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1
Batenchuk, Cory. "Transcriptional Dynamics of the Eukaryotic Cell." Thesis, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/19722.
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Gene regulatory networks are dynamic and continuously remodelled in response to internal and external stimuli. To understand how these networks alter cellular phenotype in response towards specific challenges, my first project sought to develop a methodology to explore how the strength of genetic interactions changes according to environmental context. Defined as sensitivity-based epistasis, the results obtained using this methodology were compared to those generated under the conventional fitness-based approach. By integrating this information with gene expression profiles and physical interaction datasets, we demonstrate that sensitivity-based epistasis specifically highlights genetic interactions with a dynamic component.
Having investigated how an external stimulus regulates network dynamics, we next sought to understand of how genome positioning impacts transcription kinetics. This feat was accomplished by cloning two gene-reporter constructs, representing contrasting promoter architectures, across 128 loci along chromosome III in S.Cerevisiae. By comparing expression and noise measurements for promoters with “covered” and “open” chromatin structures against a stochastic model for eukaryotic gene expression, we demonstrate that while promoter structure regulates burst frequency (the rate of promoter activation), positional effects in turn appear to primarily modulate burst size (the number of mRNA produced per gene activation event). By integrating these datasets with information describing global chromatin structure, we suggest that the acetylation state of chromatin regulates burst size across the genome. Interestingly, this hypothesis is further supported by nicotinamide-mediated inhibition of Sir2 which would appear to modulate burst size globally across the genome.
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Li, Zhaoqi Ph D. Massachusetts Institute of Technology. "Bioenergetics and metabolism of eukaryotic cell proliferation." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/130658.
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Thesis: Ph. D. in Biochemistry, Massachusetts Institute of Technology, Department of Biology, February, 2021Cataloged from the official PDF of thesis. "February 2021." Vita. Page 179 blank.
Includes bibliographical references.
Cellular growth and proliferation necessitates the transformation of cell-external nutrients into biomass. Strategies of biomass accumulation across the kingdoms of life are diverse and range from carbon fixation by autotrophic organisms to direct biomass incorporation of consumed nutrients by heterotrophic organisms. The goal of this dissertation is to better understand the divergent and convergent modes of metabolism that support biomass accumulation and proliferation in eukaryotic cells. We first determined that the underlying mechanism behind why rapidly proliferating cells preferentially ferment the terminal glycolytic product pyruvate is due to an intrinsic deficiency of respiration to regenerate electron acceptors. We tested this model across an assorted array of proliferating cells and organisms ranging from human cancer cells to the baker's yeast Saccharomyces cerevesiae. We next determined that a major metabolic pathway of avid electron acceptor consumption in the context of biomass accumulation is the synthesis of lipids. Insights from this work has led to the realization that net-reductive pathways such as lipid synthesis may be rate-limited by oxidative reactions. Lastly, we established the green algae Chlorella vulgaris as a model system to study the comparative metabolism of photoautotrophic and heterotrophic growth. We determined that heterotrophic growth of plant cells is associated with aerobic glycolysis in a mechanism that may be suppressed by light. Collectively, these studies contribute to a more holistic understanding of the bioenergetics and metabolic pathways employed by eukaryotic cells to accumulate biomass and lay the foundation for future studies to understand proliferative metabolism.
by Zhaoqi Li.
Ph. D. in Biochemistry
Ph.D.inBiochemistry Massachusetts Institute of Technology, Department of Biology
3
Vezzoli, A. "Analysis of bacterial proteins interfering with eukaryotic cell proliferation." Doctoral thesis, Università degli Studi di Milano, 2006. http://hdl.handle.net/2434/56617.
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Descriptions of a role of some bacterial pathogens in counteracting tumour progression in humans date back to the end of XIX century. Recently, it was reported that two members of the family of cupredoxins, azurin from P. aeruginosa and rusticyanin from A. ferrooxidans, are able to enter in and to induce apoptosis and/or cell cycle arrest in a number of different cancer cell-lines. The apoptotic effect mediated by azurin was confirmed also in vivo. One of the specific aims of this thesis was to assess whether the expression of cupredoxins in S. cerevisiae could recapitulate the effects observed in mammalian cancer cell-lines. The induction of cupredoxins expression in yeast caused a strong growth delay accompanied by high cytotoxicity and alterations in cellular morphologies and bud emergence. These findings validated S. cerevisiae as a system to screen the plethora of bacterial proteins with potential anti-tumoral activity. Since, at least in the case of azurin, the pro-apoptotic effects on cancer cells was traced to direct interaction with the tumor suppressor protein p53, we also investigated the possibility to find new bacterial-derived interactors of p53 through Yeast Two Hybrid System. During this screening we observed the strong interaction of a bacterial protein with murine and human p53. This interaction was extremely specific as P. aeruginosa and E. coli homologs did not exhibit a similar behaviour.
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Cosulich, Sabina Chiara. "Modulators of the cell cycle in fibroblasts." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259439.
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Rindler, Paul Michael. "Eukaryotic replication, cis-acting elements, and instability of trinucleotide repeats." Oklahoma City : [s.n.], 2009.
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6
Beltramini, Amanda Michelle. "Eukaryotic-like serine/threonine kinase signaling in Staphylococcus aureus." Columbus, Ohio : Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1243368504.
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Schreiner, Patrick. "Structural investigation of two supramolecular complexes of the eukaryotic cell." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-92522.
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Doostdar, Hamed. "Stable expression of eukaryotic p450 cDNA in mammalian cell lines." Thesis, University of Aberdeen, 1992. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU602073.
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The P450 profile of HepG2 cells was studied using specific enzyme assays and well characterised monoclonal and polyclonal antibodies. These cells were shown to endogenously express cytochrome P450 1A1, P450 1A2 and P450 3A5 when the cells were treated with benz(a)anthracene or rifampicin respectively. HepG2 cells were adapted to grow in Williams' E medium supplemented with only 5% (v/v) fetal calf serum. These cells showed a significantly higher concentration of P450 1A2 protein when treated with benz(a)anthracene and could be detected by P450 1A2 specific antibodies on Western blots. Stable cell lines of HepG2 and Cos-7 cells capable of continuous expression of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) were constructed. P450 2A6 cDNA was successfully expressed in the EBNA-1 transformed Cos-7 cell line for over 6 weeks. The rate of expression of this cDNA and the enzyme activity of the resulting protein was very low. No stable HepG2 EBNA-1 transformed cells capable of P450 2A6 expression was obtained.
9
Wilson, Timothy Craig. "The role of mRNA stability and Fos protein in transient c-fos mRNA accumulation." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304567.
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Kipling, 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.
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Murant, Susan J. "A molecular biological study of protein disulphide isomerase." Thesis, University of Kent, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.238542.
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Hodges, Matthew Edmiston. "The evolution of eukaryotic cilia." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:47784632-76c9-42ff-a517-76f6b51675b5.
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Eukaryotic cilia are complex, highly conserved microtubule-based organelles with a broad phylogenetic distribution. Cilia were present in the last eukaryotic common ancestor and many proteins involved in cilia function have been conserved through eukaryotic diversification. The evolution of these ciliary functions may be inferred from the distribution of the molecular components from which these organelles are composed. By linking protein distribution in 45 diverse eukaryotes with organismal biology, I define an ancestral ciliary inventory. Analysis of these core proteins allows the inference that the cenancestor of the eukaryotes possessed a cilium for motility and sensory function. I show that the centriolar basal body function is ancestral, whereas the centrosome is specific to the Holozoa, and I use this information to predict a number of roles for proteins based on their phylogenetic profile. I also show that while remarkably conserved, significant divergence in ciliary protein composition has occurred in many lineages, such as the unusual centriole of Caenorhabditis elegans and the transitional changes throughout the land plants. I exemplify this divergence through ultrastructural studies of the fern Ceratopteris richardii and the liverwort Marchantia polymorpha both of which have cilia that exhibit a number of distinctive morphological features, the most conspicuous of which is a general breakdown of canonical microtubule arrangements. Cilia have also been lost multiple times in different lineages: at least twice within the land plants. During these evolutionary transitions proteins with ancestral ciliary functions may be lost or co-opted into different functions. I have interrogated genomic data to identify proteins that I predict had an ancestral ciliary role, but which have been maintained in non-ciliated land plants. I demonstrate that several of these proteins have a flagellar localisation in protozoan trypanosomes and I use expression data correlation to predict potential non-ciliary plant roles.
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Livingstone, Mark. "A nuclear role for the eukaryotic translation initiation factor 4E-binding proteins." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97024.
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The regulation of mRNA translation is crucially important in determining which cellular proteins are produced in response to intracellular and extracellular cues. The resulting collection of functional proteins determines which physiological processes a given cell will carry out; therefore, the deregulation of protein production is strongly implicated in diseases, such as cancer, in which cells fail to appropriately respond to stimuli. The mammalian target of rapamycin(mTOR) signaling pathway, which links amino acid, growth factor, and energy availability to mRNA translation resulting in cellular growth and proliferation, is frequently deregulated in cancer and is an active target for drug discovery. Among the effectors of mTOR signaling are the eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs), which when phosphorylated bymTOR release the mRNA 5'-cap protein eIF4E to promote translation of progrowth/proliferation mRNAs. While previous biochemical fractionation experiments have suggested that 4E-BP1 is exclusively cytoplasmic, we show using immunoassays that this protein is also present in the nuclei of mammalian cells, where it sequesters eIF4E upon mTOR inhibition. This nuclear eIF4E accumulation is useful as a biomarker for mTOR signaling, as we use it as the read-out for a chemical genetic screen for novel mTOR pathway inhibitors. This discovery of a nuclear eIF4E:4E-BP1 complex opens the door to the potential for4E-BPs to impact nuclear RNA processing events. Evidence for and against iv such a nucleus-specific function for 4E-BPs is evaluated and the nuclear function of 4E-BPs is assessed experimentally.La régulation de la traduction des ARN messagers (ARNm) est d'une importance cruciale afin de contrôler quelles protéines sont produites en réponseaux signaux intra- et extracellulaires. La collection de protéines fonctionnelles quien résulte détermine quels processus physiologiques seront effectués par la cellule. Conséquemment, la dérégulation du contrôle traductionnel est fortement impliquée dans plusieurs pathologies, incluant le cancer, ceci dû au fait que les cellules ne répondent pas de manière appropriée aux stimuli qu'elles reçoivent. Une voie de signalisation impliquée dans la croissance et la prolifération cellulaire qui est souvent dérégulée dans les cancers, la voie de la cible mammifère de la rapamycine (mTOR), et qui intègre la disponibilité en acides aminés, facteurs de croissance et énergie avec la traduction des ARNm, est une cible pharmacologique préférentielle. Parmi les effecteurs de la voie mTOR, on retrouve les protéines s'associant au facteur d'initiation de la traduction eIF4E, les 4E-BP, qui lorsqu'elles sont phosphorylées relâchent la protéine liant la coiffe5' des ARNm, eIF4E, promouvant ainsi la traduction des ARNm encodant des protéines impliquées dans la croissance et la prolifération. En contraste avec les résultats de fractionnement subcellulaires reportés précédemment dans la littérature suggérant que 4E-BP1 est une protéine exclusivement cytoplasmique, nous montrons ici, par essais immunologique, que cette protéine est également v résidente du noyau des cellules mammifères où elle séquestre eIF4E suivant l'inhibition de mTOR. Cette accumulation nucléaire de eIF4E est un biomarqueur de choix que nous avons utilisé comme lecture du niveau d'activité de la voie mTOR lors d'un criblage chimio-génétique entrepris dans le but de trouver de nouveaux inhibiteurs de la voie mTOR. Cette découverte d'un complexe nucléaire eIF4E :4E-BP1 à ouvert la porte à une possible fonction des 4E-BP dans certains processus nucléaires. Les évidences en faveur et en opposition àun tel rôle nucléaire spécifique des 4E-BPs sont évaluées et la fonction nucléaire des 4E-BPs est testée expérimentalement.
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Taylor, Bradley Jay. "Development and Characterization of Eukaryotic Biomimetic Liposomes." DigitalCommons@USU, 2004. https://digitalcommons.usu.edu/etd/5508.
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This study developed and characterized phospholipid vesicles, or liposomes, that mimic cell surfaces. Microemulsified liposomes contained biotinylated phosphatidylethanolamine, allowing them to be immobilized to avidin-coated glass. Laminin (LN), glycosphingolipids (GMl and GM3), and Escherichia coli's mechanosensitive channel of large conductance (EcoMscL) were embedded into liposome membranes. It was determined whether these embedded molecules exhibited their physiological roles of adhesion, cell recognition, and mechanosensation, respectively. Confocal laser scanning microscopy (CLSM) was employed to examine the interaction of fluorescently probed proteins, toxins, and bacteria with the immobilized microemulsified liposomes. Capture of individual and simultaneous multiple species of bacteria by GMl, GM3, or LN liposomes was quantified using ELISA and PCR.
Surface-bound liposomes were unilamellar and immovable, allowing removal of unincorporated probes and biomolecules. Liposomes remained intact and stable against leakage of encapsulated sulforhodamine B for several months after immobilization. Functional reconstitution of EcoMscL was examined using CLSM during modulations in the immursing solution.
Cholera toxin(Β subunit) (CTB), bovine lactoferrin (BLF), and E. coli O157:H7 were co-localized proximate to the surface of GMl liposomes. ELISAs determined E. coli O157:H7 and Salmonella enteritidis were captured on GMl liposomes containing GMI at 8.9 molar percent of total lipid. Listeria monocytogenes and Listeria innocua were not captured on the same liposomes.
PCR identified the capture of specific bacterial species from individual species and mixtures of several species on liposomes. Simultaneous assays with mixtures of multiple species showed that the receptor-associated binding of bacteria, described with PCR assays of an individual species, were independent of competitive microorganisms. L. monocytogenes and L. innocua were more frequently bound to LN liposomes than other liposomes, indicating LN promotes adhesion of both the pathogenic and a nonpathogenic strain of Listeria. E. coli O157:H7 was more frequently captured on GMI liposomes than other liposomes, indicating a specificity for this bacteria. S. enteritidis bound to all liposomes, indicating a non-specific interaction.
Known eukaryotic biomolecules implicated in cell recognition, adhesion, and mechanosensation were embedded in a system of artificial bilayers immobilized on a solid support. Liposomes constitute a biomimetic capable of specifically interacting and capturing proteins, toxins, and bacteria in solution.
15
Ford, Jack Ragnar. "Cyclin dependent kinases and cell cycle control in Trypanosoma brucei." Thesis, University of Glasgow, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312512.
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Tan, 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.
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Safaie, Mehran. "Genetic control of hyphal cell growth and polarity in Aspergillus nidulans." Thesis, University of Sheffield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341792.
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Doughty, Tyler W. "Levels of YCG1 Limit Condensin Function during the Cell Cycle: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/861.
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For nearly five decades, the simple eukaryote Saccharomyces cerevisiae has been used as a model for understanding the eukaryotic cell cycle. One vein of this research has focused on understanding how chromosome structure is regulated in relation to the cell cycle. This work characterizes a new mechanism that modulates the chromatin organizing condensin complex, in hopes of furthering the understanding of chromosome structure regulation in eukaryotes.
During mitosis, chromosomes are condensed to facilitate their segregation through a process mediated by the condensin complex. Upon interphase onset, condensation is reversed, allowing for efficient transcription and replication of chromosomes. This work demonstrates that Ycg1, the Cap-G subunit of budding yeast condensin, is cell-cycle regulated with levels peaking in mitosis and decreasing as cells enter G1 phase. The cyclical expression of Ycg1 is unique amongst condensin subunits, and is established by a combination of cell cycle-regulated transcription and constitutive proteasomal degradation. Interestingly, when cyclical expression of Ycg1 is disrupted, condensin formation and chromosome association increases, and cells exhibit a delay in cell-cycle entry. These results demonstrate that Ycg1 levels limit condensin function, and suggest that regulating the expression of an individual condensin subunit helps to coordinate chromosome conformation with the cell cycle. These data, along with recent corroborating results in Drosophila melanogaster suggest that condensin regulation through limiting the expression of a single condensin subunit may be broadly conserved amongst eukaryotes.
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Doughty, Tyler W. "Levels of YCG1 Limit Condensin Function during the Cell Cycle: A Dissertation." eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/861.
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For nearly five decades, the simple eukaryote Saccharomyces cerevisiae has been used as a model for understanding the eukaryotic cell cycle. One vein of this research has focused on understanding how chromosome structure is regulated in relation to the cell cycle. This work characterizes a new mechanism that modulates the chromatin organizing condensin complex, in hopes of furthering the understanding of chromosome structure regulation in eukaryotes.
During mitosis, chromosomes are condensed to facilitate their segregation through a process mediated by the condensin complex. Upon interphase onset, condensation is reversed, allowing for efficient transcription and replication of chromosomes. This work demonstrates that Ycg1, the Cap-G subunit of budding yeast condensin, is cell-cycle regulated with levels peaking in mitosis and decreasing as cells enter G1 phase. The cyclical expression of Ycg1 is unique amongst condensin subunits, and is established by a combination of cell cycle-regulated transcription and constitutive proteasomal degradation. Interestingly, when cyclical expression of Ycg1 is disrupted, condensin formation and chromosome association increases, and cells exhibit a delay in cell-cycle entry. These results demonstrate that Ycg1 levels limit condensin function, and suggest that regulating the expression of an individual condensin subunit helps to coordinate chromosome conformation with the cell cycle. These data, along with recent corroborating results in Drosophila melanogaster suggest that condensin regulation through limiting the expression of a single condensin subunit may be broadly conserved amongst eukaryotes.
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Linka, Marc. "Understanding the origin and function of organellar metabolite transport proteins in photosynthetic eukaryotes Galdieria sulphuraria and Arabidopsis thaliana as model systems /." Diss., Connect to online resource - MSU authorized users, 2008.
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Marshall, Philip. "Expression of measles fusion protein in insect and human cells using Eukaryotic expression vectors." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61265.
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Measles virus is an animal enveloped virus that is a member of the genus morbillivirus in the paramyxoviridae family. Its envelope contains two surface glycoproteins H and F which are required for viral attachment and entry respectively. Virus penetration occurs via a process which involves fusion of the viral membrane with the plasma membrane at the cell surface. Replication of the virus thus follows and leads to giant cell (syncytia) formation.The infectivity of measles virus is dependent upon a host proteolytic cleavage of the F$ sb0$ glycoprotein into two active subunits F$ sb1$ and F$ sb2$. This cleavage was later shown to expose a hydrophobic sequence at a NH$ sb2$ terminal of the F$ sb1$ which is directly involved in cell fusion and virus penetration.
In order to increase our knowledge concerning cell mediated fusion events we have expressed the fusion glycoprotein of measles virus in insect and human cells by using recombinant baculo- and adenoviruses respectively. Analysis by SDS-PAGE demonstrated that our protein was first synthesized as a 60 Kd protein and cleaved subsequently into its two respective subunits F$ sb1$ and F$ sb2$ of 40 Kd and 20 Kd respectively. Hemolysis assays confirmed the biological activity of this protein in both systems. However, the fusion protein was unable to fuse insect cells.
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Stephens, Matthew Jon Craig. "DNA aptamers that selectively label eukaryotic cells depending on the expression of the cell surface protein, P2X7." Thesis, University of Portsmouth, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.618284.
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The ATP gated cation channel P2X7, is a cell surface protein whose function and activation is linked with several human diseases. The aim of this project was to raise DNA Aptamers targeting specifically the extracellular domain of the P2X7 protein with the hope to use these new ligands as a method for the study of the biological functions of the P2X7 protein and its potential as a therapeutic target. The strategy involved isolating Aptamers that would bind to human em~ryonic kidney cells transformed with a construct which forced the cells to express native mouse P2X7 on its cell surface (POSITIVE SELECTION), whilst not binding to untransformed non-P2X7 expressing cells (NEGATIVE SELECTION). Two candidate sequences from this selection emerged as, what we called, "winners" which were the most likely sequences to putatively bind mP2X7 (work ongoing). Interestingly using the procedure in reverse, that is to create Aptamers that specifically bind to non-transformed cells and not transformed cells, also produced viable putative Aptamers, which, once labelled, showed selective binding. These results are discussed in relation to future applications and the potential insights and implications for the study of cell surface protein expression patterns. 3
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Amasino, Audra Leigh. "Keep the ORCs at bay : how eukaryotic cells ensure one round of DNA replication per cell cycle." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/128988.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2020Cataloged from student-submitted PDF of thesis.
Includes bibliographical references.
During each cell cycle, eukaryotic cells must faithfully replicate their genome, ensuring exactly one full copy is made. Both under-replicating or over-replicating the genome can have deleterious consequences including cell death, genome instability and cancer. Thus, this process is tightly regulated. The major mechanism to ensure that DNA is replicated once per cell cycle entails the temporal separation of two key replication events: helicase loading and helicase activation. Helicase loading occurs during the G1 phase of the cell cycle. In S. cerevisiae cells, Cyclin-Dependent Kinases (CDKs) prevent helicase loading outside of G1 by phosphorylating three of the four helicase-loading proteins: Mcm2-7, Cdc6, and the Origin Recognition Complex (ORC). Phosphorylation of free Mcm2-7 and Cdc6 leads to their removal from the nucleus (Mcm2-7 by nuclear export and Cdc6 by protein degradation). However, phosphorylated ORC remains in the nucleus bound to origins.
ORC phosphorylation intrinsically inhibits the helicase loading reaction. In in vitro reconstituted helicase loading reactions, CDK phosphorylation of ORC is sufficient to completely inhibit helicase loading. However, the precise event(s) during helicase loading that are affected by ORC phosphorylation were not known prior to this study. To identify the steps of helicase loading that are inhibited by ORC phosphorylation, we used single-molecule microscopy to compare the progression of helicase loading with phosphorylated versus unphosphorylated ORC. Successful helicase loading results in two head-to-head Mcm2-7 helicases encircling DNA. We show that ORC phosphorylation prevents loading of both the first and second Mcm2-7 complexes. An initial intermediate in helicase loading containing origin DNA and all four proteins (the OCCM) still forms when ORC is phosphorylated, albeit slower.
Focusing on events after OCCM formation, we found that ORC phosphorylation alters Cdt1 dissociation kinetics and inhibits successful Mcm2-7 ring closing. ORC is phosphorylated on both the Orc2 and Orc6 subunits in vivo; we find that in vitro phosphorylation of either single subunit leads to nearly identical effects as phosphorylation of both subunits. My studies suggest a model in which ORC directly controls Mcm2-7 ring closing through physical interactions with both Cdt1 and Mcm2-7 and these interactions, and thus ring closing, are inhibited by ORC phosphorylation.
by Audra Leigh Amasino.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology
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March, John Clifton. "Metabolic engineering of eukaryotic signal transduction in Drosophila Schneider 2 (S2) cell culture." College Park, Md. : University of Maryland, 2005. http://hdl.handle.net/1903/2450.
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Thesis (Ph. D.) -- University of Maryland, College Park, 2005.Thesis research directed by: Chemical Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Coles, Lucy Clare. "Regulation of eukaryotic translational initiation in transformed and differentiation inducible haemopoietic cell lines." Thesis, University of Leicester, 1998. http://hdl.handle.net/2381/29712.
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Mechanisms which regulate gene expression at the level of protein synthesis play an important role in the control of cell proliferation, and de-regulation of translation has been shown to cause cell transformation. Haemopoiesis is a complex and highly regulated process of cell proliferation, differentiation and apoptosis which is subject to a variety of neoplastic disorders. Control of translation is usually effected through modulation of the eukaryotic initiation factors (eIFs) and cultured cells of haemopoietic origin were chosen to study eIFs and their regulation. The eIFs were examined in cell lines derived from patients with the B-cell tumours Burkitt's lymphoma and Multiple Myeloma. Study of the expression of the proto-oncogene c-myc and its transcriptional targets, eIF4E and eIF2 demonstrated no correlation between the eIFs and c-myc expression. Investigation of another component of the eIF4F complex revealed that eIF4G is cleaved into specific N- and C-terminal fragments in the Multiple Myeloma cell line GM2132. This suggests that in these cells there may be an advantage for mRNAs translated by internal ribosome entry. HL60, a differentiation inducible leukaemic cell line, was used to investigate alterations in protein synthesis during granulocytic differentiation. A 2-fold increase in eIF4E phosphorylation was observed which does not affect the protein synthesis rate. This implies that the increase in eIF4F phosphorylation may specifically affect the translation of a sub-set of mRNAs. Differentiating HL60 cells were also used to study the relationship between c-myc, eIF4E and eIF2 expression. No correlation between expression of c-myc and eIF4E was found in this system. However, eIF2 expression did appear to be responsive to c-Myc during the initial stages of granulocytic differentiation. Investigation of a serum response in HL60 cells demonstrated that association of eIF4G with eIF4E is modulated by alterations in eIF4G protein levels. This suggests that eIF4E is not a limiting factor in the initiation of translation in HL60 cells.
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Jilkine, Alexandra. "A wave-pinning mechanism for eukaryotic cell polarization based on Rho GTPase dynamics." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/17453.
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In response to chemical stimulation, many eukaryotic cells are able to sense the direction of the stimulus and initiate movement. To do so, the cell must break symmetry and develop a front and back in a process known as polarization.
During polarization, members of the Rho GTPase family (Cdc42, Rac, and Rho) are recruited to the plasma membrane and localize to form a front and a back of the polarizing cell. These proteins exist in both active forms (on the inner surface of the membrane of the cell), and inactive forms (in the cytosol).
In earlier work, I have shown that the property of membrane-cytosol interconversion, together with appropriate feedbacks, endows the Rho proteins with the ability to initiate cell polarity, resulting in a high Cdc42/Rac region, which will become the front, and a high Rho region, which will become the back of the cell.
Here I show that this property of polarizability can be explained using a simplified model system comprising of a single active/inactive protein pair with positive feedback.
In this model, a travelling wave of GTPase activation is initiated at one end of the domain, moves across the cell, and eventually stops inside the domain, resulting in a stable polar distribution.
The key requirements for the mechanism to work include conservation of total amount of protein, a sufficiently large difference in diffusion rates of the two forms, and nonlinear positive feedback that allows for multiple homogeneous steady states to exist.
Using singular perturbation theory, I explain the mathematical basis of wave-pinning behaviour, and discuss its biological and mathematical implications. I show that this mechanism for generating a chemical pattern is distinct from Turing pattern formation. I also analyze the transition from a spatially heterogeneous solution to a spatially homogeneous solution as the diffusion coefficient of the active form is increased, and show the existence of other unstable stationary wavefronts. Finally, I argue that this wave-pinning mechanism can account for a number of features of cell polarization such as spatial amplification, maintenance of polarity, and the sensitivity to new stimuli that is typical of polarization of eukaryotic cells.
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Tarrant, Daniel S. J. "Investigating the effects of increased levels of the translation elongation factor eEF1A within eukaryotic cells." Thesis, University of Kent, 2015. https://kar.kent.ac.uk/48089/.
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The highly conserved eukaryotic elongation factor 1A (eEF1A) plays a canonical role in translation elongation, where it is responsible for delivering the aminoacylated tRNA to the A-site of the 80S ribosome. Further to this essential role it is reported to be involved in a plethora of moonlighting functions that are not fully characterised or understood. One of the human isoforms, eEF1A2, is known to induce cancer when expressed in non-native tissues, although the mechanism by which it promotes tumour growth is not yet known. In this study we have characterised eEF1A overexpression in yeast and provided evidence to suggest that elevated levels of eEF1A result spindle defects which lead to chromosomal abnormalities that have the potential to induce uncontrolled cell growth in human cells. Moreover, we have confirmed conservation of this chromosomal abnormality in human cell lines suggesting that the mechanism that eEF1A utilises to induce these effects are highly conserved. We have also observed that in yeast, eEF1A overexpression results in increased metabolic activity, a hallmark of cancer cells. We hypothesise that eEF1A interacts with the dynactin complex, a regulator of spindle dynamics, resulting in aberrant spindle formation. This in turn leads to chromosomal abnormalities that appear toxic to the cell. Cells appear to overcome the toxicity induced by eEF1A by suppressing plasmid copy number to the lowest levels possible. This however, brings its own problems and appears to result in synthetic effects together with eEF1A overexpression.
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Conradie, Riaan. "A comparative analysis of the G1/S transition control in kinetic models of the eukaryotic cell cycle." Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/1236.
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Thesis (PhD (Biochemistry))--University of Stellenbosch, 2009.ENGLISH ABSTRACT: The multiplication of cells proceeds through consecutive phases of growth and division (G1, S, G2 and M phases), in a process known as the cell cycle. The transition between these phases is regulated by so-called checkpoints, which are important to ensure proper functioning of the cell cycle. For instance, mutations leading to faulty regulation of the G1/S transition point are seen as one of the main causes of cancer. Traditionally, models for biological systems that show rich dynamic behavior, such as the cell cycle, are studied using dynamical systems analysis. However, using this analysis method one cannot quantify the extent of control of an individual process in the system. To understand system properties at the process level, one needs to employ methods such as metabolic control analysis (MCA). MCA was, however, developed for steady-state systems, and is thus limited to the analysis of such systems, unless the necessary extensions would be made to the framework. The central question of this thesis focuses on quantifying the control in mathematical models of the G1/S transition by the individual cell cycle processes. Since MCA was never applied to the cell cycle, several new methods needed to be added to the framework. The most important extension made it possible to follow and quantify, during a single cell cycle, the control properties of the individual system processes. Subsequently, these newly developed methods were used to determine the control by the individual processes of an important checkpoint in mammalian cells, the restriction point. The positioning of the restriction point in the cell cycle was distributed over numerous system processes, but the following processes carried most of the control: reactions involved in the interplay between retinoblastoma protein (Rb) and E2F transcription factor, reactions responsible for the synthesis of Delayed Response Genes and Cyclin D/Cdk4 in response to growth signals, the E2F dependent Cyclin E/Cdk2 synthesis reaction, as well as the reactions involved in p27 formation. In addition it was shown that these reactions exhibited their control on the restriction point via the Cyclin E/Cdk2/p27 complex. Any perturbation of the system leading to a change in the restriction point could be explained via its e ect on the Cyclin E/Cdk2/p27 complex, showing a causal relation between restriction point positioning and the concentration of the Cyclin E/Cdk2/p27 complex. Finally, we applied the new methods, with a modular approach, to compare a number of cell cycle models for Saccharomyces cerevisiae (budding yeast) and mammalian cells with respect to the existence of a mass checkpoint. Such a checkpoint ensures that cells would have a critical mass at the G1/S transition point. Indeed, in budding yeast, a correction mechanism was observed in the G1 phase, which stabilizes the size of cells at the G1/S transition point, irrespective of changes in the specific growth rate. This in contrast to the mammalian cell cycle models in which no such mass checkpoint could be observed in the G1 phase. In this thesis it is shown that by casting specific questions on the regulation and control of cell cycle transition points in the here extended framework of MCA, it is possible to derive consensus answers for subsets of mathematical models.
AFRIKAANSE OPSOMMING: Die selsiklus bestaan uit agtereenvolgende groei- en delingsiklusse wat tot selvermeerdering lei. Die siklus word gekenmerk deur onderskeie fases (G1, S, G2 en M) wat deur sogenaamde beheerpunte gereguleer word. Hierdie beheerpunte verseker dat selvermeerdering nie ongekontroleerd kan plaasvind nie en mutasies wat lei tot foutiewe regulering van die G1/S transisiepunt word as een van die hoofoorsake van kanker beskou. Die hoofdoel van hierdie studie was om die beheer wat selsiklusprosesse op die G1/S transisie uitoefen met behulp van wiskundige modelle te kwantifiseer. Omdat biologiese sisteme soos die selsiklus ryk dinamiese gedrag vertoon, word hulle tradisioneeldeur middel van dinamiese sisteemanalise bestudeer. Die analisemetode beskik egter nie oor die vermoë om die hoeveelheid beheer wat afsonderlike sisteemprosesse op 0n sisteemeienskap uitoefen te kwantifiseer nie. Om sisteemeienskappe op prosesvlak te verstaan moet metodes soos metaboliese kontrole analise (MKA) ingespan word. MKA was egter ontwikkel om sisteme in 0n bestendige toestand te analiseer en aangesien MKA nog nooit vantevore vir selsiklus analises gebruik was nie, moes nuwe MKA tegnieke gedurende die studie ontwikkel word. Die belangrikste van die metodes maak dit moontlik om beheer (soos uitgeoefen deur die onderskeie sisteemprosesse) oor 0n enkele selsiklus na te volg en te kwantifiseer. Die nuut-ontwikkelde metodes was vervolgens gebruik om te bepaal hoe een so 0n beheerpunt in soogdierselle - die restriksiepunt - deur die onderskeie sisteemprosesse beheer word. Die studie het aangedui dat die posisie van die restriksiepunt tydens die selsiklus deur ’n verskeidenheid sisteemprosesse beheer word. Die bevinding was dat vier prosesse beduidend meer beheer op die posisie van die restriksiepunt uitoefen: Reaksies wat betrekking het op die wisselwerking tussen retinoblastoma proteïen (Rb) en E2F transkripsiefaktor; reaksies verantwoordelik vir die sintese van vertraagde responsgene en Siklien D/Cdk4 in respons tot groeiseine; die E2F afhanklike Siklien E/Cdk2 sintesereaksie; sowel as die reaksies betrokke in p27 vorming. Daar was ook aangetoon dat hierdie reaksies hul beheer op die posisie van die restriksiepunt deur die Siklien E/Cdk2/p27 kompleks uitoefen, siende enige sisteemversteuringe (wat tot veranderinge in die restriksiepuntposisie aanleiding gee) deur veranderinge in die kompleks verklaar kon word - 0n observasie wat aandui dat daar 0n kousale verhouding is tussen die posisie van die restriksiepunt en die Siklien E/Cdk2/p27 kompleks. Die nuut-ontwikkelde metodes was verder gebruik om 0n verskeidenheid selsiklusmodelle van Saccharomyces cerevisiae (bakkersgis) en soogdierselle met 0n modulêre aanpak te vergelyk om te bepaal of daar 0n massa beheerpunt in beide soogdier- en bakkersgisselle bestaan. Daar word gepostuleer dat hierdie beheerpunt verseker dat selle 0n kritiese massa by die G1/S transisiepunt bereik. Die resultate van die studie dui daarop dat bakkersgis, anders as soogdierselle, oor so 0n korreksiemeganisme beskik. Die meganisme stabiliseer die grootte van selle in die G1 fase ondanks veranderinge in die groeitempo van die selle, sodat massa homeostaties by die G1/S transisiepunt gehandhaaf word. Die studie het getoon dat moeilike vrae met betrekking tot die selsiklus beantwoord kan word deur van wiskundige modelle gebruik te maak en die probleme in die nuut-ontwikkelde metaboliese kontrole analise raamwerk te giet.
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Tam, Chun-yee. "A novel role of the E3 ubiquitin ligase as a transcription regulation in eukaryotic cell nucleus." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43278528.
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Steinmetz, Ralf Dirk. "Functional expression of recombinant N-methyl-D-aspartate (NMDA) receptors in eukaryotic cell lines." [S.l.] : [s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=961238070.
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Thompson, Michael Todd. "Quantifying effects of substrata chemomechanical properties on eukaryotic and prokaryotic cell adhesion and morphology." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/45915.
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Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008.Includes bibliographical references (p. 193-201).
It is now widely accepted that cells are capable of processing both mechanical and chemical signals from the extracellular environment. Exactly how these two factors affect the cell biology in the context of physiological circumstances is an area of intense interest that has given rise to an entire field of study called cell mechanotransduction. The unambiguous decoupling of mechanical and chemical properties that stimulate cell development and phenotypic change is challenging from an experimental standpoint. This thesis describes some of the first studies of chemomechanical coupling arising from anchorage-dependent forces between cells and a versatile class of chemically and mechanically tunable polymer thin films, termed polyelectrolyte multilayers. Specifically, investigation of the effects of extracellular chemomechanical stimulation on cell morphology and adhesion in the eukaryotic cells such as vascular endothelial cells and fibroblasts; and the adhesion of prokaryotic cells S. epidermidis and E. coli are presented. Endothelial cells (EC) comprise a major portion of the cell population in the human body. Because of the extensive distribution of endothelial cells in various tissues, they function across a broad range of mechanical and chemical environments. Furthermore, a general understanding of how mechanical forces contribute to the development of cellular function is an important aspect in the development of therapeutic techniques and materials capable of addressing a wide spectrum of human diseases and injuries. Cell adhesion to extracellular matrices and tissues can be indicative of underlying molecular processes in both healthy and disease states.
(cont.) Through the use of a mechanically tunable class of polymer thin films called polyelectrolyte multilayers (PEMs) developed by Rubner et al., we have demonstrated that the adhesion and morphology of human microvascular endothelial cells depend directly on the mechanical stiffness of these synthetic substrates, as quantified by the nominal elastic modulus E. Characterization of the mechanical properties and surface features of PEMs is attained via scanning probe microscopy (SPM) and SPM-enabled nanoindentation. Typical cellular response to increased substrata stiffness includes increased number of cells adhered per unit substratum area. We have further demonstrated that the chemical and mechanical signals imposed at the cell-substrata interface can be decoupled, thereby providing two independent parameters capable of controlling cell behavior. This capacity of the cell to sense and/or exert chemical and mechanical forces, in addition to initiating a sustained molecular response, is termed the chemomechanical response element. Finally, adhesion dependent mechanosensation in bacteria is explored, with respect to the chemomechanical response elements common to eukaryotic and prokaryotic cells. Potential applications towards the development of therapeutic materials and compounds for treatment of various disease states are discussed, with particular attention to limiting hospital acquired infections.
by Michael Todd Thompson.
Ph.D.
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Mills, Anthony David. "The use of a plasmid maintenance system to control eukaryotic cell survival and proliferation." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619947.
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Tam, Chun-yee, and 譚雋怡. "A novel role of the E3 ubiquitin ligase as a transcription regulation in eukaryotic cell nucleus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43278528.
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Solis, Eric John. "Defining the Essential Function of Yeast Hsf1 Reveals a Compact Transcriptional Program for Maintaining Eukaryotic Proteostasis." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493256.
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Despite its eponymous association with proteotoxic stress, heat shock factor 1 (Hsf1 in yeast and HSF1 in mammals) is required for viability of yeast and many human cancer cells, yet its essential role remains undefined. Here we show that rapid nuclear export of Hsf1 achieved in a matter of minutes by a chemical genetics approach results in cell growth arrest in a matter of hours, which was associated with massive protein aggregation and eventual cell death. Genome-wide analyses of immediate gene expression changes induced by Hsf1 nuclear export revealed a basal transcriptional program comprising 18 genes, predominately encoding chaperones and other proteostasis factors. During heat shock, Hsf1 increases the magnitude of its transcriptional program without expanding its breadth. Strikingly, engineered Hsf1-independent co-expression of just Hsp70 and Hsp90 chaperones enabled robust cell growth in the complete absence of Hsf1. A comparative genomic analysis of mammalian fibroblasts and embryonic stem cells revealed that HSF1 lacks a basal transcriptional program but still regulates a similar set of chaperone genes during heat shock. Our work demonstrates that basal chaperone gene expression is a housekeeping mechanism controlled by Hsf1 in yeast and serves as a roadmap for defining the housekeeping function of HSF1 in many cancers. Finally, we investigate the mechanism causing age-associated inactivation of Hsf1 during replicative aging in budding yeast. Using classic and chemical-genetic tools, we demonstrate that inactivation is due to constitutive activation of the distinct General Stress Response. These experiments reveal that stress pathway crosstalk inhibits Hsf1 activation during replicative aging and under physiological stress in young cells.Systems Biology
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Leon, 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.
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Thesis (Ph.D. in Biophysics & Genetics, Program in Molecular Biology) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 90-98). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
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Meijer, Lisa. "Signalling and activation of TLR4 by Gram-negative bacteria in epithelial cells /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-560-3/.
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Ehrenborg, Linda. "Generation and characterization of a prostate-specific membrane antigen positive eukaryotic cell system for phage selection." Thesis, KTH, Proteinvetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-302128.
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Prostate cancer is one of the most common cancer types worldwide. However, current diagnostic approaches and treatments are invasive and unspecific. Prostate-specific membrane antigen (PSMA) is an ideal biomarker for prostate cancer and can act as a target for therapeutic or diagnostic agents. Previous attempts to develop an affibody with affinity towards PSMA have been unsuccessful, therefore this thesis aimed at making the affibody selections against PSMA more efficient. In this thesis HEK293 cells expressing a modified version of PSMA containing a 3C protease cleavage site were generated, to enable extraction of the extracellular domain of PSMA during the selections. However, further analyses must be performed to determine if the extracellular domain can be successfully cleaved off. To develop an affibody that can be used both in vitro and in vivo, selections will be carried out against recombinant PSMA as well. The recombinant PSMA was previously produced incorporating an Avi tag for site-specific biotinylation and immobilization for the selections. To biotinylate the recombinant PSMA, the enzyme BirA that catalyzes the biotinylation of the Avi tag, was produced. A protein yield of 8.95 mg/liter culture was obtained and the site-specific biotinylation was highly efficient. To evaluate the proposed affibody selection strategy the next step is to determine if cleavage of the PSMA expressed on the HEK293 cells is possible, optimize the cleavage conditions and to start initial selections using the generated HEK293 cells and the produced BirA enzyme.Prostatacancer är en av de mest förekommande cancertyperna över hela världen. Nuvarande diagnostiska metoder och terapeutiska behandlingar är dock invasiva och ospecifika. Prostataspecifikt membranantigen (PSMA) är en idealisk biomarkör för prostatacancer och kan agera som en målmolekyl för terapeutiska eller diagnostiska ändamål. Tidigare försök att utveckla en affibody med affinitet mot PSMA har inte lyckats, därför var målet med detta examensarbete att effektivisera selekteringen av affibodies mot PSMA. I detta projekt har HEK293 celler som uttrycker en modifierad version av PSMA, innehållande ett 3C-proteas- klyvningsställe, genererats för att möjliggöra extraktion av den extracellulära domänen av PSMA under selekteringen. Ytterligare analyser måste dock utföras för att avgöra om den extracellulära domänen kan klyvas av. För att utveckla en affibody som kan användas både in vitro och in vivo kommer selekteringen att utföras även mot rekombinant PSMA. Rekombinant PSMA har producerats tidigare med en Avi tag för specifik biotinylering och immobilisering under selekteringen. För att biotinylera det rekombinanta PSMA producerades enzymet BirA, som katalyserar biotinyleringen av en Avi tag. Ett proteinutbyte av 8,95 mg/liter kultur erhölls och den specifika biotinyleringen var effektiv. För att utvärdera den föreslagna strategin för selektering av affibodies är nästa steg att avgöra om klyvning av PSMA uttryckt av HEK293 cellerna är möjlig, optimera klyvningsförhållandena och starta initiala selektioner med de genererade HEK293-cellerna och det producerade BirA-enzymet.
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Abdulkarim, Baroj. "The eukaryotic translation initiation factor 2, a hero turned villain in β cells." Doctoral thesis, Universite Libre de Bruxelles, 2017. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/251713.
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The prevalence of type 2 diabetes is increasing dramatically worldwide. Type 2 diabetes is a major health and socio-economic burden. Genetic predisposition and the obesity epidemic, due to sedentary life style and high caloric food intake, are associated with development of type 2 diabetes. Circulating free fatty acids (FFAs), in particular saturated FFAs, are linked with insulin resistance and β cell dysfunction. Following this background we performed RNA sequencing of human pancreatic islets treated with the saturated FFA palmitate to acquire a global image of the islet response to this insult. We identified several stress pathways induced by palmitate with a major induction of the endoplasmic reticulum (ER) stress response. The ER stress response, in particular the PKR-like ER kinase (PERK) branch, has been shown to be induced by saturated FFA. It leads to increased β cell apoptosis both in fluorescence activated cell sorter (FACS) purified rat β cells and human islets. We further clarified the role of this pathway by studying the involvement of the constitutive repressor of eIF2α phosphorylation (CReP) in a monogenic form of diabetes. CReP is a repressor of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation. A direct target of PERK, eIF2α is involved in translational attenuation and induction of apoptosis. We have shown that CReP loss-of-function leads to a new syndrome of young onset diabetes, intellectual disability and microcephaly. The identified R658C mutation abrogated CReP activity leading to increased eIF2α phosphorylation and β cell apoptosis. To further demonstrate the importance of eIF2α dysregulation in β cell demise, we used guanabenz, a chemical inhibitor of growth arrest DNA damage inducible 34 (GADD34). GADD34 is an ER stress-induced repressor of eIF2α phosphorylation. Guanabenz potentiated FFA-mediated ER stress and apoptosis in clonal and primary rat β cells and in human islets through the activation of CCAAT/enhancer binding protein homologous protein (CHOP), downstream of eIF2α. Guanabenz administration in mice impaired glucose tolerance and led to β cell dysfunction. In ex vivo experiments guanabenz also induced β cell dysfunction in mouse and rat islets.In conclusion our data demonstrate that the dysregulation of signaling in the PERK/eIF2α pathway is crucial for β cell demise. Together with previously reported monogenic diabetes caused by loss-of-function mutations in PERK in man and the eIF2αS51A mutation in mice, our findings suggest that a narrow regulation of PERK/eIF2α signaling is central for proper β cell function and survival.Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished
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Temple, Max. "The role of enzymes and binding modules in the degradation of eukaryotic, microbial and plant cell walls." Thesis, University of Newcastle upon Tyne, 2016. http://hdl.handle.net/10443/3327.
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The microbial enzymes that depolymerize complex carbohydrates are of industrial significance particularly in the biofuels and biorefinery sectors. In the human large bowel glycan utilization plays a critical role in defining the composition of the human gut microbial community (microbiota) which, in turn, has a significant impact on health. A central feature of these processes is the specificity of the enzymes and the non-catalytic carbohydrate binding modules (CBMs) that contribute to glycan degradation. This thesis describes research designed to understand the mechanisms by which CBMs and glycoside hydrolases contribute to glycan degradation and how this impacts on the structure of the microbiota. The first results chapter describes the biochemical properties and structural basis for the specificity displayed by two CBMs appended to the glucanase of a rumen bacterium. The sequence of the two CBMs are >75% identical and display essentially identical ligand specificities. Isothermal titration calorimetry revealed that the two proteins bound to a range of β1,4-glucans (cellulose) and, β1,3-β1,4-mixed linked glucans, displaying highest affinity for xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln106 is central to cellulose recognition, but is not required for binding to mixed linked glucans. This chapter reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans. In the second chapter the enzymes that contribute to the degradation of α-mannan, a prominent component of yeast cell walls, were studied. These enzymes were derived from Bacteroides thetaiotaomicron, a member of the microbiota. The data showed that the GH76 endo-α1,6-mannanases presented on the bacterial surface displayed significantly less activity against small mannooligosaccharides compared to the equivalent periplasmic enzymes. All the endo-α1,6-mannanases were only active on the linear backbone of a-mannan, the enzymes were unable to accommodate any side chains. These decorations were partially removed by a poorly expressed and slow acting surface GH92 α-mannosidase. In contrast, in the periplasm a highly active GH38 α-mannosidase rapidly debranched the imported yeast mannan oligosaccharides. The manooligosaccharides generated by the GH76 enzymes were then depolymerized into mannose by a pair of periplasmic exo-acting α1,6-mannosidases that contained only two substrate binding subsites. The biochemical characterization of these enzymes led to the selfish hypothesis in which B. thetaiotaomicron maximises deconstruction of yeast mannan in the periplasm, ensuring that the mannose generated will not be available to other organisms in the microbiota. This hypothesis were verified by showing that B. thetaiotaomicron was unable to support the growth of other Bacteroides sp. (that were able to grow on mannose and, in the case of B. xylanisolvens, also on debranched α-mannan) on yeast α-mannan. In the final results chapter the mechanism by which B. thetaiotaomicron utilized β1,6-glucan, a component of the yeast wall, was analysed. Transcriptomic analysis identified a Polysaccharide Utilization Locus (PUL) that was transcribed in response to β1,6-glucan. The PUL encoded two enzymes and two surface glycan binding proteins (SGBPs), one of which was a SusD homologue. The two SGBPs displayed tight specificity for β1,6-glucan over other β-glucans, displaying a preference for ligands that contained >3 glucose units. The surface GH30 enzyme, BT3312, was shown to be an endo-β1,6-glucanase, while the periplasmic GH3 exo-acting β-glucosidase displayed a preference for β1,6-linkages. B. thetaiotaomicron accumulated β1,6-glucobiose, which was due to the low activity of the GH3 enzyme against the disaccharide and poor expression of the β-glucosidase. The crystal structure of BT3312 revealed a deep pocket that mirrored the U-shaped typology of β1,6-glucan, revealing the mechanism of substrate specificity. Finally the catalytic amino acids of both the GH30 and GH76 enzymes were identified by site-directed mutagenesis.
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Webb, Penelope 1967. "Effects of yeast cell cycle gene expression in transgenic Nicotiana tabacum." Monash University, Dept. of Biological Sciences, 2001. http://arrow.monash.edu.au/hdl/1959.1/9084.
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Appanah, Rowin. "Replisome-mediated homeostasis of DNA/RNA hybrids in eukaryotic genomes is critical for cell fates and chromatin stability." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/100501/.
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During DNA replication, forks often stall upon encountering obstacles blocking their progression. Cells will act to speedily remove or overcome such barriers, thus allowing complete synthesis of chromosomes. This is the case for R-loops, DNA/RNA hybrids that arise during transcription. One mechanism to remove such R-loops involve DNA/RNA helicases. Here, I have shown that one such helicase, Sen1, associates with replisome components during S phase in the model organism S. cerevisiae. I demonstrate that the N-terminal domain of Sen1 is both sufficient and necessary for the interaction of the protein with the replisome. I also identified Ctf4 as one of at least two replisome interactors of Sen1. By mutational analysis, a mutant of Sen1 (Sen1-3) that cannot interact with the replisome was created. This mutant is healthy on its own but is lethal in the absence of both RNase H1 and H2. Overexpression of the sen1-3 allele from the constitutive ACT1 promoter is able to suppress this synthetic lethality, suggesting that Sen1 travels with replisomes in order to be quickly recruited at sites of R-loops that impair fork progression so as to remove those R-loops. In some cases, cells exploit fork stalling for biologically important processes. This is the case in Sz. pombe, where an imprint prevents complete DNA replication, triggering cell-type switching. This imprint is dependent on Pol1, a component of the replisome. Importantly, a single imprinting-defective allele of pol1 has been identified to date. Using in vitro assays, I have shown that this Pol1 mutant has reduced affinity for its substrates and is a correspondingly poor polymerase. By generating novel alleles of pol1, I have also demonstrated that switching-deficiency correlates with the affinity of Pol1 for its substrates in vivo. Finally, two interactors of Pol1 (Mcl1Ctf4 and Spp1Pri1 ) have been shown to have switching defects. S. cerevisiae and Sz. pombe have similar yet distinct genetic nomenclature conventions. Given that both model organisms were used in this study, it is important to highlight the conventions for both organisms to prevent confusion. In S. cerevisiae, wildtype gene names are expressed as a three letter, uppercase and italic name followed by a number (e.g. SEN1). The three letter name often corresponds to the screen through which the gene in question was originally identified. Mutants are generally designated with the same three letter but in lower case (unless the mutant is dominant) and with an allele designation (e.g. sen1∆, sen1-1 and sen1-2). Because of historical context, the allele designations vary in format (e.g. leu2-3,112 is a mutant of LEU2). Protein names are given as a three letter name with the first letter in uppercase (e.g. Sen1). This is also true for mutant proteins, with the added allele designation (e.g Sen1-1 and Sen1-2). In this study, I have generated constructs of the SEN1 gene and these constructs are referred to as SEN1 (X-Y), where X and Y refer to the first and last residues being encoded for. The corresponding proteins are referred to as Sen1 (X-Y). Different promoters have been used and, where appropriate, the promoters are expressed similarly to their wildtype gene names (e.g. GAL1, SEN1 and ACT1). In Sz. pombe, wildtype gene names are expressed as a three letter, lowercase and italic name followed by a number (e.g. pol1). Mutants are generally designated in the same format but with an allele designation. Like in S. cerevisiae, the allele designation varies widely (e.g. pol1-1, pol1-H4 and pol1-ts13). Additionally, because of the historical context, some (but not all) alleles of pol1 are referred to as swi7 to reflect the fact that they are defective for cell-type switching. Similar to the situation in S. cerevisiae, proteins names are given as a three letter name with the first letter in uppercase for both wildtype and mutants (e.g. Pol1 and Swi7-1). Sometimes, for the sake of comparison, genes or proteins are referred to their S. cerevisiae orthologues (e.g. swi1TOF1 and Swi1Tof1 , respectively). Several protein tags have been used in this study. When written in gene form, they were written in capital letters and italicized, irrespective of the host (e.g. 5FLAG) and when in protein form, they were written in capital, irrespective of the host (e.g. 5FLAG).
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Sůkal, Petr. "Výpočtové modelování mechanických zkoušek izolovaných buněk." Master's thesis, Vysoké učení technické v Brně. Fakulta strojního inženýrství, 2009. http://www.nusl.cz/ntk/nusl-228410.
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The master’s thesis deals with computational modelling of mechanical testing of isolated cells, particularly of single-axle tensile test. The aim is to imitate the real deformed shape known from experiments. At first, the structure of each cell component is described and analyzed according to their significance for mechanical behavior. The outline of basic mechanical tests used for cell testing is discussed next. A structural computational model comprising all components significant for mechanical purposes is created for the modelling. Those components are nucleus, cytoplasm, cell membrane and cytoskeleton. Due to the problems with convergence the model was divided into two parts. The first one treats separately the shape of cytoskeleton and the second one treats the shape of communicating components (nucleus, cytoplasm and cell membrane). Both of those partial models succeed in reaching the deformations according to the experiments.
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Alhassan, Hassan H. "A Genetic Approach to Identify Proteins that Interact with Eukaryotic Microtubule Severing Proteins via a Yeast Two Hybrid System." Thesis, University of North Texas, 2020. https://digital.library.unt.edu/ark:/67531/metadc1703276/.
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Microtubules (MT) are regulated by multiple categories of proteins, including proteins responsible for severing MTs that are therefore called MT-severing proteins. Studies of katanin, spastin, and fidgetin in animal systems have clarified that these proteins are MT-severing. However, studies in plants have been limited to katanin p60, and little is known about spastin or fidgetin and their function in plants. I looked at plant genomes to identify MT-severing protein homologues to clarify which severing proteins exist in plants. I obtained data from a variety of eukaryotic species to look for MT-severing proteins using homology to human proteins and analyzed these protein sequences to obtain information on the evolution of MT-severing proteins in different species. I focused this analysis on MT-severing proteins in the maize and Arabidopsis thaliana genomes. I created evolutionary phylogenetic trees for katanin-p60, katanin-p80, spastin, and fidgetin using sequences from animal, plant, and fungal genomes. I focused on Arabidopsis spastin and worked to understand its functionality by identifying protein interaction partners. The yeast two-hybrid technique was used to screen an Arabidopsis cDNA library to identify putative spastin interactors. I sought to confirm the putative protein interactions by using molecular tools for protein localization such as the YFP system. Finally, a Biomolecular Fluorescence Complementation (BiFC) assay was initiated as a proof of concept for confirmation of in vivo protein-protein interaction.
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Gradi, Alessandra. "Translational control in eukaryotes : discovery of a novel human eukaryotic translation initiation factor and its role in the shutoff of host cell protein synthesis following entero- and rhinovirus infections." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36809.
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The eukaryotic initiation factor (eIF) 4F complex is composed of three polypeptides: eIF4A, eIF4E and eIF4G. eIF4E is the cap-binding subunit; eIF4A is the component which exhibits RNA helicase activity and is thought to unwind the secondary structure present at the 5' leader sequences of mRNAs; eIF4G serves as a scaffold to bring together eIF4E, eIF4A, with eIF3, thus recruiting the mRNA to the 40S small ribosomal subunit. Here, I describe the identification and characterization of a novel homologue of eIF4G that we have termed eIF4GII. We renamed the previously discovered polypeptide, eIF4GI. eIF4GI and eIF4GII are 46% identical at the amino acid level. eIF4GII interacts directly with eIF4E, eIF4A, eIF3 and the poly(A) binding protein. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A protease to cleave endogenous eIF4G. Our findings indicate that eIF4GII is a functional homologue of eIF4GI.In contrast to cellular mRNAs, entero- and rhinovirus RNAs do not possess a cap structure, and their translation is mediated by ribosome binding to an internal ribosome entry site present within the 5' untranslated region. The cleavage of eIF4G induced by the viral protease 2A is thought to be responsible for the shutoff of host protein synthesis in entero- and rhinovirus-infected cells. Nonetheless, in virus-infected cells, a lack of correlation between eIF4GI cleavage and inhibition of host translation is observed. eIF4GII, like eIF4GI, is also cleaved upon viral infection. Here, I present data showing that the kinetics of cleavage of eIF4GII perfectly coincide with the inhibition of cellular protein synthesis observed after entero- and rhinovirus infection. Thus, proteolysis of both eIF4GI and eIF4GII appears to be required for the shutoff of host protein synthesis after entero- and rhinovirus infections.
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Eshaque, Bithi. "Characterization of Eukaryotic Translation Initiation Factor 5A isoforms (eIF-5A1 & eIF-5A2) using human cell lines as a model system." Thesis, University of Waterloo, 2006. http://hdl.handle.net/10012/1218.
Full textAbstract:
Eukaryotic translation initiation factor 5A (eIF-5A) is the only known cellular protein that contains the post-translationally derived amino acid, hypusine. Initially, eIF-5A was named as a translation initiation factor because of its capability to stimulate the formation of methionyl-puromycin, which mimics the first peptide bond formation during protein synthesis, under in vitro conditions. Subsequently, however, this proposed function of eIF-5A has been questioned because a similar effect on translation was not observed in situ. Moreover, eIF-5A appears not to be required for general protein synthesis. Rather, there is evidence that it facilitates the translation of specific subsets of mRNAs required for cell proliferation as well as apoptosis. There are two isoforms of eIF-5A in the human genome which have designated eIF-5A1 and eIF-5A2. The objective of the present study was to gain an increased understanding of the roles of eIF-5A1 and eIF-5A2 during apoptosis and cell proliferation using human cell lines as a model system. Apoptosis was induced by treating the cells with Actinomycin D or sodium nitroprusside (SNP), which initiate programmed cell death by different mechanisms. It was observed for both normal and cancer cells that eIF-5A1 protein is up-regulated during apoptosis induced by Actinomycin D or SNP, whereas eIF-5A1 mRNA is constitutively expressed and does not change in abundance during this treatment. The up regulation of eIF-5A1 protein levels in the absence of a corresponding up-regulation in eIF-5A1 mRNA suggests that eIF-5A1 may be post-transcriptionally regulated. Moreover, eIF-5A1 protein up-regulation was stronger in normal cells than in cancer cells. By contrast, eIF-5A2 protein was below detection levels during apoptosis in both normal and cancer cells, although the corresponding transcript was detectable by semi-quantitative RT-PCR. This is attributable to inefficient translation of eIF-5A2 mRNA.
The effects of eIF-5A1 and eIF-5A2 on cell proliferation were examined by modulating the levels of serum in cultures of UACC-1598 cells, which are ovarian cancer cells that express high levels of both isoforms of eIF-5A. Serum starvation, which induces cell cycle arrest and ensuing apoptosis, followed by the re-addition of serum had no effect on the transcript levels of either eIF-5A1 or eIF-5A2. However, eIF-5A1 and eIF-5A2 proteins were both up-regulated within 24 hours of the initiation of serum starvation, and this coincided temporally with the onset of apoptosis as measured by TUNEL and a subsequent decline in viable cells.
The data indicate that eIF-5A1 and eIF-5A2 are both post-transcriptionally regulated and that they have functionally redundant roles in apoptosis.
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Morrison, Alan R. "Poly(ADP)-Ribose Polymerase Activity in the Eukaryotic Mono-ADP-Ribosyl Transferase, ART2: a Dissertation." eScholarship@UMMS, 2003. https://escholarship.umassmed.edu/gsbs_diss/126.
Full textAbstract:
The glycophosphatidylinositol(GPI)-linked membrane protein ART2 is an antigenic determinant for T lymphocytes that regulate the expression of diabetes in the BB/W rat model. Though little is understood of the physiologic role of ART2 on T lymphocytes, ART2 is a member of the mono-ADP-ribosyl transferase subgroup ofthe ADP-ribosyl transferase (ART) protein family. The ART protein family, which traditionally has been divided into mono-ADP-ribosyl transferases (mono-ARTs), poly(ADP)-ribose polymerases (PARPs), and ADP-ribosyl cyclases, influences various aspects of cellular physiology including: apoptosis, DNA damage repair, chromatin remodeling, telomere replication, cellular transport, immune regulation, neuronal function, and bacterial virulence. A structural alignment of ART2.2 with chicken PARP indicated the potential for ART2.2 to catalyze ADP-ribose polymers in an activity thought to be specific to the PARP subgroup and important for their regulation of nuclear processes. Kinetic studies determined that the auto-ADP-ribosyl transferase activity of ART2.2 is multitmeric and heterogeneous in nature. Hydroxylamine-cleaved ADP-ribose moieties from the ART2.2 multimers ran as polymers on a modified sequencing gel, and digestion of the polymers with snake-venom phosphodiesterase produced AMP and the poly(ADP)ribose-specific product, PR-AMP, which was resolved by analytical HPLC and structurally confirmed by ESI-MS. The ratio of AMP to PR-AMP was higher than that of PARP raising the possibility that the ART2.2 polymers had a different branching structure than those of PARP. This alternative branching was confirmed by the presence of ribose phosphate polymers in the snake venom phophodiesterase treated samples. The site of the auto-poly(ADP)-ribose modification was determined to be R185, a residue previously proposed to influence the level of auto-ADP ribosylation of ART2.2 by mutational analysis. These data provide the first demonstration of a hybrid between mono-ARTs and PARPs and are the earliest indication that PARP-like enzymes can exist outside the nucleus and on the cell surface.
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Larsson, Ola. "Transcriptome studies of cell-fate and aging /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-296-9/.
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Muaddi, Hala. "Phosphorylation of eukaryotic initiation factor 2-alpha at serine 51 is an important determinant of cell survival and adaptation to glucose deficiency." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:8881/R/?func=dbin-jump-full&object_id=92253.
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RUSSO, ARIANNA. "Increasing Eukaryotic Initiation Factor (eIF6) gene dosage stimulates global translation and induces a transcriptional and metabolic rewiring that blocks Programmed Cell Death." Doctoral thesis, Università del Piemonte Orientale, 2018. http://hdl.handle.net/11579/97190.
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Macadangdang, Joan Karla. "Nuclear and Cytoskeletal Prestress Govern the Anisotropic Mechanical Properties of the Nucleus." Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23310.
Full textAbstract:
Physical forces in the cellular microenvironment play an important role in governing cell function. Forces transmitted through the cell cause distinct deformation of the nucleus, and possibly play a role in force-mediated gene expression. The work presented in this thesis drew upon innovative strategies employing simultaneous atomic force and laser-scanning confocal microscopy, as well as parallel optical stretching experiments, to gain unique insights into the response of eukaryotic cell nuclei to external force. Non-destructive approaches confirmed the existence of a clear anisotropy in nuclear mechanical properties, and showed that the nucleus' mechanical response to extracellular forces is differentially governed by both nuclear and cytoskeletal prestress: nuclear prestress regulates shape and anisotropic deformation, whereas cytoskeletal prestress modulates the magnitude and degree of deformation. Importantly, the anisotropic mechanical response was conserved among diverse differentiated cell types from multiple species, suggesting that nuclear mechanical anisotropy plays an important role in cell function.