Dissertations / Theses on the topic 'Saccharomyces cerevisiae Eukaryotes'
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Haider, Mustafa M. "The intracellular sorting of vacuolar proteins in the yeast Saccharomyces cerevisiae." Thesis, Durham University, 1989. http://etheses.dur.ac.uk/6495/.
Full textKubicek, Charles E. 1981. "Identifying targets and function of the ubiquitin related modifier Urm1 in Saccharomyces cerevisiae." Thesis, University of Oregon, 2009. http://hdl.handle.net/1794/10310.
Full textPost-translational modification of proteins is an important cellular method of controlling various aspects of protein activity, including protein-protein interactions, half- life, and transport. An important class of post-translational modifications involves the ubiquitin family of proteins. In these modifications, a small protein, such as ubiquitin, is conjugated to a target protein through an isopeptide bond. Conjugation by a ubiquitin family member acts as a signal to regulate the activity, function, or stability of the target protein. Urm1, a ubiquitin-like protein conserved throughout all eukaryotes, was initially identified in S. cerevisiae. Loss of Urm1 leads to the disruption of a variety of cellular processes, including oxidative stress response, filamentous growth, and temperature sensitivity. This body of work comprises efforts to identify novel targets of Urm1, the mechanism by which Urm1 is attached to target proteins, and the physiological consequences of such conjugation. To gain understanding of the function and mechanism of Urm1 conjugation, the only known conjugate of Urm1, the peroxiredoxin reductase Ahp1, was examined in an effort to identify the site of modification on Ahp1 and to evaluate the physiological consequences of urmylation of Ahp1. I then completed a series of screens--a synthetic lethal screen, a two-hybrid screen, and a protein over-expression screen--to identify novel Urm1 conjugates and cellular functions dependent on Urm1. Of particular interest were genes identified in the synthetic lethal screen, namely PTC1, which encodes a protein phosphatase, and a set of genes encoding the Elongator complex, which functions in transcriptional elongation and tRNA modification. During this time period, other groups showed that thiolation of tRNAs depends on Urm1. Thus, Urm1 does not function only in protein conjugation, but also as a sulfur carrier in the thiolation of tRNA. Interestingly, I identified Elp2, a component of the Elongator complex, as a new Urm1-conjugate. Because Elp2 is also required for tRNA modification, perhaps Urm1 plays more than one role in tRNA modification. Loss of tRNA modification may disrupt many cellular functions and could explain the variety of urm1 mutant phenotypes. I have determined that all known Urm1 dependent processes are also associated with tRNA modification.
Committee in charge: Karen Guillemin, Chairperson, Biology; George Sprague, Advisor, Biology; Alice Barkan, Member, Biology; Kenneth Prehoda, Member, Chemistry; Tom Stevens, Outside Member, Chemistry
Bartish, Galyna. "Elongation factor 2 : a key component of the translation machinery in eukaryotes : properties of yeast elongation factor 2 studied in vivo /." Stockholm : Wenner-Gren Institute for Experimental Biology, Stockholm university, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-7733.
Full textPereira, Dirce Maria Carraro. "Regulação transcricional por glicose do promotor do gene que codifica celobiohidrolase I de Trichoderma reesei em Saccharomyces cerevisiae." Universidade de São Paulo, 1998. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-27112014-152253/.
Full textThe cellulotic system of the filamentous fungus Trichoderma reesei is transcriptionally induced 1000 -fold in presence of cellulose and is strongly repressed by glucose. Using the promoter deletion approach, the upstream activating region (UARcbl) responsible for cellulose-stimulated transcription of the major member of the cellulase system, cellobiohydrolase I, was localized between -241 and -72 relative to the TATA box. In this work we show that this region controls transcription and mediates glucose repression of a reporter gene in Saccharomyces cerevisiae, a unicellular microorganism that lacks the genes required for the utilization of cellulose. Glucose-controlled transcription mediated by the UARcbl requires the product of SNF1 gene, a protein kinase, and two repressors SSN6 and TUP1, which are well estalished in controlling glucose-represible yeast genes. Our results indicate a conserved mechanism of glucose control in eukariotic microorganisms.
Chommy, Hélène. "Fidélité de la traduction chez les eucaryotes. De la molécule au génome." Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00749760.
Full textKipling, D. G. "Studies on replication origins in Saccharomyces cerevisiae." Thesis, University of Oxford, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.253151.
Full textBalyan, Prachi. "Complex genetic interactions in the model eukaryote, Saccharomyces cerevisiae." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709165.
Full textFan-Minogue, Hua. "Understanding the molecular mechanism of eukaryotic translation termination functional analysis of ribosomal RNA and eukaryotic release factor one /." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2009r/fan-minogue.pdf.
Full textJackson, Stephen Philip. "Cloning and characterisation of the RNA8 gene of Saccharomyces cerevisiae." Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/15100.
Full textKallmeyer, Adam K. "Regulatory mechanisms of eukaryotic translation termination." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2009r/kallmeyer.pdf.
Full textWicksteed, Barton. "Use of gene fusions to study the expression of PYK1 in Saccharomyces cerevisiae." Thesis, University of Aberdeen, 1994. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU068131.
Full textSweet, Deborah Jane. "The SEC20-TIP1 complex." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307092.
Full textTan, Song. "Protein-DNA interactions of transcription factors reponsible for cell-type specificity in Saccharomyces cerevisiae." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316019.
Full textFranken, Jaco. "Carnitine metabolism and biosynthesis in the yeast Saccharomyces cerevisiae." Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/4611.
Full textENGLISH ABSTRACT: Carnitine plays an essential role in eukaryotic metabolism by mediating the shuttling of activated acyl residues between intracellular compartments. This function of carnitine, referred to as the carnitine shuttle, is supported by the activities of carnitine acyltransferases and carnitine/acylcarnitine transporters, and is reasonably well studied and understood. While this function remains the only metabolically well established role of carnitine, several studies have been reporting beneficial effects associated with dietary carnitine supplementation, and some of those beneficial impacts appear not to be directly linked to shuttle activity. This study makes use of the yeast Saccharomyces cerevisiae as a cellular model system in order to study the impact of carnitine and of the carnitine shuttle on cellular physiology, and also investigates the eukaryotic carnitine biosynthesis pathway. The carnitine shuttle of S. cerevisiae relies on the activity of three carnitine acetyltransferases (CATs), namely Cat2p (located in the peroxisome and mitochondria), Yat1p (on the outer mitochondrial membrane) and Yat2p (in the cytosol), which catalyze the reversible transfer of activated acetyl units between CoA and carnitine. The acetylcarnitine moieties can be transferred across the intracellular membranes of the peroxisomes and mitochondria by the activity of the carnitine/acetylcarnitine translocases. The activated acetyl groups can be transferred back to free CoA-SH and further metabolised. In addition to the carnitine shuttle, yeast can also utilize the glyoxylate cycle for further metabolisation of in particular peroxisomally generated acetyl-CoA. This cycle results in the net production of succinate from two molecules of acetyl-CoA. This dicarboxylic acid can then enter the mitochondria for further metabolism. Partial disruption of the glyoxylate cycle, by deletion of the citrate synthase 2 (CIT2) gene, generates a yeast strain that is completely dependent on the activity of the carnitine shuttle and, as a consequence, on carnitine supplementation for growth on fatty acids and other non-fermentable carbon sources. In this study, we show that all three CATs are required for the function of the carnitine shuttle. Furthermore, overexpression of any of the three enzymes is unable to crosscomplement deletion of any one of the remaining two, suggesting a highly specific role for each CAT in the function of the shuttle. In addition, a role for carnitine that is independent of the carnitine shuttle is described. The data show that carnitine can influence the cellular response to oxidative stresses. Interestingly, carnitine supplementation has a protective effect against certain ROS generating oxidants, but detrimentally impacts cellular survival when combined with thiol modifying agents. Although carnitine is shown to behave like an antioxidant within a cellular context, the molecule is unable to scavenge free radicals. The protective and detrimental impacts are dependent on the general regulators of the cells protection against oxidative stress such as Yap1p and Skn7p. Furthermore, from the results of a microarray based screen, a role for the cytochrome c heme lyase (Cyc3p) in both the protective and detrimental effects of carnitine is described. The requirement of cytochrome c is suggestive of an involvement in apoptotic processes, a hypothesis that is supported by the analysis of the impact of carnitine on genome wide transcription levels. A separate aim of this project involved the cloning and expression in S. cerevisiae of the four genes encoding the enzymes from the eukaryotic carnitine biosynthesis pathway. The cloned genes, expressed from the constitutive PGK1 promoter, were sequentially integrated into the yeast genome, thereby reconstituting the pathway. The results of a plate based screen for carnitine production indicate that the engineered laboratory strains of S. cerevisiae are able to convert trimethyllysine to L-carnitine. This work forms the basis for a larger study that aims to generate carnitine producing industrial yeast strains, which could be used in commercial applications.
AFRIKAANSE OPSOMMING: Karnitien vervul ‘n noodsaaklike rol in eukariotiese metabolisme deur die pendel van asiel residue tussen intersellulêre kompartemente te medieer. Hierdie funksie van karnitien heet “die karnitien-pendel“ en word ondersteun deur verskeie karnitien asieltransferases en karnitine/asielkarnitien oordragsprotiëne. Die rol van die karnitien-pendel is redelik goed gekarakteriseer en is tot op hede die enigste bevestigde rol van karnitien in eukariotiese metabolisme. Verskeie onlangse studies dui egter op voordele geasosieer met karnitien aanvulling, wat in sommige gevalle blyk om onafhanklik te wees van die pendel aktiwiteit van karnitien. Hierdie studie maak gebruik van die gis, Saccharomyces cerevisiae, as ‘n sellulêre model sisteem om die impak van karnitien op sel fisiologie asook die eukariotiese karnitien biosintese pad te bestudeer. Die karnitien-pendel van S. Cerevisiae is afhanklik van die aktiwiteite van drie afsonderlike karnitien asetieltransferases (CATs), naamlik Cat2p (gelokaliseer in die peroksisoom en die mitochondria), Yat1p (op die buitenste membraan van die mitochondria) en Yat2p (in die sitosol). Die drie ensieme kataliseer die omkeerbare oordrag van asetielgroepe tussen CoA en karnitien. Die terugwaartse reaksie stel CoA-SH vry om sodoende verbruik te word in verdere metaboliese reaksies. Gis is in staat om, afsonderlik van die karnitien-pendel, gebruik te maak van die glioksilaat siklus vir verdere metabolisme van asetiel-CoA wat gevorm word in die peroksisoom. Gedeeltelike onderbreking van hierdie siklus deur uitwissing van die sitraat sintase (CIT2) geen, genereer ’n gisras wat afhanklik is van die funksie van die karnitienpendel en ook van karnitien aanvulling vir groei op vetsure en nie-fermenteerbare koolstofbronne. Hierdie studie dui daarop dat al drie CATs noodsaaklik is vir die funksionering van die karnitien-pendel. Ooruitdrukking van enige van die drie ensieme lei slegs tot selfkomplementasie en nie tot kruis-komplementasie van die ander twee CATs nie. Hieruit word ’n hoogs spesifieke rol vir elk van die drie ensieme afgelei. ’n Pendel-onafhanklike rol vir karnitien word ook in hierdie werk uitgewys in die bevordering van weerstand teen oksidatiewe stres. Dit is noemenswaardig dat karnitien ’n beskermende effek het in kombinasie met oksidante wat ROS genereer en ’n nadelige effek in kombinasie met sulfhidriel modifiserende agente. Dit word aangedui dat karnitien antioksidant funksie naboots in die konteks van ’n gis sel terwyl die molekuul nie in staat is om vry radikale te deaktiveer nie. Beide die beskermende asook die nadelige inwerking van karnitien is afhanklik van Yap1p en Skn7p, wat reguleerders is in die algemene beskerming teen oksidatiewe stres. Die resultate van ’n “microarray“ gebaseerde studie dui op ’n rol vir die sitokroom c heem liase (Cyc3p) in beide die beskermende en nadelige gevolge van karnitien aanvulling. Die vereiste vir sitochroom c dui op ’n moontlike rol vir apoptotiese prosesse. Hierdie hipotese word verder versterk deur ‘n analise van die impak van karnitien op genoomwye transkripsievlakke. ’n Afsonderlike doelwit van hierdie studie was toegespits op die klonering en uitdrukking van die vier ensieme betrokke in eukariotiese karnitien biosintese in S. cerevisiae. Die gekloneerde gene, uitgedruk vanaf die konstitutiewe PGK1 promotor, was geïntigreer in die gisgenoom om die pad op te bou. Die resultate van ’n plaat gebaseerde karnitien produksie toets dui aan dat die geneties gemanipuleerde gisrasse wel in staat is om trimetiellisien oor te skakel in Lkarnitien. Hierdie werk vorm die hoeksteen van ’n studie wat die ontwikkeling van karnitien produserende kommersiële gisrasse as doelwit stel.
Gentz, Petra Monika. "Towards understanding the mechanism of dimerisation of Saccharomyces cerevisiae eukaryotic translation initiation factor 5A." Thesis, Rhodes University, 2008. http://eprints.ru.ac.za/1161/.
Full textZanelli, Cleslei Fernando [UNESP]. "Caracterização funcional de eIF5A: análise genética e molecular utilizando o modelo de Saccharomyces cerevisiae." Universidade Estadual Paulista (UNESP), 2006. http://hdl.handle.net/11449/100606.
Full textCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O fator de início de tradução de eucariotos 5A (eIF5A) é uma proteína altamente conservada desde arquebactérias a mamíferos e sofre uma modificação póstraducional única, necessária para sua maturação funcional, chamada de hipusinação. Apesar do grau de conservação de eIF5A, e da essencialidade de sua função nos organismos estudados, seu papel no metabolismo celular ainda se encontra indeterminado. Vários mutantes condicionais de eIF5A, sensíveis ao aumento de temperatura, têm sido isolados e caracterizados na levedura Saccharomyces cerevisiae. Utilizando um desses mutantes de eIF5A, o alelo tif51A-1, foi isolado o gene PKC1 como um supressor em alto número de cópias do fenótipo de sensibilidade a temperatura deste mutante. O entendimento de como se dá esta interação genética foi um dos enfoques deste trabalho. Foi mostrado que a via de MAP quinases que atua abaixo de Pkc1 não é responsável pela supressão deste mutante e a identificação dos novos supressores do mutante tif51A-1, GIC1 e ZDS1, levou à sugestão de uma nova via de sinalização a partir de Pkc1. Com a realização de experimentos subsequentes, foi confirmado que a nova via Pkc1-Zds1-Gic1 é responsável pela supressão do mutante tif51A-1 promovida por PKC1. Além disso, estes três supressores são importantes para a polaridade celular em S. cerevisiae, um processo essencial para a progressão no ciclo celular deste organismo, e, interessantemente, os mutantes tif51A-1 e tif51A-3 de eIF5A evidenciaram defeitos na polarização do citoesqueleto de actina na temperatura não permissiva. Esses dados evidenciam uma correlação de eIF5A com progressão no ciclo celular de S. cerevisiae.
The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein from archaebacteria to mammals and undergoes hypusination, an essential unique post-translational modification. Despite the high degree of conservation of eIF5A and its essential function in the studied organisms, its cellular role remains unclear. Several temperature-sensitive eIF5A mutants have been isolated and characterized in the yeast Saccharomyces cerevisiae. Using one of these mutants, the tif51A-1 allele, PKC1 was identified as a high-copy suppressor of the temperature-sensitive phenotype shown by this mutant. The understanding of this genetic interaction was one of the aims of this work. It was shown that the MAP kinase cascade downstream Pkc1 is not responsible for this phenotypic suppression and the identification of the new tif51A-1 suppressors, GIC1 and ZDS1, suggested a new signaling pathway branching from Pkc1. Further analysis confirmed that Pkc1-Zds1-Gic1 constitute a new pathway that is responsible for tif51A-1 mutant suppression promoted by PKC1. Moreover, these three suppressors are important for cell polarity in S. cerevisiae, an essential process for cell cycle progression in yeast, and, interestingly, the eIF5A mutants tif51A-1 and tif51A-3 showed defects in actin cytoskeleton polarization at the restrictive temperature. These data supported a connection between eIF5A and cell cycle progression in S. cerevisiae. As eIF5A was originally implicated in the process of translation, in order to better investigate the specific function of this factor, polysomal profiling analysis was performed and it was demonstrated that eIF5A interacts with monosomes in a tranlation dependent manner and, besides that, eIF5A mutants show altered polysomal distribution suggesting a possible defect in the elongation step of translation.
Zanelli, Cleslei Fernando. "Caracterização funcional de eIF5A : análise genética e molecular utilizando o modelo de Saccharomyces cerevisiae /." Araraquara : [s.n.], 2006. http://hdl.handle.net/11449/100606.
Full textBanca: Maria Célia Bertolini
Banca: Gustavo Henrique Goldman
Banca: Carla Columbano de Oliveira
Banca: Nilson Ivo Tonin Zanchin
Resumo: O fator de início de tradução de eucariotos 5A (eIF5A) é uma proteína altamente conservada desde arquebactérias a mamíferos e sofre uma modificação póstraducional única, necessária para sua maturação funcional, chamada de hipusinação. Apesar do grau de conservação de eIF5A, e da essencialidade de sua função nos organismos estudados, seu papel no metabolismo celular ainda se encontra indeterminado. Vários mutantes condicionais de eIF5A, sensíveis ao aumento de temperatura, têm sido isolados e caracterizados na levedura Saccharomyces cerevisiae. Utilizando um desses mutantes de eIF5A, o alelo tif51A-1, foi isolado o gene PKC1 como um supressor em alto número de cópias do fenótipo de sensibilidade a temperatura deste mutante. O entendimento de como se dá esta interação genética foi um dos enfoques deste trabalho. Foi mostrado que a via de MAP quinases que atua abaixo de Pkc1 não é responsável pela supressão deste mutante e a identificação dos novos supressores do mutante tif51A-1, GIC1 e ZDS1, levou à sugestão de uma nova via de sinalização a partir de Pkc1. Com a realização de experimentos subsequentes, foi confirmado que a nova via Pkc1-Zds1-Gic1 é responsável pela supressão do mutante tif51A-1 promovida por PKC1. Além disso, estes três supressores são importantes para a polaridade celular em S. cerevisiae, um processo essencial para a progressão no ciclo celular deste organismo, e, interessantemente, os mutantes tif51A-1 e tif51A-3 de eIF5A evidenciaram defeitos na polarização do citoesqueleto de actina na temperatura não permissiva. Esses dados evidenciam uma correlação de eIF5A com progressão no ciclo celular de S. cerevisiae.
Abstract: The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein from archaebacteria to mammals and undergoes hypusination, an essential unique post-translational modification. Despite the high degree of conservation of eIF5A and its essential function in the studied organisms, its cellular role remains unclear. Several temperature-sensitive eIF5A mutants have been isolated and characterized in the yeast Saccharomyces cerevisiae. Using one of these mutants, the tif51A-1 allele, PKC1 was identified as a high-copy suppressor of the temperature-sensitive phenotype shown by this mutant. The understanding of this genetic interaction was one of the aims of this work. It was shown that the MAP kinase cascade downstream Pkc1 is not responsible for this phenotypic suppression and the identification of the new tif51A-1 suppressors, GIC1 and ZDS1, suggested a new signaling pathway branching from Pkc1. Further analysis confirmed that Pkc1-Zds1-Gic1 constitute a new pathway that is responsible for tif51A-1 mutant suppression promoted by PKC1. Moreover, these three suppressors are important for cell polarity in S. cerevisiae, an essential process for cell cycle progression in yeast, and, interestingly, the eIF5A mutants tif51A-1 and tif51A-3 showed defects in actin cytoskeleton polarization at the restrictive temperature. These data supported a connection between eIF5A and cell cycle progression in S. cerevisiae. As eIF5A was originally implicated in the process of translation, in order to better investigate the specific function of this factor, polysomal profiling analysis was performed and it was demonstrated that eIF5A interacts with monosomes in a tranlation dependent manner and, besides that, eIF5A mutants show altered polysomal distribution suggesting a possible defect in the elongation step of translation.
Doutor
Charlton, Jane Laura. "Understanding the biomolecular interactions involved in dimerisation of the Saccharomyces cerevisiae eukaryotic translation initiation factor 5A." Thesis, Rhodes University, 2012. http://hdl.handle.net/10962/d1004118.
Full textLeon, Ronald P. "Structural and functional analysis of MCM helicases in eukaryotic DNA replication /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2007.
Find full textTypescript. Includes bibliographical references (leaves 90-98). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
Sabouri, Nasim. "Structure of eukaryotic DNA polymerase epsilon and lesion bypass capability." Doctoral thesis, Umeå : Univ, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1477.
Full textGhosh, Arnab. "Coevolution of Ribosomes and The Translational Apparatus: The Structure and Function of Eukaryotic Ribosomal Protein uS7 from Yeast, Saccharomyces cerevisiae." Cleveland State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=csu1435159279.
Full textNeal, Andrea C. "Lipid biosynthesis in eukaryotic cells : studies on enzyme activities involved in fatty acid activation and acylation /." Uppsala : Dept. of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, 2006. http://epsilon.slu.se/200678.pdf.
Full textLlinares, Elisa. "Function, regulation and intracellular trafficking of the vacuolaryeast pq-loop (Ypq) proteins." Doctoral thesis, Universite Libre de Bruxelles, 2012. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209704.
Full textDuring this thesis work, we have studied three LCT proteins of the yeast Saccharomyces cerevisiae, named Ypq1, Ypq2 and Ypq3 (Yeast PQ-loop proteins 1, 2 and 3). We first showed that these proteins localize to the vacuolar membrane. We next studied the roles of these proteins, the regulation of their genes and the mechanisms and signals implicated in their delivery to the vacuolar membrane. We also contributed to the functional characterization of a mammalian homologue of yeast Ypq proteins, named rPqlc2.
In the first part of this work, we report that the Ypq proteins are most probably implicated in the export of basic amino acids from the vacuole to the cytosol. More precisely, Ypq2 and Ypq3 behave like vacuolar arginine and lysine exporters, respectively. Interestingly, the mammalian rPqlc2 protein expressed in yeast reaches the vacuolar membrane and functions as an orthologue of the Ypq proteins. Our results also reveal that the expression of the YPQ3 gene is regulated by the Lys14 transcription factor, responsible for the transcriptional activation of the LYS genes encoding enzymes implicated in the biosynthesis of lysine. We have also noted that, in general, the expression of the expression of the YPQ genes is regulated according to the quality of the nitrogen source available in the extracellular medium, eg. YPQ3 is sensitive to the nitrogen catabolite repression regulatory mechanism.
In the last part of this thesis work, we investigated the intracellular trafficking of the Ypq proteins and show that these predominantly reach the vacuolar membrane via the ALP (alkaline phosphatase) pathway due to the presence of a dileucine-based sorting signal in their sequences. Interestingly, a similar mechanism seems responsible for targeting to the yeast vacuole of the mammalian rPqlc2 protein.
Une caractéristique des cellules eucaryotes est leur organisation en compartiment internes délimité par une membrane lipidique, appelé organelles. Ces compartiments intracellulaires présentent une composition lipidique et protéique particulaire conforme à leur identité et fonction. Les lysosomes de cellules de mammifères et la vacuole fongique jouent un rôle clé dans la digestion intracellulaire de macromolécules et de ce fait leurs lumières sont enrichis d’enzymes hydrolytiques nécessaires à cette action. Des disfonctionnements du lysosome peuvent être la conséquence de pathologie chez l’homme, regroupé sous le nom de maladie lysosomale, lié à un à une accumulation de macromolécules non digéré ou un default d’export des produits d’hydrolysé depuis la lumière du lysosome. La cystinose est une maladie autosomale récessive avec une faible fréquence d’incidence (1/200 000) qui regroupe trois formes cliniques :deux formes rénales graves et une forme extra-rénale. Cette maladie est due à une accumulation et cristallisation de cystine dans la lumière du lysosome qui est corrélé à des mutations ponctuelles dans le gène CTNS qui code pour l’exporteur de cystine, la cystinosine. Cette protéine est un membre de la famille LCT (Lysosomal Cystine Transporter) qui possède des représentants chez les cellules animales, végétales et fongiques. Les protéines de la famille possèdent une taille et une topologie prédite similaire (7 segments transmembranaires) et on retrouve aussi au sein de ces protéines deux exemplaires de motifs PQ. Lors de ce travail de thèse nous nous sommes intéressés à trois membres de la famille LCT chez Saccharomyces cerevisiae que nous avons nommé Ypq1, Ypq2 et Ypq3 pour Yeast PQ-loop proteins. Ces protéines n’ayant pas fait l’objet de nombreuses études, nous nous sommes orientés vers une analyse fonctionnelle et transcriptionnelle. De plus, nous avons également étudié les mécanismes et signaux impliqué dans leur adressage vers la vacuole. Finalement, nous avons également inclus dans notre étude un homologue mammalien de ces protéines, rPqlc2.
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Doctorat en Sciences
info:eu-repo/semantics/nonPublished
Trucks, Sven [Verfasser], Martin [Akademischer Betreuer] Hengesbach, Martin [Gutachter] Hengesbach, and Harald [Gutachter] Schwalbe. "Structure and dynamics of eukaryotic H/ACA RNPs from saccharomyces cerevisiae / Sven Trucks ; Gutachter: Martin Hengesbach, Harald Schwalbe ; Betreuer: Martin Hengesbach." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2021. http://d-nb.info/1231911301/34.
Full textShamsah, Sara. "A gene deletion strategy to identify the function of a non-coding RNA in the eukaryotic genome using the model organism Saccharomyces cerevisiae." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/a-gene-deletion-strategy-to-identify-the-function-of-a-noncoding-rna-in-the-eukaryotic-genome-using-the-model-organism-saccharomyces-cerevisiae(f4f84f7b-845e-4d31-b16c-50e34f69604d).html.
Full textIsoz, Isabelle. "Role of yeast DNA polymerase epsilon during DNA replication." Doctoral thesis, Umeå : Umeå University, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1932.
Full textMurphy, Patrick. "Characterisation of critical interactions between translation factors eIF2 and eIF2B." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/characterisation-of-critical-interactions-between-translation-factors-eif2-and-eif2b(9138d7c8-34b1-4489-8048-a2ac45ef8533).html.
Full textGaur, Jiyoti Verma. "Elucidation Of Differential Role Of A Subunit Of RNA Polymerase II, Rpb4 In General And Stress Responsive Transcription In Saccharomyces Cerevisiae." Thesis, 2008. http://hdl.handle.net/2005/866.
Full textMunro, Edith M. "Characterization of two eukaryote-specific peptide inserts in thymidylate synthase of Saccharomyces cerevisiae." Thesis, 1995. http://spectrum.library.concordia.ca/6214/1/MM10880.pdf.
Full textFitz, Gerald Jonathan Nesbit. "The G1 DNA damage checkpoint in S. cerevisiae /." 2001. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3039067.
Full textSingh, Rajkumar Sunanda. "Studies On Saccharomyces Cerevisiae RNA Polymerase II Subunit Rpb7 And Its Eukaryotic Orthologs." Thesis, 2008. http://hdl.handle.net/2005/703.
Full textMunro, Edith M. "Functional analysis of mutations in a Eukaryote-conserved surface loop in thymidylate synthase of Saccharomyces cerevisiae." Thesis, 2002. http://spectrum.library.concordia.ca/1619/1/NQ68192.pdf.
Full textShetty, Ameet S. "Regulation of the Saccharomyces cerevisiae INO1 gene: Novel insights into a hallmark of eukaryotic transcription regulation." 2011. https://scholarworks.umass.edu/dissertations/AAI3482726.
Full textBrandes, Nicolas. "Oxidative Thiol Modifications in Pro- and Eukaryotic Organisms." Doctoral thesis, 2010. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-46542.
Full textCysteines play important roles in the biochemistry of many proteins. The high reactivity, redox properties, and ability of the free thiol group to coordinate metal ions designate cysteines as the amino acids of choice to form key catalytic components of many enzymes. Also, cysteines readily react with reactive oxygen and nitrogen species to form reversible oxidative thiol modifications. Over the last few years, an increasing number of proteins have been identified that use redox-mediated thiol modifications to modulate their function, activity, or localization. These redox-regulated proteins are central players in numerous important cellular processes. First aim of this study was to discover nitric oxide (NO) sensitive proteins in E. coli, whose redox-mediated functional changes might explain the physiological alterations observed in E. coli cells suffering from NO-stress. To identify E. coli proteins that undergo reversible thiol modifications upon NO-treatment in vivo, I applied a differential thiol trapping technique combined with two-dimensional gel analysis. 10 proteins were found to contain thiol groups sensitive to NO-treatment. Subsequent genetic studies revealed that the oxidative modifications of AceF & IlvC are, in part, responsible for the observed NO-induced growth inhibition. Noteworthy, the majority of identified protein targets turned out to be specifically sensitive towards reactive nitrogen species. This oxidant specificity was tested on one NO-sensitive protein, the small subunit of glutamate synthase. In vivo and in vitro activity studies demonstrated that glutamate synthase rapidly inactivates upon nitric oxide treatment but is resistant towards other oxidative stressors. These results imply that reactive oxygen and nitrogen species affect distinct physiological processes in bacteria. The second aim of my study was to identify redox-sensitive proteins in S. cerevisiae and to use their redox state as in vivo read-out to assess the role of oxidative stress during the eukaryotic aging process. I first determined the precise in vivo thiol status of almost 300 yeast proteins located in the cytosol and sub-cellular compartments of yeast cells using a highly quantitative mass spectrometry based thiol trapping technique, called OxICAT. The identified proteins can be clustered in four groups: 1) proteins, whose cysteine residues are oxidation resistant; 2) proteins with structurally or functionally important cysteine modifications 3) proteins with highly oxidation-sensitive active site cysteines, which are partially oxidized in exponentially growing yeast cells due to their exquisite sensitivity towards low amounts of ROS; 4) proteins that are reduced in exponentially growing cells but harbor redox-sensitive cysteine(s) that affect the catalytic function of the protein during oxidative stress. These oxidative stress sensitive proteins were identified by exposure of yeast cells to sublethal concentrations of H2O2 or superoxide. It was shown that the major targets of peroxide- and superoxide-mediated stress in the cell are proteins involved in translation, glycolysis, TCA cycle and amino acid biosynthesis. These targets indicate that cells rapidly redirect the metabolic flux and energy towards the pentose phosphate pathway in an attempt to ensure the production of the reducing equivalent NADPH to counterattack oxidative stress. These results reveal that the quantitative assessment of a protein’s oxidation state is a valuable tool to identify catalytically active and redox-sensitive cysteine residues. The OxICAT technology was then used to precisely determine extent and onset of oxidative stress in chronologically aging S. cerevisiae cells by utilizing the redox status of proteins as physiological read-out. I found that chronological aging yeast cells undergo a global collapse of the cellular redox homeostasis, which precedes cell death. The onset of this collapse appears to correlate with the yeast life span, as caloric restriction increases the life span and delays the redox collapse. These results suggest that maintenance of the redox balance might contribute to the life expanding benefits of regulating the caloric intake of yeast. Clustering analysis of all oxidatively modified proteins in chronological aging yeast revealed a subset of proteins whose oxidative thiol modifications significantly precede the general redox collapse. Oxidation of these early target proteins, which most likely results in a loss of their activity, might contribute to or even cause the observed loss of redox homeostasis (i.e., thioredoxin reductase) in chronologically aging yeast. These studies in aging yeast expand our understanding how changes in redox homeostasis affect the life span of yeast cells and confirm the importance of oxidative thiol modifications as key posttranslational modifications in pro- and eukaryotic organisms
Cazzanelli, Giulia. "Study the roles of human galectin-3 using the yeast Saccharomyces cerevisiae and colorectal cancer cells as eukaryotic models." Doctoral thesis, 2017. http://hdl.handle.net/1822/48637.
Full textGalectins are lectins characterized by a conserved CRD with a high affinity for β-galactosides. When localized extracellularly, they mainly interact with glycans on the surface of the cells, promoting cell-cell adhesion, the onset of immune response, and the recognition and clearance of pathogens. When intracellular, they interact with other proteins, modulating cell proliferation, cell cycle progression and apoptosis. The focus of the present work was galectin-3, the only chimera type galectin, aiming at turning the yeast Saccharomyces cerevisiae into a suitable model to study the roles and mechanisms of action of gal-3. Specifically, the work intended to assess the ability of extracellular gal-3 to recognize and bind yeast cells, as well as the ability of intracellular gal-3 to interfere with yeast cell survival and proliferation, in particular when interacting with the oncoprotein KRAS. The work also aimed at uncover the role of gal-3/KRAS/p16INK4a axis regulation in colorectal cancer (CRC) cells. Thus, both S. cerevisiae and human cancer cell lines were used as eukaryotic models. The effects caused by gal-3 on yeast biological processes were compared with those caused by other galectins with different structure and number of CRDs (the proto-type gal- 1 and gal-7, and the tandem repeat gal-4). S. cerevisiae and Candida albicans were used, as species with a very different biology, and because C. albicans was the only yeast in the literature tested in regard to gal-3. Each galectin caused a different pattern of effects, generally stronger on S. cerevisiae than on C. albicans. Gal-3 decreased viability and increased cell size, ROS level and DNA alterations, but did not induce membrane rupture, plasmatic nor mitochondrial, indicating that the stress it induces is not associated with cell death (necrotic or programmed). Gal-3 effects were mostly caused by its CRD (as shown using the truncated version of the galectin), and mediated by the Ras/cAMP/PKA pathway (demonstrated using the S. cerevisiae mutants). Gal-4 and gal-7 increased the levels of ROS and membrane rupture, without affecting viability. Gal-1 did not induce any significant alteration. A microarray-based analysis of the binding ability of galectins, in accordance with the results above, showed that all galectins except gal-1 bound to whole cells of S. cerevisiae and C. albicans, more efficiently to S. cerevisiae, in particular the correspondent Δras2 mutant and cell wall and membrane sub-cellular fractions. A S. cerevisiae-based high throughput platform (HTP) expressing human gal-3 and KRAS was built with the purpose of achieving the expression of both human cDNAs in the same strain. This is meant to enable in vivo study of the functional relations between the two proteins, and to serve as a HTP for pharmacological screening of drugs/molecules targeting either gal-3, KRAS or their interaction. Two different genetic backgrounds were used (W303- 1A and BY4741), wild type as well as Δras1 and Δras2 mutants. Gal-3 expression was fully achieved, altering growth rate and chronological life span. These phenotypes depended on the presence of Ras1 and/or Ras2. Human KRAS expression in wt yeast also caused phenotype variations, decreasing yeast resistance to various stress stimuli, most possibly due to the hyperactivation of the Ras/cAMP/PKA pathway. The double expression of gal-3 and KRAS in the same S. cerevisiae strain was attempted using several cloning strategies, and will be pursued in the future. Bearing this goal in mind, as well as the potential use for which the platform was built, human CRC cell lines were used to better understand the gal-3/ KRAS interaction and their role in CRC progression. The pivotal interaction of gal-3 and KRAS were confirmed, and a third protein was shown to belong to this regulation axis, p16INK4a. The three proteins physically interacted and co-localized in CRC cells, and there seems to be a reciprocal regulatory mechanism that might control their expression levels, adjusting it to the growing needs of the cancer cell. In conclusion, this work breaks through several boundaries: (i) galectins bind to yeasts, (ii) the binding is specific, (iii) it causes also specific responses from the yeast cell, (iv) these are mediated by Ras pathway, and (v) once intracellular, gal-3 induces different responses still mediated by Ras pathway. Importantly, in opposition to the scarce literature available, the work showed S. cerevisiae to be more sensitive than C. albicans, stressing the straindependent specificity of galectins recognition, at the same time discarding the suggestion that gal-3 might have the role of distinguishing between pathogenic and non- pathogenic yeasts in vivo. Moreover, p16INK4a was identified as a new member of KRAS/gal-3 regulation axis in CRC cells. All taken, the work launched the foundations for assuming and using S. cerevisiae as a model to study gal-3.
As galectinas são proteínas caraterizadas por possuírem um domínio de reconhecimento de carbohidratos (CRD) com elevada afinidade para β-galactosídeos. Quando no meio extracelular, as galectinas interagem com glucanos na superfície das células, promovendo reconhecimento e adesão, a resposta imunitária e a eliminação de patogéneos. Intracelularmente, as galectinas interagem com outras proteínas modulando a proliferação e o progresso do ciclo celular e a morte celular programada. O trabalho focou na galectina-3, o único membro da família das galectinas quiméricas. O objectivo do trabalho foi transformar a levedura Saccharomyces cerevisiae num modelo celular para estudar os papéis da gal-3. Para isso foi verificada a habilidade da gal-3, quando extracelular, de reconhecer à célula de levedura, quando intracelular, de interferir com a sobrevivência e capacidade proliferativa da levedura, muito em particular em relação à oncoproteína KRAS. O trabalho tinha também como objetivo perceber o papel da regulação do eixo of gal-3/KRAS/p16INK4a em células de cancro colorretal (CCR). Neste contexto foram usados a S. cerevisiae e células de cancro humano como modelos eucariotas. Os efeitos da presença extracelular de gal-3 foram comparados com os causados por outras galectinas com diferentes estrutura, as gal-1 e gal-7 do grupo de galectinas proto-tipo, e a gal-4 do grupo de galectinas repetição em tandem. Os efeitos destas galectinas foram comparados em estirpes de S. cerevisiae e Candida albicans, que foram escolhidas pela sua distinta biologia, e porque C. albicans era a única espécie de levedura referida na literatura sobre efeitos de galectinas em leveduras. Cada galectina provocou uma combinação de efeitos diferente, que foram genericamente mais fortes em S. cerevisiae do que em C. albicans. Em particular, gal-3 diminuiu a viabilidade da levedura e aumentou o tamanho das células, os níveis de ROS e as alterações no DNA, mas não induziu ruptura de membranas, plasmática ou mitocondrial, indicando que gal-3 induz stress sem acionar nenhum processo de morte celular, necrótica ou programada. A utilização de uma gal-3 truncada no terminal N mostrou que o CRD é suficiente para induzir os efeitos observados. A utilização de leveduras mutadas nos genes RAS mostrou que a gal-3 opera através da via Ras/cAMP/PKA. Em comparação, gal-1 não induziu qualquer efeito significativo, enquanto as gal-4 e gal-7 provocaram respostas que sugerem uma resposta de stress genérica. Através de um ensaio de binding microarrays com células de levedura inteiras e fracções sub-celulares, verificou-se que todas as galectinas, excepto gal-1, se ligam à parede e membrana de S. cerevisiae e C. albicans, mais eficientemente de S. cerevisiae, e mais ainda ao mutante Δras2 desta levedura. Foi construída uma plataforma de estirpes de levedura exprimindo os cDNA de gal-3 e KRAS humanos, com o objectivo de obter a expressão conjunta destas duas proteínas. A plataforma destina-se ao estudo das suas funções e interação, bem como a servir de ferramenta para ensaios em larga escala de moléculas e drogas farmacológicas dirigidas para a gal-3, o KRAS, ou para a sua interação. Foram usadas leveduras de dois fungos genéticos distintos (W303- 1A e BY4741), selvagens e mutantes defectivos nos genes RAS1 e RAS2. A expressão da gal-3 em levedura alterou a taxa de crescimento e o envelhecimento cronológico na dependência da via Ras/cAMP/PKA. A expressão do KRAS em levedura também provocou alterações de comportamento na extirpe selvagem devidas possivelmente à sobre-ativação da mesma via. A obtenção de uma estirpe exprimindo simultaneamente gal-3 e KRAS foi tentada usando várias estratégias de clonagem, e será possivelmente alcançada no futuro. Tendo em conta os objectivos a que se destina a plataforma, foram usadas células de CCR. A interação da gal-3 e KRAS foi confirmada e demonstrou-se interação deste complexo com a p16INK4a. Estas três proteínas interagem fisicamente e colocalizam em células de CCR, parecendo haver um mecanismo de regulação reciproca que parece controlar os seus níveis de expressão, ajustando-os mediante as necessidades de crescimento da célula de cancro. Em conclusão, o trabalho quebra barreiras mostrando: (i) que as galectinas se ligam às células de levedura, (ii) de forma específica, (iii) induzindo respostas também específicas, (iv) mediadas pela via Ras, (v) igualmente quando a gal-3 é expressa intracelularmente em S. cerevisiae. Em oposição à escassa literatura disponível, mostrou-se que S. cerevisiae é mais sensível que C. albicans, sublinhando a influência da estirpe na especificidade da resposta e ligação às galectinas, e afastando a sugestão de que gal-3 possa exercer in vivo uma função discriminatória entre leveduras patogénicas e não-patogénicas. Além disso, foi identificada a p16INK4a como um novo membro do eixo de regulação KRAS/gal-3 em CCR. Globalmente o trabalho lançou as fundações para a utilização da levedura S. cerevisiae como modelo para estudar gal-3.
This work was support by the GLYCOPHARM Marie Curie Initial Training Network PITN-GA-2012-317297 and by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI). The work presented in this thesis was performed in the Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho and in the Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC), Instituto de Química-Física Rocasolano, Madrid, Spain.
Barker, Megan. "Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae." Thesis, 2010. http://hdl.handle.net/1807/32660.
Full textdo, Couto Bordignon Pedro. "An analysis of translation heterogeneity in ribosome profiling data." Thesis, 2019. http://hdl.handle.net/1866/24470.
Full textProteins are responsible for virtually all functions performed within and in the surroundings of a cell. The control of gene expression, which determines the amount, localisation and timing of protein production in the cell, is the central processes in the regulation of cellular physiology and function. Any disturbance in this complex system can generate important consequences on cellular integrity, sometimes leading to incurable diseases. The translation of messenger RNA into a protein product is the last step of the gene expression mechanism. It can be regulated in manifold ways, both intrinsically and extrinsically to the transcript sequence. It is also the costliest cellular process in terms of energy. Ribosome profiling (Ribo-Seq) is one of the recent and promising technologies making it possible to better study the mechanisms of translation regulation. Its results have however been shown to display variability in reproducibility and to contain noise of uncharted sources. This work presents the implementation of a strategy for dissociating signals of biological origin from those of technical origin. This is performed by the computation of a consensus profile of ribosomal density derived from a comparative analysis of several Ribo-Seq experiments in yeast (Saccharomyces cerevisiae). The biological signals derived by the consensus profiles correspond with signatures of known ribosomal pauses, such as mRNA folding strength and amino acid charges. Amazingly, our strategy also enabled the identification of differentially transcribed (DT) sequences. The latter have shown an over-representation of codons associated with modifications of transfer RNAs (tRNAs). They are also involved in the control of cellular homeostasis, exhibiting a marked presence in genes involved in ribosome biosynthesis as well as in mRNAs with precise translation sub-localization, particularly in mitochondria and the endoplasmic reticulum (ER). In addition to demonstrating the possibilities of discovery offered by the Ribo-Seq technique, this study also presents evidence of the dynamic and heterogeneous nature of the translation process in the eukaryotic cell. It also showcases its diverse regulatory mechanisms and the role of information directly encoded in the sequence in the general optimization of cellular homeostasis.
Samaddar, Madhuja. "Understanding in vivo Significance of Allosteric Regulation in mtHsp70s : Revealing its Implications in Parkinson's Disease Progression." Thesis, 2015. http://hdl.handle.net/2005/3034.
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