowie virale und zelluläre Interaktionspartner untersucht. Die Ergebnisse weisen auf eine Funktion von HCMV pUL71 bei der Reifung und beim Transport der Virionen im Zytoplasma hin. „In silico“-Vorhersagen zeigten ein „Leuzin Zipper“-Motiv in pUL71, das als mögliche Oligomerisationsdomäne dienen könnte. Mutationen wurden in dieses Motiv eingebracht und die resultierenden Proteine auf ihre Oligomerisationsfähigkeit mit „in vitro“-Methoden und in rekombinanten Viren untersucht. Zusammenfassend konnten wir zeigen, dass das „Leuzin Zipper“-Motiv wichtig für die Funktion von pUL71 ist und diese mit einer unbeeinträchtigten Oligomerisation des Proteins zusammen hängt.The morphogenesis of Human cytomegalovirus (HCMV) virions starts with the capsid assem-bly and DNA insertion in the nucleus followed by maturation during transport through the cytoplasm prior to release of virus progeny. In this study we are functionally characterising two proteins that are involved in those steps. The function of essential HCMV protein pUL77 is characterised in the first part of the study. HCMV pUL77 was shown to be a structural protein associated with capsids. Furthermore, our experiments demonstrated that HCMV pUL77 interacts with DNA packaging motor compo-nents and capsid proteins. The ability of HCMV pUL77 to bind double-stranded DNA was studied in “in vitro” assays designed for this study. The homologue α-Herpesvirinae protein pUL25 is described to be involved in processes connected with DNA packaging. Data ob-tained in this study demonstrates that HCMV pUL77 might serve a similar function. In the second part of the study HCMV pUL71, conserved throughout the Herpesvirus family but to date unclassified, was functionally characterised. HCMV pUL71 was defined a struc-tural tegument protein with early-late expression kinetics. We studied the sub-cellular local-isation and interactions of pUL71 with a subset of cellular and viral proteins. Thereby we could show that HCMV pUL71 function might be connected with processes of viral egress. By in silico analyses we identified a leucine zipper motif in pUL71 that might serve as a puta-tive oligomerisation domain. In order to investigate the function of the leucine zipper motif, we performed in vitro assays and investigated the alterations of the motif in the viral context. Taken together we can conclude that (i) an intact leucine zipper motif is crucial for the func-tion of pUL71 and (ii) this function is dependent upon undisturbed oligomerisation of the pro-tein.
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Atherton, Ruth Elizabeth. "Catalytic activity and maturation of the metalloprotease-disintegrin protein, MDC9 /". Access full-text from WCMC, 1999. http://proquest.umi.com/pqdweb?did=733095101&sid=8&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Cotta, Doné Stefania. "Nephrin - intracellular trafficking and podocyte maturation /". Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-411-2/.
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Tominaga, Taiga. "Structural studies on cyano group biosynthesis by [NiFe] hydrogenase maturation proteins". 京都大学 (Kyoto University), 2013. http://hdl.handle.net/2433/180637.
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Popescu, Costin-Ioan. "Maturation and processing of endogenous and viral proteins in the endoplasmic reticulum". Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422667.
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Hodson, D. J. "The RNA-binding proteins TIS11b and TIS11d regulate lymphocyte development and maturation". Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604134.
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Over-expression studies in cultured B cells showed that both TIS11b and TIS11d were able to block plasma cell differentiation in vitro. This action was specific as class switch recombination was not affected. TIS11b conditional knockout mice showed normal lymphocyte development and maturation and a normal humoral immune response. TIS11b conditional knockout mice lacked marginal zone B cells and had reduced numbers of peritoneal B1 cells. However they mounted a normal humoral response to immunisation with NP-KLH, with normal affinity maturation and a normal memory response. Single knockouts were inter-crossed to create double knockout (dKO) mice. Unexpectedly, close to 100% of dKO mice developed T-lymphoblastic leukaemia from three months of age. Pre-leukaemic mice showed arrested B cell development at the pro-B stage and significantly perturbed thymic development. This included the aberrant passage of thymocytes through the β-selection checkpoint in the absence of expression of a functional T cell receptor β chain. A genome-wide approach to identify deregulated TIS11b/d targets in pre-leukaemic mice showed elevated expression of the oncogenic transcription factor Notch1. Bioinformatic analysis revealed potentials binding sites for TIS11b and TIS11d within a highly conserved region of the Notch1 3’UTR. The ability of TIS11b and TIS11d to interact and suppress expression of Notch1 was demonstrated by electromobility shift assay (EMSA) and luciferase reporter assays. Furthermore, development and proliferation of T-ALL was Notch1 dependent, both in vivo and in vitro.
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Lyall, Natalie. "The role of RAB2 in the maturation of macrophage phagosomes containing Candida albicans". Thesis, University of Aberdeen, 2018. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=238253.
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Phagosome maturation is a dynamic process involving the engulfment and degradation of pathogens by phagocytic cells. However, several pathogens have employed mechanisms that facilitate their survival and escape from the phagosome. The fungal pathogen, Candida albicans, is capable of switching from yeast to hyphal form to facilitate its pathogenicity and escape from the phagosome. Rab GTPases are key regulators in phagosome maturation by mediating interactions with the endocytic pathway and leading to biogenesis of the phagolysosome. The temporal localisation dynamics of Rab2 on maturing phagosomes containing live C. albicans was investigated. Live-cell imaging revealed green fluorescent protein (GFP)-tagged Rab2 was recruited to C. albicans-containing phagosomes. Rab2 appeared on phagosomes within 2 min following complete engulfment of C. albicans. Rab2 persisted transiently on macrophage phagosomes and this correlated with the length of C. albicans hyphae at the time of uptake, suggesting C. albicans morphology modulates Rab2 localisation dynamics. Expression of dominant negative or dominant active Rab2 did not affect macrophage migration, the rate of engulfment or phagosome acidification during the early stages of phagosome maturation. Furthermore, altered expression of Rab2 did not interfere with C. albicans ability to escape from and kill macrophages, suggesting Rab2 is not involved in the outcome of the host-pathogen interaction. However, altered expression of Rab2 reduced the acquisition of the late-stage phagosome maturation markers, cathepsin B and LAMP1, suggesting Rab2 impacts upon phagosome-lysosome fusion. Finally, the uptake of other particles by macrophages revealed Rab2 recruitment, as well as localisation dynamics on phagosomes, may be cargo-dependent. Through the use of live-cell imaging, real-time dynamics of phagosome biogenesis in live cells was examined, offering unique insight at the host-pathogen interface.
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Nangola, Sawitree. "The interference of human immunodeficiency virus assembly and maturation by ankyrin repeat proteins". Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112044.
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Le but de ce travail est de découvrir des nouvelles protéines suceptibles d'interférer avec le cycle vital du virus HIV. De par leur repliement, les protéines à motifs ankyrines peuvent constituer une ossature protéique trés bien adaptée à cet objectif. Plusieurs interacteurs spécifiques de la protéine MA-CA du HIV ont été sélectionnées par exposition sur phage à partir d'une bibliothèque de variants d'ankyrines. Trois protéines isolées ont été produites à partir de clones ayant une forte activité de liaison. Le meilleur interacteur protéique (1D4) interagit avec un épitope situié sur le domaine CA. La constante de dissociation entre 1D4 et la protéine HACA a été déterminée par Calorimétrie de Titrage Isotherme (ou ITC) et est égale à 0,45M. La protéine 1D4 n'a pas d'effet détectable sur la maturation virale suivie par une technique ELISA de dosage de la Protease du HIV. En revanche, cette protéine interfere avec l'assemblage viral dans des cellules supT1 qui exprime de façon stable la protéine 1D4 sous forme myristoylée. Ce resultat ouvre une perspective d'appoche pour interferer avec le cycle vital du HIVPresently, the standard regimen for antiretroviral treatment is highly active antiretroviral therapy (HAART). However, this strategy inherits the well-known side effects and is prone to promote the HIV drug-resistant strains. As a consequence, gene therapy has been introduced as an alternative approach. In this study, we aimed to discover the novel protein-based agents for intervening viral replication by gene targeting procedure. Regarding the efficient folding dynamic in cytoplasm, ankyrin repeat protein was considered to be a candidate scaffold. Several engineered ankyrin binders specific to HIV MA-CA domain were successfully retrieved from the ankyrin-displayed phage library. Three positive clones with high binding activity by ELISA were selected for further analyzing their binding property in soluble form. The best binder, 1D4, recognized its epitope located on CA domain as shown by Western immunobloting and ELISA. The affinity of 1D4 against H6MA-CA was 0.45 μM with one to two moles of target molecule determined by isothermal titration calorimetry (ITC). Although 1D4 exhibited no effect on viral maturation as verified by an ELISA based HIV protease assay technique, it disturbed the viral assembly process in Sup-T1 cells which stably expressed the myristoylated 1D4. This finding has provided a concrete prospect for HIV life cycle interruption by stem cell gene therapy in the future
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Hua, Ethan Wei. "Maturation of single retinogeniculate projections visualized by in vivo electroporation of fluorescent proteins". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p1459290.
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Thesis (M.S.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed Nov. 10, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references.
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Sasaki, Daisuke. "Functional and structural studies on nickel insertion process by archaeal [NiFe] hydrogenase maturation proteins". 京都大学 (Kyoto University), 2012. http://hdl.handle.net/2433/157802.
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Kaymak, Ebru. "Understanding the Sequence-Specificity and RNA Target Recognition Properties of the Oocyte Maturation Factor, OMA-1, in Caenorhabditis elegans: A Dissertation". eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/852.
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Maternally supplied mRNAs encode for necessary developmental regulators that pattern early embryos in many species until zygotic transcription is activated. In Caenorhabditis elegans, post-transcriptional regulatory mechanisms guide early development during embryogenesis. Maternal transcripts remain in a translationally silenced state until fertilization. A suite of RNA-binding proteins (RBP’s) regulate these maternally supplied mRNAs during oogenesis, the oocyte-to-embryo transition, and early embryogenesis. Identifying the target specificity of these RNA-binding proteins will reveal their contribution to patterning of the embryo. We are studying post-transcriptional regulation of maternal mRNAs during oocyte maturation, which is an essential part of meiosis that prepares oocytes for fertilization. Although the physiological events taking place during oocyte maturation have been well studied, the molecular mechanisms that regulate oocyte maturation are not well understood.
OMA-1 and OMA-2 are essential CCCH-type tandem zinc finger (TZF) RBP’s that function redundantly during oocyte maturation. This dissertation shows that I defined the RNA-binding specificity of OMA-1, and demonstrated that OMA-1/2 are required to repress the expression of 3ʹUTR reporters in developing oocytes. The recovered sequences from in vitro selection demonstrated that OMA-1 binds UAA and UAU repeats in a cooperative fashion. Interestingly, OMA-1 binds with high affinity to a conserved region of the glp-1 3ʹUTR that is rich in UAA and UAU repeats. Multiple RNA-binding proteins regulate translation of GLP-1 protein, a homolog of Notch receptor. In addition to previously identified RBP’s, we showed that OMA-1 and OMA-2 repress glp-1 reporter expression in C. elegans oocytes.
Mapping the OMA-1 dependent regulatory sites in the glp-1 mRNA and characterizing the interplay between OMA-1 and other factors will help reveal how multiple regulatory signals coordinate the transition from oocyte to embryo but the abundance of OMA-1 binding motifs within the glp-1 3ʹUTR makes it infeasible to identify sites with a functional consequence. I therefore first developed a strategy that allowed us to generate transgenic strains efficiently using a library adaptation of MosSCI transgenesis in combination with rapid RNAi screening to identify RBP-mRNA interactions with a functional consequence. This allowed me to identify five novel mRNA targets of OMA-1 with an in vivo regulatory connection. In conclusion, the findings in this dissertation provide new insights into OMA-1 mediated mRNA regulation and provide new tools for C. elegans transgenesis. Development of library MosSCI will advance functional mapping of OMA-1 dependent regulatory sites in the target mRNAs. Extending this strategy to map functional interactions between mRNA targets and RNAbinding proteins in will help reveal how multiple regulatory binding events coordinate complex cellular events such as oocyte to embryo transition and cell-fate specification.
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Kaymak, Ebru. "Understanding the Sequence-Specificity and RNA Target Recognition Properties of the Oocyte Maturation Factor, OMA-1, in Caenorhabditis elegans: A Dissertation". eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/852.
Texto completoResumen
Maternally supplied mRNAs encode for necessary developmental regulators that pattern early embryos in many species until zygotic transcription is activated. In Caenorhabditis elegans, post-transcriptional regulatory mechanisms guide early development during embryogenesis. Maternal transcripts remain in a translationally silenced state until fertilization. A suite of RNA-binding proteins (RBP’s) regulate these maternally supplied mRNAs during oogenesis, the oocyte-to-embryo transition, and early embryogenesis. Identifying the target specificity of these RNA-binding proteins will reveal their contribution to patterning of the embryo. We are studying post-transcriptional regulation of maternal mRNAs during oocyte maturation, which is an essential part of meiosis that prepares oocytes for fertilization. Although the physiological events taking place during oocyte maturation have been well studied, the molecular mechanisms that regulate oocyte maturation are not well understood.
OMA-1 and OMA-2 are essential CCCH-type tandem zinc finger (TZF) RBP’s that function redundantly during oocyte maturation. This dissertation shows that I defined the RNA-binding specificity of OMA-1, and demonstrated that OMA-1/2 are required to repress the expression of 3ʹUTR reporters in developing oocytes. The recovered sequences from in vitro selection demonstrated that OMA-1 binds UAA and UAU repeats in a cooperative fashion. Interestingly, OMA-1 binds with high affinity to a conserved region of the glp-1 3ʹUTR that is rich in UAA and UAU repeats. Multiple RNA-binding proteins regulate translation of GLP-1 protein, a homolog of Notch receptor. In addition to previously identified RBP’s, we showed that OMA-1 and OMA-2 repress glp-1 reporter expression in C. elegans oocytes.
Mapping the OMA-1 dependent regulatory sites in the glp-1 mRNA and characterizing the interplay between OMA-1 and other factors will help reveal how multiple regulatory signals coordinate the transition from oocyte to embryo but the abundance of OMA-1 binding motifs within the glp-1 3ʹUTR makes it infeasible to identify sites with a functional consequence. I therefore first developed a strategy that allowed us to generate transgenic strains efficiently using a library adaptation of MosSCI transgenesis in combination with rapid RNAi screening to identify RBP-mRNA interactions with a functional consequence. This allowed me to identify five novel mRNA targets of OMA-1 with an in vivo regulatory connection. In conclusion, the findings in this dissertation provide new insights into OMA-1 mediated mRNA regulation and provide new tools for C. elegans transgenesis. Development of library MosSCI will advance functional mapping of OMA-1 dependent regulatory sites in the target mRNAs. Extending this strategy to map functional interactions between mRNA targets and RNAbinding proteins in will help reveal how multiple regulatory binding events coordinate complex cellular events such as oocyte to embryo transition and cell-fate specification.
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Cole, Jason Nicklaus. "Characterisation of cell wall proteins, virulence factor maturation and invasive disease trigger of Group A streptococcus". Access electronically, 2006. http://www.library.uow.edu.au/adt-NWU/public/adt-NWU20070130.144214/index.html.
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Fairman, Peter. "The Effect of HIV-1 and Accessory Proteins on Monocyte Derived Dendritic Cell Maturation and Function". Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/24046.
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Dendritic cells (DCs) are specialized members of the innate immune system that are responsible for the initiation of primary adaptive immune responses whose purpose is to resolve infection and inflammation. During most viral infections, mature dendritic cells present critical viral antigens to naïve T-cells within secondary lymphoid organs, resulting in the generation of an antigen-specific adaptive immune response and clearance of the virus. During infection with HIV-1 however, the virus is not cleared and a chronic systemic infection develops characterized by immune dysfunction, CD4+ T-cell depletion, systemic inflammation, and opportunistic infections. A growing body of evidence indicates that HIV-1 subversion of DCs contributes to both HIV-1 pathologies and viral dissemination. A number of similar effects by accessory HIV-1 peptides on DC physiology have also been reported. In vitro studies demonstrate that HIV-1 inhibits DC maturation and function. Ex vivo studies on the other hand describe partially mature, dysfunctional DCs collecting in secondary lymphoid organs. In vitro studies examining the effects of HIV-1-Tat and HIV-1-Vpr have described opposing effects on DC maturation. Therefore we undertook experiments to comprehensively describe the effects of HIV-1 and the Tat and Vpr accessory peptides on DC maturation and function.
To understand the contributions of individual viral proteins to DC dysfunction we infected DCs with a dual tropic HIV-1 and examined phenotypic and functional changes after maturation with inflammatory cytokines. Following this we examined the influence of exogenous and endogenous HIV-1-Tat and HIV-1-Vpr on MDDC maturation and function using recombinant proteins and deletion mutant lab adapted HIV-1 strains.
Live dual tropic HIV-1 was found to selectively inhibit aspects of phenotypic maturation as well as antigen capture and presentation functions. MDDC MAPK responsiveness to bacterial LPS remained intact however. Exogenous accessory HIV-1 Tat and Vpr did not affect MDDC phenotype but inhibited dextran endocytosis and viral peptide presentation. HIV-1-gp120 increased iMDDC maturation while blunting cytokine induced decreases in MDDC antigen capture abilities. The deletion of HIV-1-Tat did not affect MDDC phenotype, but was found to affect antigen capture decreases by R5 tropic HIV-1BaL. Deletion of HIV-1-Vpr likewise did not affect MDDC phenotype, however it was found to be influential in HIV-1 induced decreases in MDDC antigen presentation to autologous T-cells. These accumulated results indicate that HIV-1 subverts DC maturation and function through whole virus effects and individual accessory peptide influences.
Understanding the mechanisms of DC dysfunction in HIV infection may provide some insight into infection prevention strategies and therapies leading to adaptive immune system activation and viral clearance.
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Pursell, Natalie W. "Hsp90-Mediated Maturation of Kinases and Nuclear Steroid Hormone Receptors: A Dissertation". eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/535.
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Among heat shock proteins, Hsp90 is unusual because it is not required for the proper folding of most cellular proteins but rather is disproportionally linked to the activation of signal transduction proteins including over forty kinases and many steroid hormone receptors. Mutated forms of many Hsp90 clients are causative agents in cancer, making Hsp90 a promising pharmacological target. Many small molecular inhibitors have been identified that competitively bind to the ATP binding site of Hsp90, some of which are in clinical trials as anticancer agents. Although the activation of kinase and hormone receptor clients by Hsp90 and its co-chaperones has been extensively studied, the molecular mechanism of client protein activation is poorly understood.
Hsp90 is a dimeric chaperone containing three domains: the N-terminal (N) and middle (M) domains contribute directly to ATP binding and hydrolysis and the C-terminal (C) domain mediates dimerization. At physiological concentration, Hsp90 predominantly forms dimers, but the possibility that full-length monomers might also function in cells has not been tested. In Chapter 3, we used a single-chain strategy to design a full-length Hsp90 monomer (NMCC). The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. NMCC Hsp90 was also defective at ATP hydrolysis and the activation of kinase and steroid hormone receptor clients in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric.
After finding that a full-length Hsp90 monomer containing only one ATPase site was unable to support yeast viability or activate Hsp90 clients, we set out to further explore the role of ATPase activity in client protein activation. Approximately 10 % of the yeast proteome binds to Hsp90 making it important to study Hsp90 function in the cellular environment where all binding partners are present. In Chapter 4, we observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered super-stabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. We utilized these super-stabilized dimers to analyze the ability of ATPase mutant homodimers to activate known Hsp90 client proteins in yeast cells. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of the glucocorticoid hormone receptor (GR) and v-src confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo.
In addition to its role in the activation of signal transduction client proteins, Hsp90 has been shown to suppress the in vitro aggregation of numerous hard-to-fold proteins. In Chapter 5, we examine the role of charge in Hsp90 anti-aggregation activity. The charge on Hsp90 is largely concentrated in two highly acidic regions. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. Addition of an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct rescued the anti-aggregation activity of Hsp90 indicating that the net charge contributes to its anti-aggregation activity.
The in vitro anti-aggregation activity of Hsp90 studied in Chapter 5 occurs in the absence of ATP. However, all of the biologically important functions of Hsp90 in cells identified to date, including the maturation of kinases and nuclear steroid hormone receptors, clearly require ATP hydrolysis. Why does Hsp90 robustly hinder the aggregation of hard-to-fold proteins without ATP in vitro, but in vivo uses ATP hydrolysis for all of its essential functions? By utilizing separation of function Hsp90 variants (that specifically lack in vitro anti-aggregation activity) we have begun to address this question. We find that anti-aggregation deficient Hsp90 is unable to support yeast growth under stressful conditions, potentially due to reduced cellular expression. Interestingly, the ATP-independent anti-aggregation activity of Hsp90 has no measureable impact on cellular function. Thus, hindering the aggregation of most hard-to- fold proteins by Hsp90 (independent of ATP hydrolysis) does not appear to be important for cell function. These results suggest a cellular model where the Hsp40/60/70 machinery is responsible for hindering the aggregation of most hard-to-fold proteins while Hsp90 assists in the maturation of a select set of clients in an ATP-dependent fashion, potentially aided by its inherent anti-aggregation properties.
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Chen, Hui-Chen. "Role for cyclic adenosine monophosphate (cAMP) response element binding proteins in B lymphocyte development and functional maturation". Connect to this title online, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1061213266.
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Thesis (Ph. D.)--Ohio State University, 2003.Document formatted into pages. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2005 Aug. 19.
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Innarella, Maria Rosaria [Verfasser]. "Investigation of the effect of human Argonaute proteins on the maturation of short haipin RNAs / Maria Rosaria Innarella". Lübeck : Zentrale Hochschulbibliothek Lübeck, 2015. http://d-nb.info/106640139X/34.
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Braganza, Annabel M. H. (Mary Helen). "Characterization of nascent enamel proteins translated in vitro from mRNA specific for the secretory and maturation stages of amelogenesis". Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=22851.
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Enamel proteins are extracellular matrix proteins expressed throughout enamel formation. However, questions concerning their numbers and origins are still somewhat ambiguous. To characterize the nascent enamel proteins, cell free translation and immunoprecipitation procedures were performed using poly(A)$ sp{+}$RNA isolated from freeze-dried segments of rat incisor enamel organs at the secretory, early maturation, and mid maturation stages of amelogenesis. Phosphoimaging revealed that the enamel organ produces enamel proteins continuously throughout enamel development albeit in decreasing number and intensity. Ten enamel proteins were translated at the secretory stage and ranged in molecular weight from 80 to 18 kDa, resembling the number (Simmer et al., 1994) and the size (DenBesten et al., 1992) of RNA transcripts recently described for murine and rat. At the early and mid maturation stages, the enamel proteins span a 27 to 68 kDa region. This selected expression of enamel proteins at each stage of development suggests that specific proteins may be important for the maturation process.
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Cannone, Giuseppe. "Structural investigation of the archaeal replicative machinery by electron microscopy and digital image processing". Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/17070.
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Previous studies suggest a degree of homology between eukaryotic replication, transcription and translation proteins and archaeal ones. Hence, Archaea are considered a simplified model for understanding the complex molecular machinery involved in eukaryotic DNA metabolism. DNA replication in eukaryotic cells is widely studied. In recent years, DNA replication studies expanded on the archaeal DNA replication machinery. P. abyssi was the first archaeon whose genome was fully sequenced. Genome sequencing and comparative genomics have highlighted an MCM-like protein in P. abyssi. In this study, I report the biochemical and structural characterisation of PabMCM. PabMCM is explored as model for understanding more complex eukaryotic MCM proteins and unravelling the biochemical mechanism by which MCM proteins release their helicase activity. The crenarchaeon Sulfolobus solfataricus possesses a simplified toolset for DNA replication compared to Eukaryotes. In particular, S. solfataricus has a subset of the eukaryotic Okazaki fragment maturation factors, among which there are a heterotrimeric DNA sliding clamp, (the proliferating cell nuclear antigen, PCNA), the DNA polymerase B1 (PolB1), the flap endonuclease (Fen1) and the ATP-dependent DNA ligase I (LigI). PCNA functions as a scaffold with each subunit having a specific binding affinity for each of the factors involved in Okazaki fragment maturation. Here, the 3D reconstruction of PCNA in complex with the Okazaki fragment maturation proteins PolB1, LigI and Fen1 is reported.
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Bohassan, Maruah Hejey. "Role of GPR17 in Thrombocyte Aggregation in Adult Zebrafish". Thesis, University of North Texas, 2015. https://digital.library.unt.edu/ark:/67531/metadc822797/.
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GPR17, a uracil nucleotide cysteinyl leukotriene receptor, belongs to the GPCR (G protein coupled receptor) family. It has been shown recently that inhibiting this protein in the nervous system in mice can lead to blockage of oligodendrocyte maturation, which supports myelin repair. Interestingly, our laboratory found GPR17 in thrombocytes. However, we do not know whether it has any function in thrombocyte aggregation or the nature of the ligand. In this paper, we studied the role of GPR17 in hemostasis, which is a fundamental defense mechanism in the event of injury. Using zebrafish as a model system, our laboratory has studied specifically thrombocytes, which play a significant role in hemostasis. The major reasons to use zebrafish as a model system are that their thrombocytes are functionally equivalent to human platelets, the adult fish are amenable to knockdown experiments, and they are readily available in the market. This study was performed by using a piggy back knockdown method where we used a chemical hybrid of control morpholino and an antisense oligonucleotide sequence leads to the degradation the mRNA for GPR17. After knockdown GPR17 in thrombocytes, the percent difference of the thrombocytes aggregation between the control and knockdown blood samples was measured by flow cytometry. We used various thrombocyte agonists to study differences in aggregation between the control and knockdown blood samples. The study showed that knockdown of GPR17 resulted in no significant differences in percent thrombocyte aggregation between control and agonist treated samples except for a slight increase in collagen-treated samples. Thus, it appears that GPR17 has no significant role in hemostasis.
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Fehlker, Marion. "Studien zur Funktion des Proteasomen-assoziierten Proteins Blm3 in Saccharomyces cerevisiae". Doctoral thesis, [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972596860.
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Xia, Wei y 夏炜. "The tango between two proteins: insight into the nickel delivery process exerted by HypA and HypB during [Ni, Fe]-hydrogenase maturation in helicobacter pylori". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46188666.
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Meissner, Christina Sylvia [Verfasser], Elke [Akademischer Betreuer] Bogner, Martin [Akademischer Betreuer] Messerle y Thorsten [Akademischer Betreuer] Wolff. "Influence of HCMV proteins pUL71 and pUL77 on viral maturation / Christina Sylvia Meissner. Gutachter: Elke Bogner ; Martin Messerle ; Thorsten Wolff". Berlin : Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://d-nb.info/1017797013/34.
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Meissner, Christina Sylvia [Verfasser], Elke [Gutachter] Bogner, Martin [Gutachter] Messerle y Thorsten [Gutachter] Wolff. "Influence of HCMV proteins pUL71 and pUL77 on viral maturation / Christina Sylvia Meissner ; Gutachter: Elke Bogner, Martin Messerle, Thorsten Wolff". Berlin : Humboldt-Universität zu Berlin, 2011. http://d-nb.info/120807671X/34.
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Pinker, Franziska. "Structural characterization of proteinaceous RNase P from Arabidopsis thaliana". Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ093/document.
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La maturation des ARNt en 5' est réalisée par RNase P. C'est un ribozyme chez les bactéries, les fungi et les nuclei des mammifères et un enzyme protéique dans les plantes ou des organelles des mammifères qui s’appelle PRORP. Il y a trois PRORP dans A. thaliana. PRORP contiennent deux domaines : un domaine PPR qui reconnaît spécifiquement des séquences d'ARN et un domaine nucléase qui assure la coupure endonucléolytique 5' des précurseurs d’ARNt. Pendant ma thèse j'ai pu montré par des méthodes biophysiques et structurales comme SRCD et SAXS que PRORP1 et 2 sont composées en majorité des hélices alpha Elles ont un rayon de giration de 33 Å et contiennent deux domaines distincts avec et une dimension maximale de 110 Å. Pour le complex entre un substrat d'ARNt et PRORP une constante de dissociation de 1 uM a pu être confirmé par la microcalorimétrie, la thermophorèse et l'ultracentrifugation analytique. Ces analyses nous ont permis de construire un modèle PRORP et un substrat d'ARNtRNase P cleaves 5’ leaders of precursor tRNAs. RNase P is a ribozyme in bacteria, fungi and animal nuclei and a protein in animal organelles, plants and many other organism. There are three PRORPs in A. thaliana. MALS, SRCD and SAXS provided first structural information: 1) PRORPs are monomers in solution. 2) PRORP 1-2 have a high alpha-helical content. 3) PRORPs are composed of two distinct domains with a radius of gyration of 33 A. These results together with homology modelling enabled us to build a first model of PRORPs in complex with tRNA. Using three different methods, isothermal titration calorimetry, microscale thermophoresis and analytical ultracentrifugation, a binding constant of about 1 µM could be determined for the system PRORP2mDD and L5T0 tRNA. This helped us conducting a SAXS experiment taking into account the low resolution affinity and designed to provide the direct structural data of a complex of proteinaceous RNase P with a substrate tRNA
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Nabhan, Myriam. "Agrégation et immunisation contre les protéines thérapeutiques : étude de la maturation des cellules dendritiques et de la réponse lymphocytaire". Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS576.
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L’immunogénicité des biothérapies constitue une limitation majeure au traitement des patients atteints de maladies chroniques et se traduit par la production d’anticorps dirigés contre le biomédicament (anti-drug antibodies, ADA). La détection d’ADA de haute affinité et d’isotypes divers chez les patients suggère la mise en place d’une réponse immunitaire adaptative classique orchestrée par les cellules dendritiques. Par ailleurs, la présence d’agrégats protéiques dans les spécialités administrées serait un des facteurs favorisant l’immunogénicité, ces agrégats pouvant jouer le rôle de signal de danger.L’objectif de notre travail était de mieux comprendre les interactions des agrégats de protéines avec les cellules dendritiques et les lymphocytes T aboutissant au déclenchement d’une réponse immunitaire adaptative spécifique nécessaire à la production d’ADA.Dans un premier temps, nous avons montré que des agrégats d’infliximab, anticorps monoclonal anti-TNFa;, induisaient la maturation de cellules dendritiques humaines dérivées de monocytes (moDC). Celle-ci se traduit par l’augmentation de l’expression de marqueurs membranaires d’activation et de costimulation et la sécrétion de cytokines et chimiokines pro-inflammatoires. Nous avons également montré que ces modifications phénotypiques induites par les agrégats favorisaient la prolifération de lymphocytes T CD4+ et la production de cytokines. Par la suite, nous avons décrit les mécanismes cellulaires précoces impliqués dans l’activation de ces cellules en montrant que la neutralisation du récepteur FcgRIIa et de la tyrosine kinase Syk inhibait la maturation des moDC ainsi que l’activation des lymphocytes T CD4+.Par ailleurs, nous avons évalué le rôle des agrégats d’infliximab dans la génération de néo-épitopes, en mettant en évidence l’existence d’un répertoire de lymphocytes T CD4+ naïfs reconnaissant spécifiquement les agrégats d’infliximab, chez le sujet sain. Ainsi, grâce à des préparations d’agrégats bien caractérisées et d’un modèle de co-cultures autologues de lymphocytes T CD4+ naïfs et de moDC chargées avec les agrégats, nous avons montré que la fréquence de LT CD4+ naïfs spécifiques des agrégats d’infliximab est plus importante que celle retrouvée pour l’anticorps natif. Ces résultats suggèrent une présentation accrue d'épitopes et de néo-épitopes dérivant des agrégats d'infliximab.In fine, ce travail contribue à une meilleure compréhension des conséquences biologiques de l’agrégation des protéines à visée thérapeutique sur le déclenchement de la réponse immunitaire adaptative spécifique en mettant l’accent sur les rôles adjuvant et antigénique des agrégats protéiquesImmunogenicity of biotherapeutic proteins is a major drawback in the treatment of patients with chronic diseases characterized by the production of anti-drug antibodies (ADA). The detection of ADA with high affinity and of various isotypes suggests a CD4 T cell-dependent adaptive immune response with a pivotal role for dendritic cells. Among other factors, the presence of protein aggregates in the administered products can promote immunogenicity, as aggregates seem to act as danger signals.The aim of our work is to better understand the interactions of protein aggregates with dendritic cells and T cells, leading to the establishment of a specific adaptive immune response needed for the production of ADA.We first showed that aggregation of infliximab, a monoclonal anti-TNFa; antibody, induced the maturation of human monocyte-derived dendritic cells (moDC) via an increase in the expression of activation and costimulatory surface markers and the secretion of pro-inflammatory cytokines and chemokines. Moreover, we showed that these phenotypic changes induced by aggregates promote CD4 T-cell proliferation and cytokine production. Subsequently, we described the early events involved in moDC and T-cell response by showing that the neutralization of the FcgRIIa receptor and the tyrosine kinase Syk inhibited moDC maturation and CD4 T-cell activation.Furthermore, we evaluated the involvement of infliximab aggregates in the generation of neo-epitopes by identifying a naïve CD4 T-cell repertoire recognizing infliximab aggregates in healthy subjects. By testing well characterized aggregate preparations and using an autologous co-culture model of naïve CD4 T cells and moDC loaded with aggregates, we showed that the frequency of naïve CD4 T cells specific for infliximab aggregates was higher than the one found for the native antibody. These results suggested an increased presentation of epitopes and neo-epitopes derived from infliximab aggregates.In resume, our work contributes to a better understanding of the biological consequences of therapeutic protein aggregation on the onset of the specific adaptive immune response by focusing on the adjuvant and antigenic role of protein aggregates
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Costa, Mariana Fernandes Alves. "Molecular remodelling of the spindle architecture during metaphase arrest in oocytes". Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31255.
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Oocytes of most species assemble and maintain a functional bipolar spindle in the absence of centrosomes. Strikingly, after bipolar spindle formation, oocytes arrest in metaphase for several hours before fertilisation. How the dynamic spindle maintains its bipolarity during this long arrest is poorly understood. I hypothesise that the bipolar spindle is stably maintained by changes in the distribution of microtubule-associated proteins (MAPs) on the spindle during the long oocyte arrest. To test this, I generated transgenic flies expressing GFP-tagged microtubule-associated proteins (MAPs), and found that 13 out of 24 proteins change localisation between early and late oocytes. I refer to these changes in MAP localisation after establishment of bipolarity as 'spindle maturation'. In order to identify the molecular mechanisms triggering MAP relocalisation, I manipulated the kinase activity of the cell cycle regulator Cdk1 by over-expressing non-degradable cyclin A or B, the major activators of Cdk1. Their expression prevented re-localisation of distinct sets of MAPs, and disrupted spindle bipolarity and accurate chromosome segregation in oocytes. Kinesin-6 Pavarotti/MKlp1 localised strongly to the spindle equator in late oocytes, whilst nearly always absent from this region in early oocytes. The localisation of Pavarotti to the spindle equator in late oocytes was reduced when cyclin B is over-expressed in oocytes, suggesting a role for Cdk1/cyclin B complex in regulating Pavarotti localisation. Indeed, a Pavarotti/Mklp1 mutant non-phosphorylatable by Cdk1 prematurely localised to the meiotic spindle and disrupted spindle bipolarity. Moreover, removal of Pavarotti from the metaphase-I spindle by RNAi induced spindle defects in oocytes. Therefore, it is likely that the microtubule cross-linking activity of Pavarotti enhances the stability of the metaphase-I spindle during the long arrest. Consistent with this, I found that the microtubule density in the spindle equator is higher in late oocytes. Altogether, I propose that remodelling the molecular architecture of the spindle during the long oocyte arrest is important to stabilise the bipolar spindle without centrosomes.
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Pagnier, Adrien. "Maturation de sites métalliques de protéines par les machineries d'assemblage des centres fer-soufre ISC et Hyd". Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV016.
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De nombreuses protéines possèdent des cofacteurs inorganiques contenant des métaux de transition. Les propriétés physico-chimiques de ces métaux permettent aux enzymes qui les portent de catalyser des réactions impossibles par l'utilisation des seules potentialités chimiques des vingt-deux acides aminés. Cependant, ces métaux sont toxiques pour la cellule lorsqu'ils sont libres. La synthèse et l'incorportion de ces cofacteurs dans les enzymes nécessitent alors des machineries protéiques complexes d'assemblage. Au cours de cette thèse, les mécanismes de synthèse des centres FeS par les machineries ISC (Iron-Sulfur Cluster) et Hyd (Hydrogenase) ont été étudiés. Le système ISC correspond à la machinerie primaire d'assemblage des centres FeS chez les bactéries, et un système équivalent existe chez les eucaryotes au niveau de la mitochondrie. Le système Hyd est la machinerie de maturation de l'hydrogénase à FeFe chez plusieurs eucaryotes inférieurs (algues et protistes) et dans une grande variété de bactéries. Dans un premier temps, nous nous sommes intéressés à la machinerie ISC d'Archaeoglobus fulgidus dont le coeur est composé de la cystéine désulfurase IscS et de la protéine échafaudage IscU ; IscS apportant le soufre nécessaire à l'assemblage du centre FeS sur IscU. Au cours de cette étude, il est apparu que IscS d'Archaeoglobus fulgidus ne possède pas d'activité cystéine désulfurase, mais qu'elle joue tout de même un rôle fondamental dans la synthèse du centre FeS sur le complexe IscSU en fournissant sa cystéine active en tant que ligand de l'agrégat. Dans un second temps, nous avons étudié la protéine à radical S-adénosyl-L-méthionine HydG, responsable de la synthèse des ligands CN- et CO du sous-agrégat à 2 Fe des hydrogénases à FeFe, qui était la seule maturase du système Hyd dont la structure n'était pas connue. Nos résultats structuraux et fonctionnels suggèrent que HydG synthétise successivement le ligand CN- dans un site actif basique, puis le ligand CO sur le cinquième Fe de son agrégat [5Fe-4S] C-terminal. Ce dernier pourrait être stabilisé par un ligand cystéine ou homocystéineMany proteins have inorganic cofactors containing transition metals. The physicochemical properties of these metals allow the enzymes, which carry them to catalyze reactions not possible when only using the chemical properties of the twenty-two amino acids. However, these metals are toxic to the cell when they are free. Consequently, the synthesis and incorporation of these cofactors into enzymes requires complex protein assembles. In this thesis, the FeS clusters synthesis mechanisms by the ISC (Iron-Sulfur Cluster) and Hyd (Hydrogenase) machineries were studied. The ISC system corresponds to the primary FeS clusters assembly machinery in bacteria, and a homologous system exists in mitochondria. The Hyd system is FeFe-hydrogenase active site maturation machinery found in several lower eukaryotes (algae and protists) and in a wide variety of bacteria. Initially, we studied the ISC machinery from Archaeoglobus fulgidus whose core is composed of the cysteine desulfurase IscS and the scaffold protein IscU; IscS delivers the sulfur needed for the FeS assembly to IscU. From this study we conclude that IscS from Archaeoglobus fulgidus has no cysteine desulfurase activity, but it still plays a fundamental role in FeS cluster synthesis by IscSU complex by providing a cysteine ligand to the nascent cluster. Secondly, we studied the radical S-adenosyl-L-methionine HydG, responsible for the synthesis of CN- and CO ligand of the active site [FeFe] subcluster, which was the only Hyd system maturase for which the structure was unknown. Our structural and functional results suggest that HydG successively synthesizes the CN- ligand at a basic site, and then the CO ligand at the unique fifth Fe ion of its C-terminal [5Fe-4S] cluster. The latter could be stabilized by either a cysteine or a homocysteine ligand
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Cruz, Larissa Prado da. "Caracterização do proteoma de entrenós maduros e imaturos de cana-de-açúcar durante o estádio de maturação". Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-05112012-092946/.
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Grandes esforços dos programas de melhoramento genético da cana-de-açúcar são dedicados ao desenvolvimento de novas variedades com maior rendimento e conteúdo de sacarose. As etapas do metabolismo da sacarose têm sido foco de pesquisas há muito tempo, porém apenas recentemente têm-se buscado compreender os processos de transporte e acúmulo da sacarose no colmo da cana-de-açúcar. O objetivo deste trabalho é a caracterização do proteoma de entrenós maduros (entrenó 9) e imaturos (entrenó 5) de dois genótipos de cana-de-açúcar (Co 740 e SP 80-3280), ambos com 12 meses de idade, visando à identificação de proteínas relacionadas aos processos de transporte e acúmulo de sacarose. A altura, diâmetro do colmo, área foliar, umidade dos entrenós, trocas gasosas, potencial hídrico da planta e índice de maturação foram obtidos no momento da coleta do material vegetal. A quantificação dos açúcares glicose, frutose e sacarose foi realizada por cromatografia líquida e não mostrou a existência de diferenças no acúmulo desses carboidratos entre as duas variedades. Porém observamos diferenças entre os entrenós maduros e imaturos, indicando que o entrenó considerado imaturo encontra-se em estádio de pleno acúmulo de sacarose. O perfil de expressão proteica, gerado por 2D-PAGE, mostrou 87 e 85 proteínas diferencialmente expressas nos entrenós 5 e 9, respectivamente. Nos entrenós imaturos 12 proteínas foram identificadas como preferencialmente expressas em cada variedade e nos entrenós maduros 11 e 16 proteínas foram identificadas como preferencialmente expressas em Co 740 e SP 80-3280, respectivamente. As proteínas totais dos dois entrenós das duas variedades foram separadas e identificadas via UPLC acoplado a espectrômetro de massas LC-MS/MS com auxílio da base de dados do SUCEST. A caracterização do proteoma dos entrenós foi complementada pela avaliação da localização celular, função molecular e processo biológico a que pertencem todas as proteínas identificadas. Ao todo, foram identificadas 1.493 proteínas localizadas em 19 componentes celulares, com 20 funções metabólicas diferentes, atuantes em 24 processos biológicos e integrantes de 84 vias metabólicas. Deste total, 71 foram classificadas como participantes do metabolismo de carboidratos e encontram-se distribuídas em 21 vias metabólicas, segundo a base de dados KEGG.Great efforts have been put into sugarcane breeding programs in order to develop new varieties with increased sucrose yield and content. The steps of sucrose metabolism have been focus of research for a long time, but only recently have researchers sought to understand the processes of transport and accumulation of sucrose in sugarcane stem. The objective of this study is characterize the proteome of mature (internode 9) and immature (internode 5) internodes in two genotypes of sugarcane (Co 740 and SP 80-3280), both 12 months old, focusing on the identification of proteins related to the processes of transport and accumulation of sucrose. The height, stem diameter, leaf area, internode moisture, gas exchange, plant water potential and maturation index were all recorded at the time the plant material was collected. The quantification of sugars glucose, fructose and sucrose was performed by liquid chromatography and showed no difference in carbohydrates accumulation between the two varieties. Nevertheless, we observed differences between the mature and immature internodes, indicating that the internode considered immature was actively accumulating sucrose. The protein profile, produced by 2D-PAGE, showed 87 and 85 differentially expressed proteins in internodes 5 and 9, respectively. In immature internodes 12 proteins were identified as being preferentially expressed in each of the varieties and in the mature internodes 11 and 16 proteins were identified as preferentially expressed in Co 740 and SP 80- 3280, respectively. Total proteins of the two internodes of two varieties were separated and identified by UPLC coupled to a mass spectrometer LC-MS/MS using the SUCEST database. The characterization of the internodes proteome was further complemented by evaluating cellular localization, function and molecular biological processes to which all identified proteins belong. Altogether, 1,493 proteins were identified, located in 19 cellular components, with 20 different metabolic functions, operating in 24 biological processes and involving 84 metabolic pathways. Of this total, 71 have been classified as being involved in carbohydrate metabolism and are distributed in 21 metabolic pathways, according to the KEGG database.
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Valário, Bárbara Panoff [UNESP]. "Estudo da tolerância à dessecação e longevidade em sementes de soja (Glycine max (L.) MERR.)". Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/144345.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Não existe consenso sobre qual o estádio de desenvolvimento que a tolerância à dessecação e longevidade são adquiridos em sementes de soja e quais proteínas estão associadas com estes eventos. Portanto, o presente trabalho teve o objetivo caracterizar a aquisição da tolerância à dessecação e longevidade em sementes de soja e identificar proteínas associadas. O estudo foi realizado na Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Faculdade de Ciências Agronômicas-UNESP-Botucatu em parceria com o Centro Virtual de Toxicologia (CEVAP), Campus de Botucatu-SP e com o Laboratório de Sementes Florestais da Universidade Federal de Lavras (UFLA). As sementes foram produzidas na safra 2013/2014 seguida da coleta e caracterização morfofisiológica (caracterização visual, germinação e teor de água) das sementes nos estádios reprodutivos R5.1, R5.2, R5.3, R5.4, R5.5, R6, R7.1, R7.2, R7.3, R8.1, R8.2, R8.3 e R9. Posteriormente, realizou-se a determinação do teor de água, matéria seca e fresca das sementes. Em seguida, as sementes foram secas e armazenadas de 5 a 85 dias à 35°C e 75% umidade relativa (UR), para caracterizar a aquisição de longevidade. Para tolerância à dessecação, as sementes foram secas em gerbox contendo carbonato de potássio à 42% de umidade e 35°C até 0.10g água por grama de massa seca. Para se conhecer o perfil proteômico, foram extraídas proteínas de cada estádio de desenvolvimento, separadas em fitas de pI e géis de acrilamida e analisados no ImageMaster Platinum 7.0. Os spots significativos e exclusivos de cada estádio foram recortados e analisados por espectrometria de massas ESI-QToF. Foram sequenciadas e identificadas 167 proteínas, sendo que trinta e cinco tiveram a expressão diferencial ao longo da fase tardia da maturação. A tolerância à dessecação foi adquirida no estádio R7.2, porém a longevidade foi adquirida em estádios fenológicos posteriores. As proteínas LEAs MAT1, SBP65 e MP2 estão relacionadas com tolerância à dessecação e as pertencentes ao grupo 3 (51 kDa, SBP65 e MP2) juntamente com a MAT9 e algumas LEAs do grupo 5 (Small hydrophilic plant seed protein) estão relacionadas com aquisição de longevidade.
There is no consensus regarding when desiccation tolerance and longevity are acquired in soybean seeds. Therefore, this study aimed at to characterize the acquisition of desiccation tolerance and longevity in soybean seeds and identify proteins associated. The study was performed at the Universidade Estadual Paulista Julio de Mesquita Filho (UNESP), Colegue of Agricultual Science-UNESP-Botucatu in collaboration with the Virtual Center for Toxicology (CEVAP), Botucatu-SP and with the Forest Seeds Laboratory at the Federal University of Lavras (UFLA). Seed production was carried out in the crop year 2013/2014 followed by the collection and characterization of seeds at the reproductive stages R5.1, R5.2, R5.3, R5.4, R5.5, R6, R7.1, R7.2, R7.3, R8.1, R8.2, R8.3 and R9. Subsequently, it was performed the determination of water content, dry and fresh weight of the seeds. Then, seeds were dried and stored at 35 °C and 75% relative humidity (RH), to characterize the acquisition of longevity. To know the proteomic profile, proteins were extracted from each stage of development, separated in acrylamide gels and analyzed by ImageMaster Platinum 7.0. The significant spot and unique to each stadium were cut and analyzed by mass spectrometry ESI-QTOF mass. They were sequenced and identified 167 proteins, and thirty-five had the differential expression along the late stage of maturation. The desiccation tolerance was acquired at the stage R7.2 but longevity was acquired at later during seed development. Proteins LEAs MAT1, SBP65 and MP2 are related to desiccation tolerance and those belonging to group 3 (51 kDa, SBP65 and MP2) together with MAT9 and some LEAs group 5 (Small hydrophilic plant seed protein) are related to acquisition of longevity.
FAPESP: 2014/14638-0
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Ghamrawi, Sarah. "Modifications de la paroi au cours de la maturation et de la germination des conidies de Scedosporium boydii". Thesis, Angers, 2014. http://www.theses.fr/2014ANGE0031/document.
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Les espèces du complexe Scedosporium apiospermum sont des agents pathogènes émergents qui se situent au deuxième rang parmi les champignons filamenteux rencontrés au cours de la mucoviscidose. Ils sont omniprésents et particulièrement rencontrés dans les zones polluées. En dépit de leur importance clinique, nos connaissances sur leur biologie moléculaire et leur physiologie restent limitées. Chez les champignons, la paroi constitue un bouclier protecteur face à des conditions environnementales défavorables, et joue un rôle essentiel dans la pathogénicité. Ici, nous avons étudié les changements dynamiques de la paroi des conidies de S. boydii, l’une des deux espèces majeures de ce complexe avec S. apiospermum, avec pour objectif d'identifier des facteurs de virulence potentiels. En utilisant une large variété de techniques, allant de la microscopie électronique à balayage ou à transmission à l’analyse protéomique des protéines à ancre glycosylphosphatidylinositol (GPI) en passant par la microélectrophorèse et la partition de phase, la cytométrie en flux, la microscopie de force atomique, la résonance paramagnétique électronique, ou encore des techniques moléculaires, nous avons mis en évidence diverses modifications qui se produisent dans la paroi pendant la maturation et la germination des conidies de S. boydii et nous avons identifié la DHN-mélanine ainsi qu'un nombre important de protéines à ancre GPI. Enfin, nous avons fourni la première séquence complète du génome de S. apiospermum qui appuierait les différents domaines de la recherche sur ces champignons que ce soit pour l’étude des mécanismes pathogènes ou pour des applications biotechnologiquesSpecies of the Scedosporium apiospermum complex are emerging human pathogens which rank the second, after Aspergillus fumigatus, among the filamentous fungi colonizing the airways of patients with cystic fibrosis. These fungi are ubiquitous in nature and particularly encountered in polluted areas. Despite their clinical relevance, our knowledge about their molecular biology and physiology remains rather limited. In fungi, the cell wall forms a protective shield against adverse environmental conditions, and therefore plays a key role in pathogenesis, which makes it an interesting target for antifungal drug development. Here, in an attempt to identify potential virulence factors, we investigated the dynamic changes of the cell wall of conidia in S. boydii, one of the main pathogenic species within this species complex with Scedosporium apiospermum. Using various techniques, ranging from scanning and transmission electron microscopy to proteomic analysis of glycosylphosphatidylinositol (GPI)- anchored proteins, through two-phase partitioning and microelectrophoresis, atomic force microscopy and chemical force spectroscopy, flow 5 cytometry, electron paramagnetic resonance and molecular techniques, we highlighted various modifications occurring in the cell wall during maturation and germination of S. boydii and we identified DHN-melanin as well as a substantial number of GPI-anchored proteins in the cell wall. Finally, we provided the first publicly available genome sequence of S. apiospermum that would support various research fields on these fungi whether for understanding their pathogenic mechanisms or for various biotechnological applications
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Snow, Alan J. "Phosphorylation-dependent interaction of tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein (14-3-3) with PADI6 following oocyte maturation in mice". [Kent, Ohio] : Kent State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=kent1208796428.
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Thesis (Ph.D.)--Kent State University, 2008.Title from PDF t.p. (viewed May 26, 2009). Advisor: Douglas W. Kline. Keywords: oocyte, egg, ovum, PADI6, PAD6, 14-3-3, peptidylarginine deiminase, phosphorylation, mouse, oocyte maturation, gamete biology. Includes bibliographical references (p. 91-97).
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Lebrette, Hugo. "Etude structurale de l'import de nickel par les protéines extracytoplasmiques de systèmes ABC chez les bactéries : le nickel voyage-t-il seul ou accompagné ?" Thesis, Grenoble, 2013. http://www.theses.fr/2013GRENV086.
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Chez les procaryotes, les systèmes ABC (ATP-binding cassette) canoniques permettent un import efficace et de haute affinité du nickel, en grande partie via l'action de la Ni-BP (nickel-binding protein) qui va jouer le rôle de récepteur extracytoplasmique du nickel avant son passage à travers la membrane interne. Dans ces travaux, nous avons cherché à mieux caractériser les stratégies d'import de nickel par les bactéries, notamment par l'étude cristallographique des Ni-BP. La résolution des structures de cinq de ces protéines, en interaction avec du nickel, nous a permis d'identifier les sites de fixation et ainsi de mettre en évidence les différents modes d'interaction de ce métal avec les Ni-BP. Nos résultats montrent notamment que la coordination du nickel requiert toujours la présence d'un métallophore exogène, appelé nickelophore. En parallèle, des études in vivo ont été conduites sur Escherichia coli, montrant que cette bactérie semble être capable de synthétiser son propre nickelophore. D'autre part, ces travaux ont permis la résolution de la structure de HypB de Helicobacter pylori en interaction avec du nickel. Celle-ci permet de mieux appréhender le rôle central de cette protéine au sein des voies de maturation des enzymes à nickelIn prokaryotes, canonical ABC (ATP-binding cassette) importers allow an efficient uptake of nickel with high affinity through the inner membrane, largely via the action of the extracytoplasmic nickel-binding protein (Ni-BP). In this work, we intended to better understand the strategies developed by bacteria to scavenge nickel in the environment, especially through the structural studies of several Ni-BP. The resolution of the crystal structures of five Ni-BPs from diverse bacteria, in interaction with nickel, led us to identify their nickel-binding sites and shed light on the different binding modes. Our results show that the presence of an exogenous metallophore, called nickelophore, is always required. In vivo studies in Escherichia coli were also conducted, showing that this bacteria seems to be able to synthesize its own nickelophore. In addition, we have solved the crystal structure of Helicobacter pylori HypB in complex with nickel. This result provides insight into its cellular function in nickel enzyme maturation pathways
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Lachance, Véronik. "L’association du récepteur β2-Adrénergique (β2AR) avec les protéines RGGT et HACE1 module son trafic intracellulaire en régulant les mécanismes de maturation et d’activation de la protéine Rab11a". Thèse, Université de Sherbrooke, 2014. http://savoirs.usherbrooke.ca/handle/11143/160.
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Résumé : L’expression de surface des récepteurs couplés aux protéines G (GPCRs) est un processus hautement régulé et très important dans le maintien de l’homéostasie cellulaire. En effet, un déséquilibre dans leur niveau d’expression est souvent relié à différentes pathologies comme le cancer, le diabète, l’obésité, les maladies cardiovasculaires et les maladies neurodégénératives. C’est pourquoi la compréhension des mécanismes moléculaires influençant ce phénomène est si importante et nous permettra d’élaborer et/ou d’améliorer les médicaments ciblant la régulation de ce processus.
Il est bien connu qu’un des acteurs importants dans le trafic vésiculaire des GPCRs est représenté par la famille des Rab GTPases. Effectivement plusieurs de ces dernières, soit les Rabs 1, 2, 4, 5, 6, 7, 8 et 11 pour ne nommer que les plus connues, modulent l’expression de surface des GPCRs. De plus, certaines études soulèvent la possibilité qu’un GPCR soit lui-même capable de réguler son propre trafic intracellulaire, et ce grâce à son interaction avec les Rab GTPases. Toutefois, le mécanisme emprunté par le GPCR pour atteindre cette fin reste à élucider.
Dans le présent travail, je démontre que le GPCR, β2AR, module non seulement la maturation de la petite protéine G Rab11a grâce à son interaction avec la Rab GéranylGéranylTransférase (RGGT), mais influence également son activation en modulant son ubiquitination via son association avec la E3-ubiquitine ligase, HACE1. De plus, je révèle que la sous-unité alpha de la RGGT (RGGTA) accroît significativement la maturation et le transport antérograde du récepteur β2AR, ce qui souligne ainsi un nouveau rôle cellulaire pour cette protéine. L’ensemble des résultats générés appuie l’hypothèse qu’un GPCR puisse contrôler son propre routage intracellulaire, et éclaircit les mécanismes utilisés pour réguler l’activé de la Rab GTPase avec laquelle il interagit.
// Abstract : Cell surface expression of G Protein-Coupled Receptors (GPCRs) is a highly regulated and very important phenomenon for keeping cellular homeostasis. In fact, dysregulation of their cell expression is related to many diseases like cancer, neurological disorders, obesity, diabetes and cardiovascular diseases. These facts illustrate how important understanding the molecular mechanisms involved in cell surface transport of those receptors is, which will help us in designing or improving drugs which actually target this pathway.
Rab GTPases are proteins known for being essential regulators of GPCR vesicular trafficking. Indeed, an increasing number of studies report the implication of Rab1, 2, 4, 5, 6, 7, 8 and 11 (to cite the most frequently studied) cell surface transport of GPCRs. Moreover, some studies also put forward the possibility that a GPCR might be able to regulate its own cellular trafficking by interacting and controlling activation of Rab GTPases. However, the mechanism involved in this process remains to be clarified.
In the present study, I demonstrate that the prototypic GPCR, β2AR, not only modulates prenylation/maturation of the small G protein Rab11a by interacting with Rab GeranylGeranylTransferase (RGGT), but also influences Rab11a activation by modulating its ubiquitination via its association with the E3-ubiquitin ligase, HACE1. Furthermore, I reveal that the α subunit of the RGGT (RGGTA) also promotes the maturation and anterograde transport of the receptor, which highlight a new cellular role for this protein. Altogether, those results support the hypothesis that GPCRs control their own trafficking, and shed light on some of the mechanisms that might be employed by those receptors in activation of Rab GTPases.
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Duval, Manuel. "La semence et la biotine : découverte d'une protéine à biotine chez Pisum sativum L., marqueur moléculaire de la maturation des semences et des phases précoces de la germination". Grenoble 1, 1995. http://www.theses.fr/1995GRE10133.
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L'etat de vie ralentie caracterise la semence orthodoxe des spermaphytes. Cet etat, induit au cours de la maturation, est leve pendant la germination. La biotine est une molecule necessaire a toute forme de vie. Le mecanisme moleculaire determinant l'etat de vie ralentie est encore inconnu. La biotine peut-elle intervenir dans ce phenomene biologique ? cette these tend a le demontrer. La biotine est decrite comme etant le cofacteur d'enzymes catalysant des reactions de carboxylations. Or, dans les cellules de tous les organes de la plante et a tout moment de son cycle, la biotine libre est en exces par rapport a la biotine liee aux carboxylases. La semence mature est l'unique exception. Deuxieme caracteristique de la semence: elle contient une proteine biotinylee specifique, liant la biotine de maniere covalente. Sa masse moleculaire apparente est de 65 kda, d'ou sa denomination de sbp65 pour seed biotinyl protein (proteine de semence). Depourvue d'activite enzymatique a biotine, sa cinetique d'accumulation au cours de la maturation est concomitante avec l'epuisement de la biotine libre des cellules embryonnaires. Elle est localisee dans le cytoplasme, lieu de synthese de la biotine (baldet, 1993). Tout se passe comme si la sequestration de la biotine par sbp65 de maniere physiologique en fin de maturation chez le pois reproduisait le phenotype du mutant bio1 de l'arabette (shellammer et meinke, 1990). Cette mutation letale correspond a une deficience de synthese de biotine qui entraine une interruption precoce du developpement embryonnaire. Ceci suggere que la regulation du niveau de biotine libre, i. E. Celle disponible pour les carboxylases a biotine indispensables a un metabolisme normal, est un mecanisme de modulation de l'activite metabolique globale. D'ou les observations suivantes: sbp65 est degradee aussitot que le metabolisme reprend au cours de la germination et son gene n'est jamais exprime en dehors de l'embryon en fin de maturation. Sbp65 a son equivalent dans les especes agronomique. Au niveau moleculaire, elle presente deux caracteristiques: elle est tres hydrophile et possede plusieurs motifs repetes, ce qui l'apparente au groupe des proteines lea (late embryogenesis abundant i. E. Abondante en fin d'embryogenese). D'autre part, la structure primaire de sa region biotinylee est radicalement differente de celle de toutes les autres proteines a biotine d'ou son absence d'activite enzymatique
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Seaayfan, Elie. "Régulation du contrôle de qualité de NKCC2 par les interactions protéine-protéine". Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB026.
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Le co-transporteur Na+-K+-2Cl- spécifique du rein et sensible au bumétanide, NKCC2, joue un rôle essentiel dans l’homéostasie hydro-électrolytique et acido-basique de l’organisme. Les mutations inactivatrices de NKCC2 induisent le syndrome de Bartter anténatal de type 1, une grave maladie rénale caractérisée par une hypotension artérielle associée à des anomalies électrolytiques. À l’opposé, une activité accrue de NKCC2 est associée à une hypertension artérielle sensible au sel. Pourtant, peu est connu sur la régulation moléculaire de NKCC2. Le but de ces travaux de thèse a donc été l’identification des déterminants moléculaires impliqués dans la régulation de l’expression et du trafic intracellulaire de NKCC2, plus spécifiquement dans le contrôle de qualité de ce co-transporteur. Suite au criblage par la technique de double hybride chez la levure d’une banque d’ADNc de rein humain, nous avons identifié OS-9 en tant que partenaire de NKCC2. La léctine OS-9 est un facteur clé de régulation du contrôle de qualité des protéines au niveau du RE. Les analyses de co-immunoprécipitation dans les cellules rénales ont montré qu’OS-9 interagit principalement avec la forme immature de NKCC2. De plus, les expériences d’immunofluorescence ont révélé que cette interaction aurait lieu au niveau du RE. La surexpression d’OS-9 diminue l’abondance totale de NKCC2. Cet effet est aboli suite à l’inhibition de la voie de dégradation protéique par le protéasome par le MG132. De plus, les expériences pulse-chase et cycloheximide-chase ont montré que cette diminution est secondaire à l’augmentation de la dégradation de la forme immature de NKCC2. A l’inverse, le knock-down d’OS-9 endogène augmente l’expression du co-transporteur en augmentant la stabilité de sa forme immature. Enfin, la mutation du domaine MRH (Mannose 6-phosphate Receptor Homology) d’OS-9 n’altère pas son effet sur NKCC2, alors que la mutation des deux sites de N-glycosylation de NKCC2 abolie l’effet d’OS-9. L’ensemble de nos résultats démontre l’implication de la lectine OS-9 dans le système ERAD de NKCC2. Le deuxième volet de ce travail a porté sur l’identification de nouveaux mécanismes Moléculaires impliqués dans le Syndrome de Bartter. Nous avons découvert des mutations dans le gène MAGE-D2, situé sur la chromosome X, responsables d’une nouvelle et très sévère forme du syndrome de Bartter anténatal, caractérisé par un polyhydramnios très précoce avec un risque élevé d’accouchement prématuré et de mortalité. Nous avons montré que les anomalies de MAGE-D2 entraînent un défaut de maturation et d’expression membranaire de NKCC2 ainsi que celle du co-transporteur Na-Cl, NCC, du tubule distal. La comparaison in vitro de l’interactome de MAGED2 sauvage et mutée a révélé que la protéine MAGE-D2 sauvage interagit spécifiquement avec DNAJB1 (HSP40) et/ou GNAS, suggérant l’implication de ces deux partenaires protéiques dans la régulation de NKCC2 et NCC par MAGE-D2 pendant la grossesse. Le troisième volet de ce travail a porté sur l’étude de l’effet de DNAJB1/HSP40, partenaire de MAGE-D2, sur l’expression de NKCC2. HSP40 a été identifiée aussi comme partenaire de NKCC2 par la technique de double hybride réalisée par notre équipe. Nous avons montré que HSP40 et son co-chaperon HSPA1A (HSP70) interagissent avec la forme immature de NKCC2 au niveau du RE. La co-expression de HSP40 et HSP70 augmente l’expression de NKCC2 en augmentant sa stabilité et sa maturation. De plus, ces deux co-chaperons régulent l’expression de NCC de la même manière. Ces observations suggèrent que MAGE-D2 coopère avec DNAJB1/HSP40 et HSPA1A/HSP70 pour protéger NKCC2 et NCC contre la rétention et la dégradation de NKCC2 au niveau du RE durant la grossesse, révélant ainsi une nouvelle voie de régulation du trafic intracellulaire de NKCC2 et NCC. (...)The kidney-specific Na + -K + -2C1 co-transporter, sensitive to bumetanide, NKCC2, plays an essential role in the body's fluid, electrolyte and acid-base homeostasis. Mutations of NKCC2 cause antenatal type 1 Bartter syndrome, a life-threatening kidney disease characterized by arterial hypotension associated with electrolyte abnormalities. In contrast, an increase in NKCC2 activity is associated with salt-sensitive hypertension. Yet the mechanisms underlying the regulation of NKCC2 trafficking in renal cells are scarcely known. The aim of this work was to identify the protein partners involved in the regulation of the expression and the intracellular trafficking of NKCC2, specifically in the quality control of this co-transporter. Using the yeast tow-hybrid system, we identified OS-9 as a specific binding partner of NKCC2. Lectin OS-9 is a key factor in the regulation of protein quality control at ER. Co-immunoprecipitation assay in renal cells showed that OS-9 interacts mainly with NKCC2 immature forms. Accordingly, immunocytochemistry analysis showed co-localization of the proteins mainly in the ER. Overexpression of OS-9 decreased the total abundance of NKCC2. This effect is abolished following the inhibition of the proteasome protein degradation pathway by MG132. In addition, the pulse-chase and cycloheximide-chase assays demonstrated that the marked reduction in the co-transporter protein levels was essentially due to increased protein degradation of NKCC2 immature forms. Conversely, knock-down endogenous of OS-9 increased the expression of the co-transporter by increasing the stability of its immature form. Finally, inactivation of the Mannose 6-phosphate Receptor Homology domain had no effect on its action on NKCC2, while mutation of the two NKCC2 N-glycosylation sites abolished the effect of OS- 9. In summary, our results demonstrate the involvement of lectin OS-9 in the ERAD of NKCC2. The second part of this work focused on the identification of new molecular mechanisms involved in Bartter Syndrome. We found that MAGE-D2 mutations caused X-linked new and severe form of antenatal Bartter's syndrome, characterized by a very early polyhydramnios with a high risk of premature delivery and mortality. We have shown that MAGE-D2 abnormalities lead to a lack of maturation and membrane expression of NKCC2 as well as that of the Na-Cl co-transporter, NCC, of the distal tubule. In vitro comparison of the wild-type and mutated MAGED2 interactome revealed that wild-type MAGE-D2 interacts specifically with DNAJB1 (HSP40) and / or GNAS, suggesting involvement of these two protein partners in NKCC2 and NCC regulation by MAGE-D2 during pregnancy. The third part of this work focused on the study of the effect of DNAJB1 / HSP40, partner of MAGE-D2, on the expression of NKCC2. HSP40 was also identified as a specific binding partner of NKCC2 by the yeast two-hybrid system realized by our team. We have shown that HSP40 and its co-chaperone HSPA1A (HSP70) interact with the immature form of NKCC2 at the ER. The co-expression of HSP40 and HSP70 increased the expression of NKCC2 by increasing its stability and maturation. In addition, these two co-chaperones regulate the expression of NCC in the same way. These findings suggest that MAGE-D2 cooperates with DNAJB1 / HSP40 and HSPA1A / HSP70 to protect NKCC2 and NCC against retention and degradation of NKCC2 at ER during pregnancy, revealing a new pathway for regulating NKCC2 and NCC intracellular trafficking. A better understanding of NKCC2 and NCC regulatory pathways would help to better understand the pathophysiology of sodium retention and ultimately would provide a new target for a pharmaceutical approach to preventing and / or treating kidney disease related to sodium balance
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Puccio, François. "Influence de l'hydrocortisone, de l'egf, de la bombesine et du grf sur la proliferation cellulaire et la maturation du tractus gastrointestinal et du pancreas chez le jeune rat non sevre". Paris 6, 1988. http://www.theses.fr/1988PA066497.
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Wang, Jing. "Function of protein kinase A in Xenopus oocyte maturation". Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/29377.
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Like most other vertebrate oocytes, oocytes from Xenopus laevis are physiologically arrested at the first meiotic prophase. Re-initiation of meiosis, or oocyte maturation, is triggered by progesterone. Progesterone-induced oocyte maturation is thought to involve inhibition of cAMP-dependent protein kinase (protein kinase A or PKA). However, changes in PKA activity in live oocytes have never been demonstrated. I have developed a novel approach to monitor endogenous PKA activity in live oocytes, by expressing a PKA-specific substrate in oocytes followed by monitoring its PKA phosphorylation status during progesterone-induced oocyte maturation. I demonstrate that PKA is fully activated in prophase oocytes and that progesterone causes a rapid and permanent inhibition of PKA (Chapter 3). Utilizing this PKA activity indicator in live oocytes, I further demonstrate that progesterone-induced oocyte maturation requires persistent inhibition of PKA such that critical maturation-specific proteins can be synthesized and accumulated to threshold levels necessary for activation of maturation promoting factor (MPF) (chapter 4). In addition to analyzing PKA activity dynamics during oocyte maturation, I have also provided the first biochemical evidence supporting the notion that in prophase oocytes, a constitutively activated G protein coupled receptor system is responsible for elevated cAMP and the high levels of PKA activity (chapter 2).
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Grahl, Sabine. "Tat signal peptide recognition during protein maturation and export". Thesis, University of Dundee, 2011. https://discovery.dundee.ac.uk/en/studentTheses/d306d17d-931d-4313-8f91-5a5108fb2256.
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Nitrogen is one of the most abundant elements on Earth and mostly found in the atmosphere as the inert gas N2. Therefore the nitrogen cycle is important for maintaining the bioavailabilty of nitrogen for organisms. Denitrification is a process that closes the nitrogen cycle by subsequent conversion of nitrate to dinitrogen with reduction of nitrate to nitrite being the very first necessary step. The bacterial periplasmic nitrate reductase NapA is one of those nitrate reducing enzymes and contains a molybdenum cofactor and a [4Fe-4S] cluster as cofactors. As a periplasmic terminal reductase NapA has an N-terminal signal peptide harbouring a Tat (twin-arginine translocation) motif, which follows closely the consensus S/T-R-R-x-F-L-K. As with other proteins transported via the Tat pathway, NapA needs to be fully folded, and cofactor insertion needs to be completed, prior to export. This is assured by an individual chaperone in a process called ‘Tat-proofreading’. The proofreading chaperone for NapA is NapD, which had been previously shown to interact tightly with the signal peptide of NapA.In this work the binding epitope on the Escherichia coli NapA signal peptide recognised by NapD was mapped for the first time. The key amino acid residues (NapA R6, K10, A17) overlapped with the Tat targeting motif and were further characterized in vitro and in vivo for their importance in NapD binding, Tat transport and NapA biosynthesis. In addition, napD suppressor mutants able to re-bind the NapA A17Q variant were isolated. NMR spectroscopy revealed the 3D solution structure of NapD in complex with the NapA signal peptide. Interestingly, the signal peptide of NapA is a-helical when bound to NapD. Overall, the structure supports strongly that NapA residues R6, K10 and A17 interact with NapD. Pulsed EPR spectroscopy on the isolated signal peptide indicated structural changes of the NapA signal peptide between NapD bound and unbound states, though it was believed that an overall a-helical structure was maintained. Co-purification studies of the complete NapDA complex for crystallisation trials resulted in increased information on the behaviour of the complex and the order of cofactor insertion into NapA. Finally, an in vitro translation and cross-linking approach was attempted with the aim of addressing whether direct contact was made between NapD and the Tat translocase. In addition, functional chromosomal fusions of either NapD or NapA with fluorescent proteins were generated to form a basis for a future project based on fluorescence correlation spectroscopy in living cells. This project has therefore provided fresh insight into the NapA-NapD interaction at the molecular level and laid the foundations for future research in this area.
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Martin, Niall M. B. "Protein turnover in Salmonids : sexual maturation and hormonal control". Thesis, University of Aberdeen, 1990. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU548688.
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Aspects of tissue protein metabolism were studied in different groups of Atlantic salmon (Salmo salar). Firstly, 2 groups of sea water salmon were fasted for 3 and 5 days, prior to the measurement of tissue fractional rates of protein synthesis (ks, %day), using the method of 3H-phenylalanine flooding. The sensitivity of liver and gill to a short-term fast was indicated by their reduced rates of ks, while in other tissues such as ventricle, stomach and red muscle protein synthesis was unaffected. Both liver and gill are tissues which show the greatest capacities to synthesise protein, expressed by their high RNA contents. The response of tissue protein metabolism to sexual maturation was investigated in 2 groups of salmon undergoing river migration, tissues were analysed in both July salmon (sexually maturing) and in more mature October salmon. White muscle contributed most of the amino acids required during maturation, denoted by the high loss of protein and RNA from the flesh of the salmon by October. Red muscle, gill and ventricle were tissues which were protected during maturation, showing only slight changes in rates of protein metabolism. Liver, stomach and ovary on the other hand increased their RNA and protein contents, and showed increased rates of protein synthesis. The liver however, displayed a greater increase in its RNA concentration than protein i.e. the liver by October had increased its capacity to produce large amounts of export proteins. Despite the overall loss by stomach and other visceral tissues of protein and RNA, the stomach by October showed a dramatic 5-6 fold increase in its rate of protein synthesis. It was concluded that smooth muscle may have a particular role or function in the sexually mature fish. Factors controlling these in vivo changes in protein synthesis were investigated using rainbow trout (Oncorhynchus mykiss) isolated hepatocytes and a smooth muscle preparation in vitro . Anabolic actions on hepatocyte protein synthesis were exhibited by insulin, thyroxine and in the absence of fetal calf serum by triiodothyronine. The synthetic glucocorticoid, dexamethasone, unlike its actions in muscle, also exhibited an anabolic action on hepatocytes, by raising RNA and protein levels in cells from immature fish, while increasing protein synthesis rates in liver cells of sexually mature fish. Estradiol, like dexamethasone, stimulated rates of protein synthesis over 24 hours. However, hydroxyprogesterone caused no change in protein synthesis and decreased the effect of estradiol. Estradiol also increased protein synthesis rates in smooth muscle by some 30%. However, hydroxyprogesterone, as in liver cells, caused no stimulation of protein synthesis and again decreased the estradiol stimulated ks. It was proposed that estradiol is one of the factors involved in increasing the ks in the stomach of the sexually maturing salmon, while progesterone regulates the action of estradiol towards the end of sexual maturation, when such an effect of estradiol on liver and smooth muscle ks is unnecessary.
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Sheng, Yinglun. "G protein signaling and G protein coupled receptor (GPCR) pathway in Xenopus oocyte maturation". Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/29262.
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Xenopus laevis oocytes are physiologically arrested at the first meiotic prophase. Progesterone reinitiates meiosis (maturation) through inhibition of an oocyte adenylyl cyclase (AC) and reduction of intracellular cAMP. However, the mechanism by which progesterone regulates AC activity and cAMP level still remains unclear.
In this thesis, I summarize work I conducted that collectively helps elucidate how high levels of cAMP might be achieved in G2 arrested oocytes. In Chapter 2, I describe our finding that inhibiting endogenous G-protein betagamma subunits, through the use of two structurally distinct Gbetagamma scavengers, causes hormone-independent oocyte maturation. In contrast, overexpression of Xenopus Gbeta1, alone or together with bovine Ggamma2, inhibits progesterone-induced oocyte maturation. These results for the first time implicate that an endogenous G protein coupled receptor system releases a Gbetagamma complex as the dominant meiosis inhibitor.
Chapter 3 describes my research aiming to reveal the identity of the oocyte AC responsible for generating meiosis-inhibiting cAMP. I provide further evidence here that the ability of Gbetagamma to inhibit meiosis is attributed to the activation of an endogenous AC, rather than other possible Gbetagamma effectors. Through molecular cloning and biochemical characterization, I discovered that the likely AC candidate is Xenopus AC7, an isoform that is activated by Gbetagamma, but only in the presence of GTP-bound Gsalpha. The identification of xAC7 suggests that the maintenance of high levels of cAMP may require the cooperation of Gsalpha and Gbetagamma.
Finally, in Chapter 4, I describe our efforts in identifying the GPCR(s) responsible for activating the cAMP signaling in prophase-arrested oocytes. A screening of known antagonists of GPCR(s) led to the identification of ritanserin, a potent antagonist of serotonin receptors, as a potent maturation inducer in Xenopus oocytes. Pharmacological and molecular studies, however, have ruled out the involvement of a known serotonin receptor in meiosis arrest. Instead, the most likely candidate is a "constitutively activated" GPCR that bears structural similarities to Xenopus serotonin receptor 7.
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Morrison, Donna Lorraine. "Identification and characterization of a maturation-inhibited protein kinase (MIPK)". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/nq27206.pdf.
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Mathew, Anu. "Selective loss of protein and exosome formation during erythroid maturation". Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=28838.
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Vesicle (exosome) externalization is a mechanism for removal of obsolescent membrane proteins, such as the transferrin receptor (TFR), during reticulocyte maturation. This thesis involves further characterization of the exosomal phenomenon using avian (nucleated) and mammalian erythroid systems. Chicken reticulocytes release exosomes, indicating independence of this process from enucleation. Exosome release during chicken erythroleukemic cell (HD3) differentiation (representing pre-reticulocyte development) demonstrates that this process commences prior to the reticulocyte stage.The heat shock protein, hsp70, has been found in exosomes from every species examined (four species, including the chicken). It is shown that hsp70 is physically, but non-covalently, associated with exosomal TFR, suggesting its possible involvement in targeting proteins into exosomes during their formation.
Exosome formation is an energy-dependent process. Our observations indicate that the primary energy substrate for avian red cells is not glucose, but glutamine, inosine and guanosine. Work with the HD3 cells (capable of glucose transport), suggests there is an early differentiation-associated switch in the chicken red cell's ability to use glucose as a major energy substrate.
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Blain, Françoise 1966. "Studies on the maturation of the measles virus hemagglutinin protein". Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=59946.
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The hemagglutinin glycoprotein (HA) encoded by measles virus (MV) is a type II integral membrane protein that is expressed at the infected cell surface. The maturation of HA was studied in infected Vero cells. HA oligomerized into a disulfide-linked homodimer with a t$ sb{1/2}$ of 30 min. The rate of transport of HA to the medial Golgi complex was also examined using endoglycosidase-H and found to occur with a t$ sb{1/2}$ of 2 hr. Genes encoding two mutant HA proteins shortened at their C-terminal end by either 18 (HA$ Delta$18) or 223 (HA$ Delta$223) amino acids were constructed and studied in a transient expression system in COS cells. HA$ Delta$18 was able to assemble into homodimers while HA$ Delta$223 remained in a monomeric form in the absence of the reducing agent DTT. Hemadsorption assays revealed that neither of the mutants were functional even though HA$ Delta$18 was transported to the cell surface, as determined by chymotrypsin sensitivity. These studies showed that the C-terminal ectodomain of the HA protein is essential to its proper folding and assembly into homodimers, and for its transport to the cell surface. In addition, the last 18 amino acids were shown to be important to hemadsorption and might therefore be part of the MV HA functional domain.
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Handley, Matthew. "Mitogen-activated protein kinases in dendritic cell maturation and death". Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1446249/.
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Dendritic cells (DC) sense infection in their microenvironment and undergo a dynamic process of changes in phagocytic capacity, morphology and migratory activity in order to induce optimum T cell immunity. The acquisition of these properties is termed "DC maturation". In this study, key selected aspects of this DC maturation process have been explored. One aspect of DC maturation is the signalling pathways that DC use to respond to micro-environmental signals. DC respond to conserved microbial structures in their micro-environment via pattern recognition receptors including Toll-like receptors (TLRs). Generally it is thought that this leads to activation of the mitogen-activated protein kinase (MAPK) pathways, p38, ERK, and JNK. JNK and p38 MAPK are also activated by reactive oxygen species, including hydrogen peroxide (H202), known to be present in the inflammatory DC milieu. Therefore the ability of H2O 2 to stimulate DC, or modulate their response to TLR ligands such as lipopolysaccharide (LPS) was examined. Activation of JNK, associated with inhibition of tyrosine phosphatases, is linked to induction of DC apoptosis. JNK inhibition partially protected the DC from the pro-apoptotic effects of H2O2. Furthermore, H2O2 and LPS synergise in inducing JNK activation, and DC apoptosis. These studies suggest that excessive DC maturation, which may induce pathogenic T cell activation may be limited by DC apoptosis. The second section of the thesis explores the mechanisms leading to MAPK activation upon TLR ligation. Biochemical studies demonstrated that mixed lineage kinase 3 (MLK3), a kinase not previously implicated in DC maturation, was phosphorylated upon TLR3 and 4, but not TLR2 ligation. Studies using a selective MLK inhibitor supported a role for the mixed lineage kinase (MLK) family of MAPK kinase kinase (MAP3K) proteins in regulation of DC signalling, phenotype and cytokine secretion, in response to TLR3 and 4, but not TLR2 ligation. Selective activation of MAP3K may therefore contribute to the heterogeneity and flexibility of DC/pathogen interaction. A further feature of the DC sensory role is antigen uptake, but the relationship between morphology and phagocytic capacity has not been defined. Therefore a simple and flexible assay to examine this relationship was developed. DC with long dendritic processes were less efficient at antigen internalisation than round DCs.
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LISI, GAIA. "Spinster is an Ambra1 interacting protein required for autophagosome maturation". Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/202447.
Texto completoResumen
Autophagy is a self-degradative process involved in the turnover of cellular components in response to nutrient starvation or to organelle damage. During this process, portions of cytoplasm are sequestered by double-membrane vesicles, the autophagosomes, and degraded after fusion with lysosomes. Different protein complexes participate to autophagosome formation including various components of the class III phosphatidylinositol-3-OH kinase complex (Ambra1, Beclin 1, Vps34, Vps15, UVRAG) and most of the Atg genes. Here, I show that Ambra1 also plays a role in the regulation of the late steps of autophagy via the interaction with the lysosomal protein Spinster. Spinster is trans-membrane protein known to regulate the endosomal pathway in Drosophila neurons. Recent studies have shown that Spinster is able to bind the antiapoptotic protein Bcl2 and is involved in the execution of a caspase-independent cell death associated to autophagic vacuole formation. In this study, I demostrate that Spinster is involved in the regulation of autophagosome maturation. Spinster localizes with LC3, a marker of the autophagosomal compartment. Notably, Spinster overexpression induces autophagy, while its down-regulation leads to an alteration of autophagolysosomal acidification causing a block of autophagic degradation. Finally, I showed that Ambra1 regulates Spinster activity by regulating its levels of ubiquitination. Taken together, my data show that Spinster is a positive regulator of the autophagic process and that Ambra1 has a novel role in the regulation of autophagosome maturation.
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Heavey, Patricia. "New methodologies for studying diet and gut maturation in early life". Thesis, University of Ulster, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268569.
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Holm, Åsa. "Leishmania donovani lipophosphoglycan : effects on actin and phagosomal maturation /". Linköping : Univ, 2003. http://www.bibl.liu.se/liupubl/disp/disp2003/med800s.pdf.
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