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1
Ibrahim, Yasser Musa. "Stress response proteins in Streptococcus pneumoniae." Thesis, University of Glasgow, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412962.
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Bradley, Dominic. "The universal stress proteins of bacteria." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/6946.
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Universal stress proteins (USPs) are a widespread and abundant protein family often linked to survival during stress. However, their exact biochemical and cellular roles are incompletely understood. Mycobacterium tuberculosis (Mtb) has 10 USPs, of which Rv1636 appears to be unique in its domain structure and being the only USP conserved in M. leprae. Over-expression of Rv1636 in M. smegmatis indicated that this protein does not share the growth arrest phenotype of another Mtb USP, Rv2623, suggesting distinct roles for the Mtb USPs. Purified Rv1636 was shown to have novel nucleotide binding capabilities when subjected to UV crosslinking. A range of site-directed mutants of Rv1636 were produced, including mutations within a predicted nucleotide binding motif, with the aim of identifying and characterising key residues within the Rv1636 protein. Further putative biochemical activities, including nucleotide triphosphatase, nucyleotidylyation and auto-phosphorylation were also investigated in vitro; however Rv1636 could not be shown to definitively possess these activities, raising the possibility that addition factors may be present in vivo. Bioinformatic analysis of Rv1636 has provided an in-depth look at the protein. The crystal structure of Rv1636 shows a strand swapped dimer conformation that appears to block the predicted nucleotide binding site, providing a possible reason for the low NTPase activity previously observed. Truncated Rv1636 constructs were successfully generated, in which the strand swapped dimer was disrupted, and subjected to biophysical analysis, including analytical ultracentrifugation and size exclusion chromatography combined with multi-angle light scattering. Previous Mtb single-USP mutants are known to have no phenotype under a range of stress conditions. For this reason the P. aeruginosa USP PA3309, which does possess a stress survival phenotype, was also investigated. This provided the opportunity to investigate the role of USPs and their putative nucleotide binding motif in vivo. Site-directed mutants of PA3309 were generated to investigate the role of the nucleotide binding motif in vivo. It remains to be determined if the survival defect observed for ΔPA3309 strain can be complemented with these mutants as the vector system used in these experiments proved unable to integrate into the attB site of the genome. Through the analysis of the USPs from mycobacteria and Pseudomonas, the aim was to elucidate a greater understanding of the role of Rv1636 in Mtb and the role of USPs in general. The bioinformatic and biochemical analyses of USPs, in addition to the site directed mutants generated as a result of this work, will provide a strong foundation for future studies.
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Gregory, Mary Sarah-Jane, and n/a. "Thioredoxin and Oxidative Stress." Griffith University. School of Health Science, 2004. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20040301.082639.
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The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress. Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. Purification of tTrx from this protein solution required anion exchange chromatography followed by gel permeation in a HPLC system to obtain fully purified, recombinant tTrx which allowed further characterisation studies to be undertaken. An initial investigation into tTrx was performed to determine some basic physical, biochemical and functional aspects of this hitherto relatively undefined protein. Analysis by sedimentation equilibrium indicated that freshly prepared tTrx forms a single species with a molecular weight of 18.8kDa. This value indicates that recombinant tTrx naturally forms a dimer in solution that was shown to be non-covalent in nature and stable in solution. The capacity of tTrx to reduce protein disulphide bonds was determined using the insulin reduction assay. Results show that tTrx lacks this particular redox ability. The rate of oxidisation at 4 degrees C was analysed using free thiol determination, sedimentation equilibrium and SDS-PAGE patterning. Results indicated a steady rise in the degree of oxidation of tTrx over an eight day period. After six days the oxidated protein consistently displayed the presence of intramolecular disulphide bonds. Covalently-linked disulphide dimers and higher molecular weight oligomers were detectable after eight days oxidation. An investigation of the reducing capacity of the basic Trx system determined that fully oxidised tTrx was unable to act alone as a substrate for thioredoxin reductase (TR). However, when reduced Trx was added to the system, it appeared capable of acting as an electron donor to the oxidised tTrx in order to reduce disulphide groups. Recombinant tTrx was successfully radiolabelled with Trans 35S-methionine/cysteine for use in cell association studies. No evidence was found to indicate the presence of a receptor for tTrx on either MCF-7 or U-937 cells. Findings suggest that a low level of non-specific binding of tTrx to these cell lines rather than a classical ligand-binding mechanism occurs thus suggesting the absence of a cell surface receptor for tTrx. The role that Trx may play in the cellular responses to oxidative stress was also investigated. The chemical oxidants hydrogen peroxide (H2O2) and diamide were used to establish an in vitro model of oxidative stress for the choriocarcinoma cytotrophoblast cell line JEG-3. Cellular function was assessed in terms of membrane integrity, metabolic activity and the ability to synthesis new DNA following exposure to these oxidants. Results indicated that both agents were capable of causing cells to undergo oxidative stress without inducing immediate apoptosis or necrosis. Initially, JEG-3 cells exposed to 38μM or 75μM H2O2 or 100μM diamide were shown to display altered cell metabolism and DNA synthesis without loss to cell viability or membrane integrity. Cells were also shown to be capable of some short-term recovery but later lapsed into a more stressed state. Expression levels of Trx were studied to determine whether this type of chemical stress caused a change in intercellular protein levels. Both cELISA and western blotting results indicated that only cells exposed to 100μM diamide displayed any significant increase in Trx protein levels after 6 or 8hrs exposure to the oxidant. Further studies over a longer time-frame were also performed. These found that when JEG-3 cells were exposed to 18μM H2O2 or 200μM diamide over 12-48hrs, a positive correlation between increasing endogenous Trx protein levels and a decline in cell proliferation was observed. Cytotrophoblast cells, which are responsible for implantation and placentation, are susceptible to oxidative stress in vivo and their anti-oxidant capacity is fundamental to the establishment of pregnancy. The findings obtained during these studies suggest that Trx plays a role in this process.
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Gregory, Mary Sarah-Jane. "Thioredoxin and Oxidative Stress." Thesis, Griffith University, 2004. http://hdl.handle.net/10072/367183.
Full textAbstract:
The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress.
Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. Purification of tTrx from this protein solution required anion exchange chromatography followed by gel permeation in a HPLC system to obtain fully purified, recombinant tTrx which allowed further characterisation studies to be undertaken.
An initial investigation into tTrx was performed to determine some basic physical, biochemical and functional aspects of this hitherto relatively undefined protein. Analysis by sedimentation equilibrium indicated that freshly prepared tTrx forms a single species with a molecular weight of 18.8kDa. This value indicates that recombinant tTrx naturally forms a dimer in solution that was shown to be non-covalent in nature and stable in solution. The capacity of tTrx to reduce protein disulphide bonds was determined using the insulin reduction assay. Results show that tTrx lacks this particular redox ability.
The rate of oxidisation at 4 degrees C was analysed using free thiol determination, sedimentation equilibrium and SDS-PAGE patterning. Results indicated a steady rise in the degree of oxidation of tTrx over an eight day period. After six days the oxidated protein consistently displayed the presence of intramolecular disulphide bonds. Covalently-linked disulphide dimers and higher molecular weight oligomers were detectable after eight days oxidation.
An investigation of the reducing capacity of the basic Trx system determined that fully oxidised tTrx was unable to act alone as a substrate for thioredoxin reductase (TR). However, when reduced Trx was added to the system, it appeared capable of acting as an electron donor to the oxidised tTrx in order to reduce disulphide groups.
Recombinant tTrx was successfully radiolabelled with Trans 35S-methionine/cysteine for use in cell association studies. No evidence was found to indicate the presence of a receptor for tTrx on either MCF-7 or U-937 cells. Findings suggest that a low level of non-specific binding of tTrx to these cell lines rather than a classical ligand-binding mechanism occurs thus suggesting the absence of a cell surface receptor for tTrx.
The role that Trx may play in the cellular responses to oxidative stress was also investigated. The chemical oxidants hydrogen peroxide (H2O2) and diamide were used to establish an in vitro model of oxidative stress for the choriocarcinoma cytotrophoblast cell line JEG-3. Cellular function was assessed in terms of membrane integrity, metabolic activity and the ability to synthesis new DNA following exposure to these oxidants. Results indicated that both agents were capable of causing cells to undergo oxidative stress without inducing immediate apoptosis or necrosis. Initially, JEG-3 cells exposed to 38μM or 75μM H2O2 or 100μM diamide were shown to display altered cell metabolism and DNA synthesis without loss to cell viability or membrane integrity. Cells were also shown to be capable of some short-term recovery but later lapsed into a more stressed state.
Expression levels of Trx were studied to determine whether this type of chemical stress caused a change in intercellular protein levels. Both cELISA and western blotting results indicated that only cells exposed to 100μM diamide displayed any significant increase in Trx protein levels after 6 or 8hrs exposure to the oxidant. Further studies over a longer time-frame were also performed. These found that when JEG-3 cells were exposed to 18μM H2O2 or 200μM diamide over 12-48hrs, a positive correlation between increasing endogenous Trx protein levels and a decline in cell proliferation was observed. Cytotrophoblast cells, which are responsible for implantation and placentation, are susceptible to oxidative stress in vivo and their anti-oxidant capacity is fundamental to the establishment of pregnancy. The findings obtained during these studies suggest that Trx plays a role in this process.Thesis (Masters)
Master of Philosophy (MPhil)
School of Health Sciences
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Fladvad, Malin. "Structure and function in c-Myc and Grx4 : two key proteins involved in transcriptional activation and oxidative stress /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7357-007-9/.
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Naim, Adnan. "The Role of G3BPs in the Stress Response Pathway." Thesis, Griffith University, 2016. http://hdl.handle.net/10072/367499.
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The ras-GTPase SH3-domain Binding Proteins (G3BP) are a family of RNA-binding proteins that have been implicated in multiple cellular activities ranging from signal transduction to regulation of messenger RNA (mRNA). G3BPs were named after their interaction with the SH3 domain of Ras-GTPase-activating protein; however recent research did not find this interaction. All three members of the G3BPs family, G3BP1, G3BP2a and G3BP2b, share structural similarities with each other by having four distinct regions (1) the Nuclear Transporting Factor 2, (NTF2) domain at the N-terminal, (2) the acidic and proline-rich domain in the centre, (3) the RNA recognition motif (RRM) and (4) the arginine glycine (RGG)-rich region rich at the C-terminal. The presence of the NTF2 domain in its structure suggests G3BP might play a role in nucleocytoplasmic transportation, which was observed after serum stimulation where G3BP1 was translocated to the nucleus from the cytoplasm. The RNA recognition motif (RRM) region plays a vital role in its interaction with the target RNA. The RGG-rich box is a region rich in arginine and glycine residues, which plays a role assisting RRM in interactions with protein or RNA.
G3BP1 is found to be overexpressed in many cancers, including breast cancer, and head and neck tumours, as well as cell lines derived from human lung, prostrate, colon, thyroid and breast cancer. G3BPs have also been implicated in translational control within differentiating neurons, suggesting that G3BP may play several roles in controlling the translational fate of its cargo and that its role may be cell-specific. G3BP1 has also been found in β-integrin- induced adhesion complexes. This information highlights G3BPs as a dynamic protein that is involved in several biological functions.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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Doherty, Sean. "Apoplastic proteins, enzymes and radicals." Thesis, Durham University, 2000. http://etheses.dur.ac.uk/4376/.
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The soluble and readily extractable part of the plant extracellular matrix has been termed, the apoplast and contains a wide range of components such as, complex carbohydrates, structural proteins, enzymes and radicals that are known to be responsive to stress and developmental pressures. This thesis describes the development of a technique for the selective enrichment of apoplastic components for a range of subsequent analyses. Using this technique a number of apoplastic proteins were N-terminally sequenced and revealed 2 cell wall related enzymes, an antifungal protein and 3 auxin-binding/germin-like proteins. This technique also provided a novel approach to the further study of auxin-binding proteins via the use of affinity chromatography at their putative site of action, the apoplast. Three potential auxin-binding protiens were identified. Many attempts were made to subject the material extracted from the apoplast to the highly resolving technique of 2-dimensional electrophoresis, and during the process two unusual 2D systems were developed. These systems could be run in a small format that permitted very rapid analysis and/or using an in-gel loading strategy to subject up to 500µg of protein to 2D separation therefore permitting N-terminal sequencing from single 2D gels. Unfortunately 2D separation of apoplastic proteins was never fully achieved within the time frame of this study due to the vast degree of heterogenity present in the sample material. It did however demonstrate the very complex nature of apoplastic components. A series of experiments revealed that the tobacco leaf apoplast contained compartment specific antioxidant enzymes, some of which share physical characteristics with similar enzymes from other species. The activity of these enzymes altered in response to stress and according to the developmental age of the tissue. The reduced activity of these enzymes directly correlated to the degree of oxidative modification of apoplastic proteins illustrating that these enzymes are important in the detoxification of apoplastic radicals. Follow on experiments following the apoplastic generation of the superoxide anion and nitric oxide from impact stressed potato tuber tissue showed that radicals play important roles in the responses of plant tissue to stress, and show the first involvement of nitric oxide in plants in response to abiotic stress.
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Amara, Imen. "Abiotic stress in plants: Late Embryogenesis Abundant proteins." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/83820.
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In order to improve our understanding on LEA proteins and their molecular functions in drought tolerance, the present work analyzes in the first place, the composition of LEA subproteomes from Arabidopsis seeds and maize embryos; second, three maize embryo LEA proteins from groups 1, 2, and 3 are analyzed in order to detect functional differences among them and finally, transgenic maize plants over-expressing group 5 “rab28” lea gene are characterized. The following results are presented:
- Chapter 1. Proteomic approach to analyze the composition of LEA subproteomes from
Arabidopsis seeds by mass spectrometry. The main objective was the development and isolation method to obtain enriched LEA populations from Arabidopsis seeds. LEA subproteomes were obtained using an extraction procedure that combines heat stability and acid solubility of LEA proteins. To identify the protein content, we followed two approaches: first, a classical 1D (SDSPAGE) gel-based procedure associated with MS analysis using an electrospray ionization source coupled on-line to liquid chromatography (LC-ESI-MSMS) and second, a gel-free protocol associated with an off-line HPLC and analysis via matrix assisted laser desorption/ionization (LC-MALDI-MSMS).
- Chapter 2. Proteomic analysis of LEA proteins accumulated in maize mature seeds. Identification of LEA protein content by mass spectrometry and selection of three LEA proteins, Emb564, Rab17 and Mlg3, as representatives of groups 1, 2 and 3 for further study. Comparative functional analysis covering different aspects of maize Emb564, Rab17 and Mlg3 proteins, posttranslational modifications, subcellular localization and their properties in in-vitro and in- vivo assays.
- Chapter 3. Characterization of transgenic maize plants expressing maize group 5 rab28 LEA gene under the ubiquitin promoter. Evaluation of Rab28 transcripts and protein levels, phenotype and stress tolerance traits of transgenic plants under drought stress. Investigation of the subcellular localization of transgenic and wild-type Rab28 protein using immunocytochemical approaches.Las proteínas LEA, originalmente fueron descritas en las semillas de algodón; se acumulan en grandes cantidades en estructuras tolerantes a la desecación (semillas, polen) y en tejidos vegetativos sometidos a estrés abiótico, sequía, salinidad y frío. También se hallan en organismos anidrobióticos, en plantas de resurrección, algunos invertebrados y microorganismos. La presencia de proteínas LEA se correlaciona con la adquisición de tolerancia a la desecación. Desde un principio se les atribuyó un papel en las respuestas de las plantas en la adaptación al estrés (revisado en Bartels and Salamini 2001, Tunnacliffe 2007, Shih et al. 2010, Tunnacliffe 2010, Hand et al. 2011). Las proteínas LEA se clasifican en diversos grupos en función de dominios y secuencias de aminoácidos específicos (Wise 2010, Batagglia et al 2008, Bies-Ethève et al 2008). Los grupos 1, 2 y 3 son los más relevantes ya que abarcan la mayoría de las proteínas de la familia LEA. Una característica general de estas proteínas es su elevada hidrofilicidad, alto contenido de aminoácidos cargados y su falta de estructura en estado hidratado. A pesar de encontrarse mayoritariamente en forma de “random coil”, algunas adquieren un cierto grado de estructura durante la deshidratación o en la presencia de agentes promotores de α-hélices (Shih et al. 2010, Hand et al. 2011). A nivel celular se han hallado en todas las localizaciones, citosol, núcleo, nucleolo, mitocondria, cloroplasto, vacuola, retículo endoplásmico, peroxisoma y membrana plasmática, donde se supone ejercen su función protectora frente al estrés (Tunnacliffe and Wise 2007, Hundertmark and Hincha 2008). En relación a las modificaciones post-traduccionales, algunas se hallan fosforiladas (Jiang and Wang 2004; Plana et al. 1991, Heyen et al. 2002, Rohrig et al. 2006). Los efectos protectores de las varias proteínas LEA se han demostrado mediante ensayos in vitro y en aproximaciones transgénicas que han dado lugar a fenotipos resistentes a la sequía, sal y frío. Por lo general, se considera que estas proteínas contribuyen a la protección y a la estabilización de macromoléculas y estructuras celulares en las respuestas de adaptación al estrés en plantas; sin embargo, sus funciones específicas aún no han sido esclarecidas. A nivel molecular se ha propuesto que las funciones de las proteínas LEA pueden ser variadas: estabilización y renaturalización de proteínas, mantenimiento de membranas, en combinación, o no, con azúcares, tampones de hidratación (substitución de moléculas de agua), afinidad por iones y función antioxidante (Tunnacliffe and Wise 2007, Shih et al. 2010, Batagglia et al. 2008). Para finalizar, diremos que los objetivos principales de esta tesis consisten en ampliar los conocimientos sobre las proteínas LEA y sus funciones relativas a la tolerancia a la sequía. Los resultados están presentados en forma de capítulos.
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Kolodziejski, Jakub. "Twist proteins as oxidative and hypoxic stress regulators." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTS008/document.
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Les facteurs de transcription Twist1 et Twist2 (famille Twist) jouent un rôle majeur dans le développement embryonnaire et dans la progression tumorale. Leur potentiel oncogénique dérive directement de la combinaison de leurs nombreuses activités développementales. Les gènes Twist peuvent notamment, en induisant la transition épithélio-mésenchymateuse (EMT), promouvoir l’invasion des cellules cancéreuses et participer de ce fait aux processus métastatique. De plus, en bloquant l’activité des voies de signalisation Rb et p53, ils peuvent inhiber les deux principaux programmes de sauvegarde cellulaire que sont l’apoptose et la senescence. Enfin, ils sont également impliqués dans la résistance des cellules cancéreuses aux agents chimio-thérapeutiques. En plus de ces nombreuses activités, nos données préliminaires nous ont amené à considérer un rôle de Twist dans la réponse au stress. Les cellules cancéreuses doivent croitre dans un environnement en perpétuel changement qui génère de nombreux types de stress. Seules les cellules capables de s’adapter, peuvent survivre et acquérir de nouvelles capacités les rendant plus agressives. La résistance au stress fait donc partie intégrante de la progression tumorale. Nos travaux révèlent que Twist en induisant une résistance au stress, plus particulièrement métabolique, est un acteur essentiel de l’acquisition d’u phénotype agressif des cellules cancéreuses. Dans une première étude, nous avons montré que Twist module le stress oxydatif, une condition très fréquemment retrouvée dans les tumeurs. Ainsi, nos résultats indiquent que l’expression de Twist provoque une réduction du taux d’espèces réactives de l’oxygène (ROS) intracellulaire. Cette activité a pour conséquence directe d’induire une résistance accrue à l’apoptose déclenchée par divers traitements. Nous avons par la suite caractérisé cette activité et mis en évidence un programme génétique contrôlé par Twist impliquant divers facteurs possédant des propriétés anti-oxydantes. Dans un second temps, nous nous sommes intéressés à un autre type de stress métabolique, l’hypoxie. L’hypoxie définie par un taux insuffisant d’oxygène, est retrouvée dans la plupart des tumeurs solides du fait de l’absence ou de l’anomalité de la vascularisation. L’hypoxie mène à la stabilisation d’un facteur de transcription, HIF1α. Cette protéine est essentielle à l’adaptation hypoxique et contrôle l’expression de nombreux gènes impliqués dans le métabolisme du glucose, le transport de l’oxygène, l’angiogenèse ou l’apoptose. Dans les premiers temps d’hypoxie, l’effet d’adaptation induit par HIF1α est bénéfique pour les cellules. Cependant, si l’absence d’oxygène se prolonge, HIF1α, peut pousser les cellules vers la mort. Nos travaux démontrent que Twist est capable de rendre les cellules résistantes à une hypoxie prolongée. De plus, cette activité de protection contre le stress hypoxique agit via un effet paracrine. Enfin, nos données suggèrent que cet effet est médié par une interaction directe entre les protéines Twist et HIF1α. Au final, cette étude indique que l’expression de Twist dans les cellules cancéreuses, en conférant une résistance accrue à l’environnement hypoxique, joue un rôle essentiel dans l’adaptation au stress et à l’acquisition de nouveaux phénotypes agressifs. En résumé, L’objectif principal de ma thèse était de mettre en évidence de nouvelles propriétés cellulaires des oncogènes de la famille Twist. Nos résultats démontrent que Twist par ses capacités à contrôler le stress métabolique, permet à la cellule cancéreuse de mieux s’adapter et donc survivre dans un environnement en constante évolution. Nos travaux renforcent donc la notion de l’importance de ces facteurs dans la progression tumoraleTwist1 and Twist2 are related transcription factors that play major roles both during embryonic development and in several pathologies, including cancer. Twists' oncogenic potential arises from a combination of their multiple functions in development. Notably, both Twist induce epithelial-to mesenchymal transition, thus promoting tumour invasiveness and possibly conferring to cells self-renewal properties. Furthermore, through disruption of both Rb- and p53-driven pathways, Twist override two major oncogene-induced fail-safe programs, namely senescence and apoptosis, thereby promoting malignant conversion. Twist has also been reported to participate in acquisition of drug resistance and in promotion of neo-angiogenesis.Current knowledge of pleiotropic activities of Twist prompted us to postulate that these factors may be major regulators of stress response. Cancer cells survive and grow within a continuously changing environment that creates multiple stresses to which they must adapt in order to survive and strive. Such adaptations often give rise to the acquisition of an aggressive phenotype. Consistent with this hypothesis, we recently unveiled new activities of Twist proteins that are related to stress response. We have shown that Twist regulates response to oxidative stress, a condition exacerbated in cancer by stimuli such as inflammation, increased cellular metabolism and changes in tumour oxygenation. Our work has contributed to the understanding of molecular mechanisms through which Twist diminishes cellular ROS and thus participates in the escape from apoptosis and senescence. In the first part of my thesis, I worked on the antioxidant activity of Twist and described its molecular mechanisms.The second part of my work addressed the impact of Twist proteins on cellular response to hypoxia that is insufficient oxygen supply, frequently found in solid tumours. Cellular response to hypoxic stress relies on stabilization and activation of HIF1α, a key transcriptional mediator of the hypoxic response, regulating numerous genes involved in glucose metabolism, oxygen transport, angiogenesis, cell growth and apoptosis. HIF1α is beneficial for cancer cells in response to short hypoxic episodes, however its sustained activation in case of prolonged hypoxia may push cancer cells towards apoptosis. In this context, we have shown that Twist protects cancer cells from hypoxia-induced apoptosis. We have discovered HIF1α and Twist physically interact, suggesting a possible mechanistic basis for Twist's protective effect. These results led us to postulate that Twist plays a role in cellular response to hypoxia and thus participates in cancer cell adaptation and acquisition of aggressive phenotypes triggered by lack of oxygen.Our results reinforce the notion that Twist factors are major cellular stress modulators that might be important for adaptation of cancer cells to changing conditions in the process of tumour progression
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Di, Paolo Tiziano. "Stress response in Entamoeba histolytica." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=68169.
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The heat shock response was studied in the intestinal parasitic protozoan Entamoeba histolytica. Temperature shifts from 37$ sp circ$C to 44$ sp circ$C enhanced the synthesis of five major heat shock (or stress) proteins (HSP) of 100, 50, 42, 37, and 28 kDa. Similarly, exposure of amebae to lymphokine activated macrophages and hydrogen peroxide caused HSP expression. Heat shock caused the reversible inhibition of amebic adherence to Chinese hamster ovary cells and human colonic mucin binding to trophozoites by ${>80 %}$. This was due to a decrease in the surface expression of the Gal/GalNAc adherence lectin and a marked reduction in the lectin mRNA expression. However, the presence of target Chinese hamster ovary cells during recovery at 37$ sp circ$C augmented amebic adherence. These results suggest that E. histolytica trophozoites produce a variety of HSP in response to different stimuli and can modulate the expression of the surface adherence lectin which maybe important in pathogenesis.
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Nadarajah, Kalaivani. "Regulation of stress response in Arabidopsis thaliana." Thesis, University of East Anglia, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301940.
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Schroder, Wayne Ashley. "Cloning and Characterisation of the Human SinRIP Proteins." Thesis, Griffith University, 2003. http://hdl.handle.net/10072/366190.
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This thesis describes the cloning and characterisation of a novel human gene and its protein products, which have been designated SAPK- and Ras-interacting protein (SinRIP). SinRIP shares identity with JC310, a partial human cDNA that was previously identified a candidate Ras-inhibitor (Colicelli et al., 1991, Proc Natl Acad Sci USA 88, p. 2913). In this study, it was shown that SinRIP is a member of an orthologous family of proteins that is conserved from yeast to mammals and contains proteins involved in Ras- and SAPK-mediated signalling pathways. Comparison of this family of proteins showed that human SinRIP contains a potential Ras-binding domain (RBD; residues 279-354), a PH-like domain (PHL; 376-487), and a highly conserved novel region designated the CRIM (134-265). Several other potential targeting sites, such as nuclear localisation signals and target sites for kinases, were identified within the SinRIP sequence. The human SinRIP gene is unusually large (>280 kbp) and is located on chromosome 9 at 9q34. SinRIP mRNA was detected in a wide variety of tissue-types and cell lines by RT-PCR, and the SinRIP sequences in the EST database were derived from an diverse array of tissues, suggesting a widespread or ubiquitous expression. Northern blot analysis revealed the highest levels in skeletal muscle and heart tissue. However, the steady-state levels of SinRIP mRNA vary greatly from cell to cell, and SinRIP expression is likely to be regulated at multiple post-transcriptional levels. It was shown that SinRIP mRNA is likely to be translated inefficiently by the normal cap-scanning mechanism, due to the presence of a GC-rich and structured 5’-UTR, which also contains upstream ORFs. Alternative polyadenylation signals in the SinRIP 3’-UTR can be used, resulting in the expression of short and long SinRIP mRNA isoforms. Several potential A/T-rich regulatory elements were also identified in SinRIP mRNA, which may target specific SinRIP mRNA isoforms for rapid degradation. Importantly, it was shown that SinRIP mRNA is alternatively spliced, resulting in the production of distinct SinRIP protein isoforms. Three isoforms, SinRIP2-4, were definitively identified by RT-PCR and full-length cloning. The SinRIP isoforms contain deletions in conserved regions, and are likely to have biochemical characteristics that are different to full-length SinRIP1. SinRIP2 is C-terminally truncated and lacks the PHL domain and part of the RBD, and relatively high levels of SinRIP2 expression arelikely to occur in kidneys. The RBD is disrupted in SinRIP3, but all other domains are intact, and RT-PCR analyses suggest that SinRIP3 is present in some cells at levels comparable to SinRIP1. A rabbit polyclonal antiserum against SinRIP was generated and detected endogenous SinRIP proteins. Using the anti-SinRIP antibody in immunoblots, multiple SinRIP isoforms were observed in most cell types. SinRIP1 and another endogenous SinRIP protein, likely to be SinRIP3, were detected in most cell lines, and appear to be are the major SinRIP proteins expressed in most cells. The subcellular localisation of both recombinant and endogenous SinRIP proteins was investigated by immunofluorescence assays and biochemical fractionation. Recombinant SinRIP1 protein was found in the cytoplasm and associated with the plasma membrane. In contrast, the SinRIP2 protein was predominantly nuclear, with only low-level cytoplasmic staining observed. The endogenous SinRIP proteins, likely to comprise these and other SinRIP isoforms, were found in both the nucleus and cytoplasm. SinRIP1 interacted with GTP-bound (active) Ras, but not GDP-bound (inactive) Ras, in an in vitro assay, and also co-localised with activated H- and K-Ras in cells. The binding profile observed is typical of Ras-effectors, and SinRIP did not inhibit signalling by the Ras proteins, suggesting that it is not likely to be a Ras-inhibitor. It was also shown that SinRIP1 and SinRIP2 both interact and colocalise with c-Jun NH2- terminal kinase (JNK). Both SinRIP proteins were able to recruit JNK to their respective sub-cellular compartments. These interactions suggest an adaptor role for SinRIP in the Ras and/or JNK pathways. In addition, Sam68 was isolated as a SinRIP-binding protein in a yeast two-hybrid screen. Sam68 was shown to colocalise with SinRIP2 and endogenous SinRIP proteins, but not SinRIP1. Further colocalisation studies showed that endogenous SinRIP proteins localise in nuclear structures that may be associated with pre-mRNA splicing. Likely functions for SinRIP, as indicated by experimental results and studies of the orthologues of SinRIP in other species, are discussed.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Faculty of Science
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Longshaw, Victoria Mary. "The phosphorylation and nuclear localization of the co-chaperone murine stress-inducible protein 1." Thesis, Rhodes University, 2003. http://hdl.handle.net/10962/d1004038.
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The co-chaperone murine stress-inducible protein 1 (mSTI1), a heat shock protein 70 (Hsp70)/ heat shock protein 90 (Hsp90) organizing protein (Hop) homologue, mediates the assembly of the Hsp70/Hsp90 chaperone heterocomplex. mSTI1 is phosphorylated in vitro by cell cycle kinases, proximal to a putative nuclear localization signal (NLS), substantiating a predicted CKII-cdc2-NLS (CcN) motif at position 189-239. Stable transfectants of NIH 3T3 fibroblasts that expressed mSTI1-EGFP, NLSmSTI1-EGFP and EGFP, were prepared. Fluorescence microscopy revealed mSTI1 was cytoplasmically localized, and that this localization was not affected by the fusion of mSTI1 with the EGFP moiety. NLSmSTI1-EGFP was targeted to the nucleus compared to EGFP, suggesting that the NLSmSTI1 was a functional NLS. The localization of mSTI1 was determined under normal and heat shock conditions, inhibition of nuclear export (leptomycin B), inhibition of CKII 5,6-dichlorobenzimidazole riboside, DRB), inhibition of cdc2 kinase (olomoucine), and G1/S phase arrest (hydroxyurea). mSTI1-EGFP and mSTI1 were excluded from the nucleus in the majority of resting cells, but accumulated in the nucleus following leptomycin B treatment, implying that mSTI1 possibly undergoes a functional import process, and export via the chromosomal region maintenance 1 (CRM-1)-mediated export pathway. Hydroxyurea and olomoucine (but not DRB or heat shock) treatment increased the proportion of cells in which mSTI1-EGFP exhibited cytoplasmic and nuclear localization. 2D gel electrophoresis detected three endogenous mSTI1 isoforms, which changed following hydroxyurea treatment. Furthermore, point inactivation and mimicking of phosphorylatable residues in mSTI1 altered the translocation of the protein and the isoform composition. Modification of mSTI1 at S189 and T198 decreased the number of isoforms of mSTI1-EGFP, suggesting that the protein is modified at these sites in vivo. The removal of the in vitro cdc2 kinase site at T198 promoted a nuclear localization during G1/S phase arrest. Therefore active cdc2 kinase, but not CKII, may be required for cytoplasmic localization of mSTI1. The CKII site appears to have no regulatory role under heat shock conditions or during the cell cycle. In vitro phosphorylation studies on untagged mSTI1 further supported the prediction that S189 is the only site recognised by CKII. The cdc2 kinase site at T198, however, although the major site, was not the only site phosphorylated in vitro. However, mSTI1 and cdc2 kinase did not interact in a detectable stable complex. Bioinformatic analysis of mSTI1 revealed NLS residues were conserved in STI1 proteins, and the NLS and TPR2A motifs were in close proximity. This may have mechanistic implications for the formation of the Hsp90-mSTI1 heterocomplex. The cytoplasmic or nuclear localization of mSTI1 is predicted to be the result of a dynamic equilibrium between nuclear import and nuclear export, the fulcrum of which may be shifted under different cell cycle conditions. These data provide the first evidence of regulated nuclear import/export of a major Hsp70/Hsp90 co-chaperone, and the regulation of this nuclear import by cell cycle status and cell cycle kinases.
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Georgiev, Alexander. "Membrane Stress and the Role of GYF Domain Proteins." Doctoral thesis, Stockholm : Department of Biochemistry and Biophysics, Stockholm university, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-7764.
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Lougheed, Kathryn. "The role of universal stress proteins in mycobacterium tuberculosis." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445323.
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Owen, Gillian Audrey. "Stress induced ribosomal proteins of Streptomyces coelicolor A3(2)." Thesis, University of Sussex, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.421519.
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Ragno, Silvia. "Heat shock proteins and experimental arthritis." Thesis, Queen Mary, University of London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.281712.
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Gutierrez, Edgar. "Roles of transglutaminase and protein 4.2 in mechanical stress induced crosslinking of erythrocyte membrane proteins /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2004. http://wwwlib.umi.com/cr/ucsd/fullcit?p3137230.
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Hui, Daniel Jason. "The Mechanism of Protein Synthesis Inhibition by the P56 Family of Viral Stress Inducible Proteins." Connect to text online, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1104848977.
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Liu, Ziqiang. "Molecular analysis and functional characterization of Nucleosome Assembly Protein 1 (NAP1) family proteins in plants." Université Louis Pasteur (Strasbourg) (1971-2008), 2008. http://www.theses.fr/2008STR13132.
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Eukaryotic genomes are packaged into chromatin, a regularly repeated structure whose fundamental building block is the nucleosome. Each nucleosome core particle is composed of a histone octamer consisting of two molecules each of the core histones H2A, H2B, H3, and H4, around which approximately 146–147 bp of DNA is wrapped. The assembly of nucleosome is the first step of chromatin assembly which is important for the chromatin structure affecting a broad ranges of biological events including DNA replication, repair, recombination, transcription, cell differentiation, proliferation, and organism development, and is fulfilled with the help of histone chaperones, which are important for the organization and dynamics of chromatin templates, and are involved in the storage, translocation to the nucleus and exchange of histones and their deposition onto the DNA for replication-dependent chromatin assembly. [. . . ].
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Olsson, Björne. "Protein Expression in Baltic Sea Blue Mussels Exposed to Natural and Anthropogenic Stress : The use of stress inducible proteins in ecotoxicological studies." Doctoral thesis, Stockholm University, Department of Systems Ecology, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-542.
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The focus of this thesis is the early detection of stress in the environment. It has been proposed that studies on the cellular level would detect stress reactions earlier in time compared to common physiological methods. In a series of experiments we investigated how different stress factors, both natural and introduced by man, affect levels of stress proteins. One- and two-dimensional gels were used to determine individual proteins and families of proteins. The two-dimensional gels were also used in a proteomic approach, were the presence and absence of proteins after treatment was observed, and the protein expression signatures (PES) were identified.
Baltic Mytilus edulis was used in all experiments and it is evident that earlier observed differences in physiological rates and pollution sensitivity, compared to marine mussels, is also manifested as lower concentrations of stress proteins after exposure to copper and cadmium. When the Baltic mussels were allowed to acclimate for one month the difference decreased, suggesting an environmentally induced difference (paper I). Pre-exposure to heat before exposure to either a second heat-shock or cadmium was found to enhance the levels of HSP70 and thus tolerance, significantly (paper II). Exposure to a mixture of stress factors (PCB, copper and lowered salinity) revealed synergistic, additive and antagonistic effects in induction of 6 different stress proteins. When analyzing a large number of proteins it was shown that it is possible to identify PES with this technique, and we hypothesize that it could be possible to separate responses to mixtures of stress factors (Papers III and IV). Different techniques were also applied to analyze the protein expression pattern when mussels were exposed to PAH- and PCB-fractions extracted from Baltic Sea sediments. In this experiment the protein assays were accompanied by physiological measurements. All methods indicated stressed conditions, but the variation between individual mussels within treatments was smaller in terms of protein response than for physiological parameters (paper V). It is concluded that measuring the induction of stress proteins is a reliable way to detect stressful conditions. Proteins visualized on a one dimensional gel give a “gross” picture of an organism’s condition. The major challenge is to identify the origin and severity of the elucidated stress response. Further mapping of two-dimensional gels suggested that protein patterns are specific to type and level of stress.
A most important future step is to establish links between sub-cellular protein response to well known physiological effects. This should include long term experiments where altered protein expression signatures are linked to life history characteristics like survival, growth and reproductive success.
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Olsson, Björne. "Protein expression in Baltic Sea blue mussels exposed to natural and anthropogenic stress : the use of stress inducible proteins in ecotoxicological studies /." Stockholm : Dept. of Systems Ecology, Stockholm University, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-542.
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Schroder, Wayne Ashley, and n/a. "Cloning and Characterisation of the Human SinRIP Proteins." Griffith University. School of Biomolecular and Biomedical Science, 2003. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20030829.140754.
Full textAbstract:
This thesis describes the cloning and characterisation of a novel human gene and its protein products, which have been designated SAPK- and Ras-interacting protein (SinRIP). SinRIP shares identity with JC310, a partial human cDNA that was previously identified a candidate Ras-inhibitor (Colicelli et al., 1991, Proc Natl Acad Sci USA 88, p. 2913). In this study, it was shown that SinRIP is a member of an orthologous family of proteins that is conserved from yeast to mammals and contains proteins involved in Ras- and SAPK-mediated signalling pathways. Comparison of this family of proteins showed that human SinRIP contains a potential Ras-binding domain (RBD; residues 279-354), a PH-like domain (PHL; 376-487), and a highly conserved novel region designated the CRIM (134-265). Several other potential targeting sites, such as nuclear localisation signals and target sites for kinases, were identified within the SinRIP sequence. The human SinRIP gene is unusually large (>280 kbp) and is located on chromosome 9 at 9q34. SinRIP mRNA was detected in a wide variety of tissue-types and cell lines by RT-PCR, and the SinRIP sequences in the EST database were derived from an diverse array of tissues, suggesting a widespread or ubiquitous expression. Northern blot analysis revealed the highest levels in skeletal muscle and heart tissue. However, the steady-state levels of SinRIP mRNA vary greatly from cell to cell, and SinRIP expression is likely to be regulated at multiple post-transcriptional levels. It was shown that SinRIP mRNA is likely to be translated inefficiently by the normal cap-scanning mechanism, due to the presence of a GC-rich and structured 5-UTR, which also contains upstream ORFs. Alternative polyadenylation signals in the SinRIP 3-UTR can be used, resulting in the expression of short and long SinRIP mRNA isoforms. Several potential A/T-rich regulatory elements were also identified in SinRIP mRNA, which may target specific SinRIP mRNA isoforms for rapid degradation. Importantly, it was shown that SinRIP mRNA is alternatively spliced, resulting in the production of distinct SinRIP protein isoforms. Three isoforms, SinRIP2-4, were definitively identified by RT-PCR and full-length cloning. The SinRIP isoforms contain deletions in conserved regions, and are likely to have biochemical characteristics that are different to full-length SinRIP1. SinRIP2 is C-terminally truncated and lacks the PHL domain and part of the RBD, and relatively high levels of SinRIP2 expression arelikely to occur in kidneys. The RBD is disrupted in SinRIP3, but all other domains are intact, and RT-PCR analyses suggest that SinRIP3 is present in some cells at levels comparable to SinRIP1. A rabbit polyclonal antiserum against SinRIP was generated and detected endogenous SinRIP proteins. Using the anti-SinRIP antibody in immunoblots, multiple SinRIP isoforms were observed in most cell types. SinRIP1 and another endogenous SinRIP protein, likely to be SinRIP3, were detected in most cell lines, and appear to be are the major SinRIP proteins expressed in most cells. The subcellular localisation of both recombinant and endogenous SinRIP proteins was investigated by immunofluorescence assays and biochemical fractionation. Recombinant SinRIP1 protein was found in the cytoplasm and associated with the plasma membrane. In contrast, the SinRIP2 protein was predominantly nuclear, with only low-level cytoplasmic staining observed. The endogenous SinRIP proteins, likely to comprise these and other SinRIP isoforms, were found in both the nucleus and cytoplasm. SinRIP1 interacted with GTP-bound (active) Ras, but not GDP-bound (inactive) Ras, in an in vitro assay, and also co-localised with activated H- and K-Ras in cells. The binding profile observed is typical of Ras-effectors, and SinRIP did not inhibit signalling by the Ras proteins, suggesting that it is not likely to be a Ras-inhibitor. It was also shown that SinRIP1 and SinRIP2 both interact and colocalise with c-Jun NH2- terminal kinase (JNK). Both SinRIP proteins were able to recruit JNK to their respective sub-cellular compartments. These interactions suggest an adaptor role for SinRIP in the Ras and/or JNK pathways. In addition, Sam68 was isolated as a SinRIP-binding protein in a yeast two-hybrid screen. Sam68 was shown to colocalise with SinRIP2 and endogenous SinRIP proteins, but not SinRIP1. Further colocalisation studies showed that endogenous SinRIP proteins localise in nuclear structures that may be associated with pre-mRNA splicing. Likely functions for SinRIP, as indicated by experimental results and studies of the orthologues of SinRIP in other species, are discussed.
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Scapin, Cristina. "Risposta cellulare allo stress e proteine dello stress nei tessuti muscolari striati. Ruolo di Grp94." Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3425588.
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The purpose of this thesis was to study the stress response of muscle cells using in vivo and in vitro experimental procedures. Animal models which replicate cytoprotective (mild exercise training) or pathologic (muscle disuse atrophy) conditions were used to analyze the extent of the stress response and the cellular distribution of stress proteins in muscle tissues. Here we show that mild exercise training (up to 30m/min treadmill run for 1h/day) significantly increased Hsp70 expression in cardiac and skeletal muscles. Studies on the cellular distribution of this protein showed that the percentage of Hsp70-positive myofibers increased about 3-fold in trained fast muscles of the posterior rat hindlimb, compared to the sedentary ones (P<0.001), and involved a larger subset of both type 2A and intermediate type 2A/2X-myofibers (P<0.001), and vascular smooth muscle cells. Therefore, chronic induction of Hsp70 expression in rat skeletal muscles is not obligatory related to the slow fiber phenotype, but reveals the occurrence of a stress-response (Tarricone et al. 2008). In addition, training significantly increased protein levels of HO-1 in the myocardium, which contributed to the reduced infarct size occurring after ischemia-reperfusion (Marini et al. 2007). Muscle disuse atrophy is characterized by increased ROS production.
Experimental muscle disuse atrophy was achieved in the rat soleus muscles by means of hindlimb unloading by tail-suspension for 4 to 21 days. Our results showed significant and transient upregulation of heme oxygenase-1 at 7 days of unloading, whereas the relative amounts of the Glucose- regulated proteins Grp94 and Grp78 decreased significantly after 14 days of unloading to about 50% and 75%, respectively, of the protein levels of the non-suspended age- and sexmatched controls. In addition, protein oxidation was significantly increased in unloaded atrophic muscles. It is therefore possible that the reduced levels of the ER chaperones which bind calcium, such as Grp94 or Grp78, might adversley affect calcium homeostasis and potentially enhance oxidative stress of disused myofibers and favour progression of muscle atrophy. In vitro studies were then performed in order to analyze more precisely the contribution of Grp94 protein levels to cytoprotection of muscle cells againstapoptosis and oxidative damage, and mechanistichally to the control of calcium homeostasis. Grp94 overexpression was achieved by means of both stable and transient transfer of Grp94 cDNA into the myogenic cell line C2C12. The effects of pharmacological treatments, which selectively increased Grp94 cellular levels, were also explored. Results show that Grp94 overexpression obtained by gene transfer, protected myogenic C2C12 cells against apoptosis induced by exposure to 1µM staurosporin. Caspase-3 activation was reduced by 40-80% compared to control clones (p<0.05). Additional experiments showed that the percentage of apoptotic death, revealed by TUNEL assay, in C2C12 cells transiently transfected with grp94 cDNA was reduced by 50%, compared to empty vector-transfected cells. Furthermore, Grp94 overexpression reduced the degree of protein oxidation induced by exposure to hydrogen peroxide. A comparable effect was obtained when cells were transiently exposed, 24 hour in advance, to curcumin, an antioxidant which is also a SERCA inhibitor. Through its latter property, curcumin was effective in inducing a mild ER response, which apparently upregulated Grp94, in a selective manner, without changing protein levels of other ER chaperones, such as Grp78, calreticulin and HO-1. Subsequent exposure of curcumin- pretreated cells to hydrogen peroxide indeed showed reduced degree of protein oxidation. In order to identify the molecular mechanism through which Grp94 exerts antiapoptotic and anti-oxidant cytoprotection, we investigated changes in calcium homeostasis occurring in Grp94 overexpressing cells. By means of the fluorescent Ca2+ probe fura-2, Grp94- overexpressing clones showed a reduction ranging from 40 to 80% in ciclopiazonic acid-released Ca2+ compared to control clones. Transient co-transfection of C2C12 with both Grp94 and the cDNA coding for the recombinant luminescent Ca2+ sensor aequorin specifically targeted to the ER showed a slight (20%), but significant, decrease in [Ca2+]er (p<0.01), compared to control, void vector-transfected cells. However, when transient co-transfections were performed in HeLa or HEK-293 cells, no significant difference in [Ca2+]er was observed between Grp94 overexpressing and control cells, suggesting that Grp94 overexpression did not directly affect ER Ca2+ load. Experiments of immunoprecipitation from wild type C2C12 cells, processed with chemical cross-linking before lysis, showed that SERCA2 co-immunoprecipitated with Grp94. The Grp94-SERCA2 co-immunoprecipitation was clearly evident in C2C12 cells, whereas it was barely detectable in HeLa and HEK-293 cells, despite the higher relative amount of both Grp94 and SERCA2 in these two latter cell lines. The possibility that Grp94 overexpression affected [Ca2+]er by interaction with SERCA2 was further suggested by the analysis of the ER calcium refilling traces: the SERCA2 activity showed a significant decrease (20%) (p<0.01) in Grp94 overexpressing C2C12 cells compared to controls, while no differences were found in HeLa and HEK293. The possibility that Grp94 overexpression exerts a cytoprotective role by reducing [Ca2+]er load of myogenic cells through a specific inhibitory interaction with SERCA2 is supposed.
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Eriksson, Sylvia. "Molecular properties of disordered plant dehydrins : Membrane interaction and function in stress." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-136033.
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Dehydrins are intrinsically disordered plant stress-proteins. Repetitively in their sequence are some highly conserved stretches of 7-17 residues, the so called K-, S-, Y- and lysine rich segments. This thesis aims to give insight into the possible role dehydrins have in the stressed plant cell with main focus on membrane interaction and protection. The work includes four recombinant dehydrins from the plant Arabidopsis thaliana: Cor47 (SK3), Lti29 (SK3), Lti30 (K6) and Rab18 (Y2SK2). Initially, we mimicked crowded cellular environment in vitro to verify that dehydrins are truly disordered proteins. Thereafter, the proposal that the compulsory K-segment determines membrane binding was tested. Experiments show that only Lti30 and Rab18 bind, whereas Cor47 and Lti29 does not. As Lti30 and Rab18 binds they assembles vesicles into clusters in vitro, a feature used to characterize the interaction. From this it was shown that membrane binding of Lti30 is electrostatic and determined by global as well as local charges. Protonation of histidine pairs flanking the K-segments works as an on/off-binding switch. By NMR studies it was shown that the K-segments form a dynamic α-helix upon binding, so called disorder-to-order behaviour. Also, dehydrins electrostatic interaction with lipids can be further tuned by posttranslational phosphorylation or coordination of calcium and zinc ions. Finally, specific binding of Rab18 to inositol lipids, mainly PI(4,5)P2, is reported. The interaction is mainly coordinated by two arginines neighboring one of the K-segments. In conclusion, the K-segments are indeed involved in the binding of dehydrins to membrane but only in combination with extensions (Lti30) or modified (Rab18).At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.
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Lubaretz, Olga. "Non-stress induced small heat shock proteins in higher plants." [S.l. : s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=962714305.
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Qiu, Ye. "Modulation and roles of stress-responsive proteins in coxsackievirus infection." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/62935.
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Viral myocarditis is an inflammatory heart disease caused by viral infection, which is a major cause of sudden death in children and young adults. Among the various viruses, coxsackievirus B3 (CVB3) is a predominant pathogen of viral myocarditis. As CVB3 replication is tightly tangled with signaling pathways in host cells, an in-depth study of CVB3-host interactions would promote the understanding of the pathogenesis of viral myocarditis and provide critical drug targets for the development of therapeutics. CVB3 infection induces different types of stress in host cells, and in turn, the cells respond to the stress via expressing certain stress-responsive proteins (SRPs) to counteract the stress for cell survival. During the co-evolution of virus and host, CVB3 has developed sophisticated strategies to modulate and utilize SRPs to benefit its own replication. The main objective of this dissertation is to investigate the modulation and functional roles of SRPs in CVB3 infection and CVB3-induced myocardium damage. I hypothesize that 1) CVB3 infection differentially regulates the expression and activity of SRPs at transcription, translation or post-translation level; 2) the dysregulation of SRPs benefits CVB3 replication and promotes CVB3-induced cell damage. This dissertation mainly focuses on two SRPs, the inducible heat shock 70 kDa protein (Hsp70) and nuclear factor of activated T-cell 5 (NFAT5), during CVB3 infection. Using in vitro (cell culture) and in vivo (mouse) models, I demonstrated an increase of Hsp70 but a decrease of NFAT5 during CVB3 infection. Further studies elucidated the mechanism underlying such changes as well as the feedback effects on CVB3 replication. Hsp70 was upregulated via CaMKII-HSF1 signaling cascade activated in CVB3 infection and in turn promoted CVB3 infectivity via stabilizing viral genome and benefiting viral translation. NFAT5 was cleaved by CVB3 proteases 2A and 3C, generating a 70 kDa dominant negative truncate, which inhibited the iNOS-mediated anti-viral activity of NFAT5. Together, my findings have uncovered the new roles of SRPs in CVB3 infection and potential novel drug targets for CVB3-induced myocarditis.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
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Li, Aiqing. "Identification Of Proteins Associated With Insect Diapause And Stress Tolerance." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1211487603.
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Laberge, Marie-Kristine. "Nck1 is required for ER stress-induced insulin resistance and regulation of IRS1-dependent insulin signalling." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111950.
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Activation of the Unfolded Protein Response (UPR) following stress in the Endoplasmic Reticulum (ER) is an important mechanism by which obesity results in insulin resistance and type II diabetes. We uncovered a role for the adaptor protein Nck in modulating the UPR. In this study, we report that obese Nck1-/- mice, which show lower levels of UPR in liver and adipose tissue, present improved insulin signalling in these tissues. We established that the effect of Nck1 is cell autonomous by showing that HepG2 cells treated with Nck1 siRNA have reduced ER stress-induced UPR and Insulin Receptor Substrate-1 (IRS-1) serine phosphorylation. In these cells, we observed that the IRS-1 levels and activation of signalling components downstream of the insulin receptor were increased. This correlates with enhanced cell survival to stress and insulin stimulated glycogen synthesis. Overall, we demonstrated that Nck1 participates in ER-stress-induced insulin resistance and regulation of IRS-1-dependent signalling.
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O'Farrell, Francis J. "An investigation of the response of lymphoid cells to oxidative stress." Thesis, University of Ulster, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241678.
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Aken, Bronwen Louise. "Stress-inducible protein 1 : a bioinformatic analysis of the human, mouse and yeast STI1 gene structure /." Thesis, Rhodes University, 2005. http://eprints.ru.ac.za/160/.
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Thesis (M. Sc. (Biochemistry, Microbiology and Biotechnology))--Rhodes University, 2005.A research report submitted in partial fulfilment of the requirements for the degree of Master of Science (in Bioinformatics and Computational Molecular Biology).
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Odunuga, Odutayo Odutola. "Molecular characterization of the tetratricopeptide repeat-mediated interactions of murine stress-inducible protein 1 with major heat shock proteins." Thesis, Rhodes University, 2003. http://hdl.handle.net/10962/d1007724.
Full textAbstract:
Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human heat shock protein 70 (Hsp70)/heat shock protein 90 (Hsp90)-organizing protein (Hop). The two proteins are homologues of the highly conserved stress-inducible protein 1 (STI1) family of co-chaperones. The STI1 proteins interact directly and simultaneously at some stage, with Hsp70 and Hsp90 in the formation of the hetero-multi-chaperone complexes that facilitate the folding of signal transducing kinases and functional maturation of steroid hormone receptors. The interactions of mSTI1 with both Hsp70 and Hsp90 is mediated by a versatile structural protein-protein interaction motif, the tetratricopeptide repeat (TPR). The TPR motif is a degenerate 34-amino acid sequence a-helical structural motif found in a significant number of functionally unrelated proteins. This study was aimed at characterizing the structural and functional determinants in the TPR domains of mSTI1 responsible for binding to and discriminating between Hsp70 and Hsp90. Guided by data from Hop's crystal structures and amino acid sequence alignment analyses, various biochemical techniques were used to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal TPR domain (TPR1) of mSTI1 to bind to the C-terminal EEVD motif of heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. Substitutions in the first TPR motif of Lys⁸ or Asn¹² did not affect binding of mSTI1 to Hsc70, while double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn⁴³ lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys⁸ or Asn¹² reduced but did not abrogate binding. Steady state fluorescence and circular dichroism spectroscopies revealed that the double substitution of Lys⁸ and Asn¹² resulted in perturbations of inter-domain interactions in mSTl1. Together these results suggest that mSTI1-Hsc70 interaction requires a network of electrostatic interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70, but also outside the TPR1 domain. It is proposed that the electrostatic interactions in the first TPR motif collectively made by Lys⁸ and Asn¹² define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. In the first central TPR domain (TPR1A), single substitution of Lys³°¹ was sufficient to abrogate the mSTI1-Hsp90 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, residues predicted by crystallographic data to determine Hsp70 binding specificity were substituted in the TPR1 domain. The modified protein had reduced binding to Hsc70, but showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on the TPR2A domain. These data suggest that binding of Hsc70 to the TPR1 domain is more specific than binding of Hsp90 to the TPR2A domain. In addition, residues C-terminal of helix A in the second TPR motif of mSTI1 were shown to be important in determining specific binding to Hsc70. Binding assays using surface plasmon resonance spectroscopy showed that the affinities of binding of mSTI1 to Hsc70 and Hsp90 were 2 μM and 1.5 μM respectively. Preliminary in vivo studies revealed differences in the dynamics of binding of endogenous and exogenous recombinant mSTI1 with Hsc70 and Hsp90. The outcome of this study poses serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in the cell.
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Gray, Caroline Claudia. "Indogenous protection of the iscaemic myocardium." Thesis, Imperial College London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252345.
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Thinn, Kyi Kyi. "Induction of stress proteins and monokines in human monocyte-derived cells." Thesis, Imperial College London, 1990. http://hdl.handle.net/10044/1/46576.
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Du, Zhiyan, and 杜志岩. "Functions of arabidopsis acyl-coenzyme A binding proteins in stress responses." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hdl.handle.net/10722/208430.
Full textAbstract:
In Arabidopsis thaliana, a gene family encodes acyl-CoA-binding proteins
(ACBPs) conserved at the acyl-CoA-binding domain which facilitates the binding to
acyl-CoA esters. These ACBPs, designated ACBP1 to ACBP6, range in size from
10.4 to 73.1 kD. Previous studies have shown that the the overexpression of ACBP1
or ACBP2 in Arabidopsis likely promotes repair of lipid membranes and result in
enhanced tolerance to lead and cadmium, respectively. Microarray data
(http://bar.utoronto.ca/) revealed that the expression of ACBP1 and ACBP2 is also
regulated by other abiotic stresses, such as cold and drought, suggestive of their
association with these environmental pressures. The aim of this study is to investigate
and better understand the roles of ACBP1 and ACBP2 in different stress responses. It
has been previously observed that the expression of both ACBP1 and ACBP4 is lead
[Pb(II)]-inducible and recombinant ACBP1 and ACBP4 bind Pb(II) in vitro. In this
study, ACBP1 and ACBP4 were overexpressed in Brassica juncea to test if these
ACBPs could be extended for application in Pb(II) phytoremediation in transgenic B. juncea.
On freezing (-12 to -8 °C) treatment, ACBP1-overexpressing Arabidopsis was
freezing sensitive and accumulated more phosphatidic acid (PA), but less
phosphatidylcholine (PC), in contrast to acbp1 mutant plants which were freezing
tolerant and had reduced PA and elevated PC levels. Such changes in PC and PA were
consistent with the expression of the mRNA encoding phospholipase D1 (PLD1), a
major enzyme that promotes the hydrolysis of PC to PA. In contrast, the expression of
phospholipase D (PLD), which plays a positive role in freezing tolerance, was
up-regulated in acbp1 mutant plants and down-regulated in ACBP1-overexpressing
plants. Reduced PLD1 expression and decreased hydrolysis of PC to PA may
enhance membrane stability in the acbp1 mutant plants. Given that recombinant
ACBP1 binds PA and acyl-CoA esters in vitro, the expression of PLD1 and PLD
could be regulated by PA or acyl-CoAs maintained by ACBP1, if ACBP1 were to
resemble the yeast 10-kD ACBP by its capability to modulate gene expression during
stress responses. Interestingly, another membrane-associated ACBP, ACBP2, which
shows high (76.9%) conservation in amino acid homology to ACBP1, did not appear
to be affected by freezing treatment.
Besides freezing stress, ACBP1, as well as ACBP2, have been observed to
participate in abscisic acid (ABA) signaling. They both promote ABA signaling in
seed germination and seedling development, while only ACBP2 is involved in the
drought response. The overexpression of ACBP2 in Arabidopsis up-regulated reactive
oxygen species (ROS) production culminating in reduction in stomatal aperture and
water loss in guard cells, thereby enhancing drought tolerance.
For tests in phytoremediation, B. juncea was selected for overexpression of
ACBP1 and ACBP4 because it is fast-growing, has a higher biomass than Arabidopsis,
and is known to be a good accumulator of Pb(II). However, results of Pb(II) treatment
for two days showed that the overexpression of ACBP1 or ACBP4 in B. juncea did
not significantly improve Pb(II) tolerance. Nevertheless, B. juncea overexpressing
ACBP1 did accumulate Pb(II) in roots whereas ACBP4-overexpressing B. juncea
lines accumulated Pb(II) in both shoots and roots. Given that B. juncea has a larger
biomass than Arabidopsis, it is likely that the duration of Pb(II)-incubation tested in
this study was not drastic enough for comparison, and the incubation time should be
further extended for Pb(II) translocation. In addition, future studies on Arabidopsis
should be conducted to better understand the mechanism of ACBP4-mediated Pb(II)
accumulation using Arabidopsis acbp4 mutant and ACBP4-overexpressing plants.published_or_final_version
Biological Sciences
Doctoral
Doctor of Philosophy
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Rassadi, Roozbeh. "The effect of stress on nuclear protein transport : classical nuclear protein transport versus the nuclear transport of heat shock proteins." Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=33476.
Full textAbstract:
The stress response is conserved among eukaryotes and affects different cellular functions including protein transport. Here, we have investigated the effect of different types of stress on classical nuclear protein import as well as nuclear import of Ssa4p family of heat shock proteins in Saccharomyces cerevisiae.Under normal conditions, Aequorea victoria green fluorescent protein (GFP), carrying a classical nuclear localization sequence (cNLS-GFP) is nuclear. However, cNLS-GFP equilibrates throughout the cell upon exposure to heat, ethanol, H2O2 or starvation. Redistribution of the small GTPase Gsp1p, a soluble nuclear transport factor, correlates with cNLS-GFP equilibration. This suggests that a collapse of the Gsp1p gradient underlies the inhibition of classical nuclear protein import. In contrast to cNLS-GFP, the cytoplasmic heat shock protein Ssa4p accumulates in nuclei when classical nuclear import is inhibited. The N-terminal 236 amino acid residues of Ssa4p are sufficient for nuclear localization of Ssa4p-GFP upon heat and ethanol stress. The nuclear localization of Ssa4p(1--236)-GFP requires components of Gsp1-GTPase system, but is independent of Srp1p, the cNLS receptor.
Ssa4p(16--642)-GFP accumulates in nuclei of starving cells, mediated by a hydrophobic stretch of amino acid residues in its N-terminal domain. This nuclear localization is reversible upon addition of fresh medium and its export is sensitive to oxidants and temperature-dependent.
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Poon, Wai-hei. "The induction of cellular stress responses by specific Kappa-opioid receptor agonist." Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B31473921.
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Lee, Sang C., Jack Zhang, Josh Strom, Danzhou Yang, Thai Nho Dinh, Kyle Kappeler, and Qin M. Chen. "G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress." AMER SOC MICROBIOLOGY, 2017. http://hdl.handle.net/10150/622753.
Full textAbstract:
Inhibition of protein synthesis serves as a general measure of cellular consequences of chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of the NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in the 5' untranslated region (UTR) of NRF2 mRNA, as measured by circular dichroism, nuclear magnetic resonance, and dimethylsulfate footprinting analyses. Such a structure is important for 5'-UTR activity, since its removal by sequence mutation eliminated H2O2-induced activation of the NRF2 5' UTR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed elongation factor 1 alpha (EF1a) as a protein binding to the G-quadruplex sequence. Cells responded to H2O2 treatment by increasing the EF1a protein association with NRF2 mRNA, as measured by RNA-protein interaction assays. The EF1a interaction with small and large subunits of ribosomes did not appear to change due to H2O2 treatment, nor did post translational modifications, as measured by two-dimensional (2-D) Western blot analysis. Since NRF2 encodes a transcription factor essential for protection against tissue injury, our data have revealed a novel mechanism of cellular defense involving de novo NRF2 protein translation governed by the EF1a interaction with the G-quadruplex in the NRF2 5' UTR during oxidative stress.
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Stevanin, Tania Maria. "Bacterial flavohaemoglobins : physiological function and responses to nitrosative stress." Thesis, University of Sheffield, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340137.
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Poon, Wai-hei, and 潘偉曦. "The induction of cellular stress responses by specific Kappa-opioid receptor agonist." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B31473921.
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Menéndez, Benito Victoria. "The ubiquitin-proteasome system during proteotoxic stress /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-706-5/.
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Hladun, Suzanne Lynn. "Water stress protein is secreted, is subject to rapid proteolysis upon rehydration of dessiccated cells, and may be glycosylated." Thesis, This resource online, 1992. http://scholar.lib.vt.edu/theses/available/etd-10102009-020350/.
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Leblanc, Rosanne. "Protein synthesis and drought stress in two rapeseed cultivars." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=60487.
Full textAbstract:
Desiccation effects on rate and pattern of protein synthesis in Brassica napus (cv westar) and Brassica juncea (cv cutlass) have been examined. Results showed that while the rate of water loss was similar in the two species, the inhibition of amino acid incorporation was greater in B. napus than B. juncea at any given level of desiccation. Electrolyte leakage increased with the degree of desiccation and was greater in B. napus than in B. juncea. In both, the increase in leakage was much sharper after 12 hours of desiccation. Quantitative changes in patterns of boiling-stable protein synthesis due to desiccation stress were observed. The control level of protein radioactivity which was boiling-stable in B. napus was 16.16% and 19.96% for B. juncea. After desiccation, the percentage of boiling-stable radioactivity increased to 23.30% for B. juncea and 16.63% for B. napus. In vitro translation of total RNA indicated that desiccation alone does not induce the synthesis of new mRNA species in either cultivar, but it may change the translation pattern resulting in different levels of abundance of proteins.
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Levesque, Steve. "DNA Methylation, Cellular Stress Response and Expression of Inner Nuclear Membrane Proteins." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/19949.
Full textAbstract:
Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.
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Forsyth, Robert Bruce. "Stress proteins, phagocytes, and pathology in coho salmon with bacterial kidney disease." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0017/NQ48636.pdf.
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Waterston, Claire Louise. "Structural studies of putative general stress and related proteins from Deinococcus radiodurans." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/804/.
Full textAbstract:
This study describes the cloning, expression, purification, biophysical characterisation and crystallisation of DR_1146; a putative general stress protein from the extremophilic bacterium Deinococcus radiodurans (R1). The extraordinary ability of D. radiodurans to resist mutation or apoptosis on exposure to high does of ionising radiation has formed the basis of a structural genomics project underway at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The work presented in this study forms part of the ESRF’s D. radiodurans initiative, and was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and the ESRF as an Industrial Cooperative Award in Science and Engineering (CASE) PhD studentship. A period of one-year was spent on secondment at the ESRF, working within the Macromolecular Crystallography Group. Several constructs of the dr_1146 gene have been successfully overexpressed in E. coli cells to give high yields of target protein. Purification by immobilised metal affinity chromatography (IMAC) was facilitated by the incorporation of a 6xHis tag and supplemented by a final gel filtration step. Although high purity levels were achieved, imaging by SDS-PAGE analysis identified that DR_1146 was susceptible to stringent proteolysis. It is thought that initial crystallisation trials were unsuccessful due to inhomogeneity of the sample caused by reported degradation of the target protein. Biophysical characterisation of DR_1146 by isothermal titration calorimetry (ITC) and fluorescence spectroscopy (FS) identified a moderate affinity of 4-11 μM for the flavin molecules, riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Differential scanning calorimetry (DSC) and circular dichroism (CD) experiments demonstrated an increase in chemical and thermal stability of the protein on binding to the flavin molecule, FMN. Analytical ultracentrifugation (AUC) and Nuclear magnetic resonance (NMR) spectroscopy were employed to investigate the solution behaviour of DR_1146 in the presence of FMN. AUC results uncovered a monomer-dimer equilibrium; with DR_1146 self-associating to form a dimer at a concentration of 7.67 μM. NMR spectroscopy depicted that global changes occur within the structure of DR_1146 on binding to FMN. The high quality of spectra obtained showed potential for 3-D structure determination by NMR if ordered crystals could not be obtained for X-ray diffraction. Interestingly, analysis of NMR spectra proved to be integral to identifying a homogenous sample for successful crystallisation of DR_1146. By monitoring chemical shifts it was possible to determine the time needed for degradation of DR_1146 to cease, and the amount of FMN needed to ensure saturation of binding sites. From this particular sample, a stable 28 kDa fragment was isolated by gel filtration. Automated sitting-drop vapour-diffusion experiments resulted in the growth of yellow DR_1146-FMN crystals for which, although poor in quality, X-ray diffraction was obtained. Overall this study reflects the importance and advantage of incorporating information gained from biophysical characterisation into the strategies employed for successful protein crystallisation. The characterisation of DR_1146 as a flavoprotein points towards a possible role in electron transfer due to the extensive redox capacity of flavin. This could implicate the protein in the production of damaging reactive oxygen species (ROS) as a result of irradiation, contributing to oxidative stress levels. Alternatively, if DR_1146 is identified as a FMN-binding pyridoxine 5'-phosphate oxidase (PNPOx) enzyme, as sequence homology suggests, it could play a role in detoxification and stress response through production of pyridoxal 5'-phosphate (PLP), a known scavenger of ROS. Only further characterisation and elucidation of a 3-D structure would confirm or dispel these functional hypotheses and ultimately provide a greater understanding of how D. radiodurans is able to deal with such oxidising conditions. Simultaneously, experiments were carried out on other soluble and membrane protein targets from D. radiodurans and their corresponding homologues from Streptococcus pneumoniae (TIGR4). The aim of comparable studies was to identify key structural or functional differences between the two Gram-positive bacterial strains. Identification of features unique to D. radiodurans, but unconserved in S. pneumoniae, could contribute to further understanding of bacterial radioresistance. SP_1651 is a thiol peroxidase which forms part of the Mn-ABC transport system in S. pneumonia. Its homologue from D. radiodurans, DR_2242 is a putative thiol-specific antioxidant protein, the structure of which has been solved by Dr. Dave Hall as part of the ESRF’s structural genomics project (unpublished). The aim of this part of the project was to elucidate the structure of SP_1651 so that a comparison with DR_2242 could be made. The sp_1651 gene (psaD) was successfully expressed and purified to homogeneity by IMAC and gel filtration. After the proteolytic removal of a 6xHis tag, the purified protein was crystallised by sitting-drop vapour-diffusion. Preliminary diffraction with a resolution limit of 3.2 Å was obtained, however data showed high mosaic spread. Unfortunately, attempts to reproduce initial crystals failed and hence, structural comparisons with DR_2242 could not be made. DR_0463 is a 108 kDa maltooligosyltrehalose synthase (MTSase) which has been shown to catalyse the breakdown of maltooligosaccharide (or starch) into the disaccharide, trehalose. The full length gene was expressed in BL21(DE3)pLysS cells, producing large yields of insoluble target protein. DR_0463 was solubilised with 8 M Urea and then purified by IMAC in the presence of the denaturant. The low affinity of DR_0463 for the Ni2+ matrix of the HisTrap column proved to be problematic when trying to obtain homogeneity. However, by sequentially repeating IMAC purification up to three times with the same protein sample, a large proportion of impurities were removed. SP_1648 (PsaB) is an ATP-binding protein that forms part of the Mn-ATP transport system in S. pneumoniae and its homologue from D. radiodurans, DR_2284 is predicted to share similar function. Purification of soluble SP_1648, expressed in B834(DE3) cells, was complicated by an inability to bind the protein to the column matrix for IMAC. In the case of DR_2284, expression trials yielded only a minute amount of insoluble protein in BL21-AI competent cells. The bottlenecks in early expression and purification stages provided valuable experience in dealing with problematic proteins. As an introduction to molecular cloning, two genes predicted to encode integral membrane proteins from D. radiodurans, were cloned for preliminary expression trials. This work was carried out at the ESRF and contributed to an extension of the structural genomics project, to incorporate membrane protein targets from D. radiodurans. Full length forms of the genes thought to encode an undecaprenyl diphosphatase (UDP) and a diacylglycerol kinase (DGKA) were successfully cloned in to pET-28b, with incorporation of separate N- and C- terminal 6xHis tags.
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Yocum, George David. "The expression of stress proteins in response to temperature extremes in insects /." The Ohio State University, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487760357823428.
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Bernardo, Letizia. "IDENTIFICATION AND CHARACTERIZATION OF PROTEINS INVOLVED IN BIOTIC STRESS RESISTANCE OF CEREALS." Doctoral thesis, Università degli studi di Padova, 2010. http://hdl.handle.net/11577/3426966.
Full textAbstract:
Leaf rust is one of the most important diseases of barley (Hordeum vulgare) and is caused by the biotrophic fungal pathogen Puccinia hordei. The rust fungi penetrate barley leaves through stomata and colonize cells of the mesophyll, then growing systemically through the leaf vascular tissue.
The leaf rust resistance gene Rph15 is of outstanding interest for resistance breeding because it confers resistance to over 350 Puccinia hordei isolates collected from around the world (Weerasena et al. 2004).
Plant-pathogen interactions activate many cellular signalling processes and, most likely, changes on protein accumulation and phosphorylation pattern of proteins play a pivotal role in plant responses to biotic stress. In this work, a proteomic approach was undertaken to study changes in total proteins accumulation and protein phosphorylation pattern in response to the leaf rust pathogen infection in two barley near isogenic lines, Bowman and Rph15, which differ for the introgression of the leaf rust resistance gene Rph15. Two infection time points, 24 hours and four days, were considered for the analysis.
No statistically significant differences were identified at the early time point, 24 hours post infection, for total and phosphorylated proteins.
At four days after inoculation, total protein analysis led to the identification of twenty-one protein spots significantly up or down regulated with a fold-change equal or higher than two following pathogen infection. Most of down-regulated proteins were found in the Rph15 near-isogenic line while no significantly differential protein abundance was recovered in the susceptible line. Nineteen out of 21 protein spots were characterized by LC-MS/MS analysis and found to be involved in photosynthesis, sugar metabolism, energy balance and defence.
Phosphoproteomics analysis was performed at four day after inoculation. A phosphoprotein enrichment methodology based on MOAC (metal oxide affinity chromatography) was optimized for subsequent 2DE analyses.La ruggine fogliare è una delle malattie più importanti della coltura dell'orzo (Hordeum vulgare) ed è causata dal patogeno fungino biotrofo Puccinia hordei. Il fungo penetra attraverso gli stomi delle foglie dell’orzo e colonizza le cellule del mesofillo, crescendo poi per via sistemica nei tessuti vascolari della foglia. Il gene Rph15 di orzo è di considerevole importanza per il miglioramento genetico della resistenza in quanto conferisce resistenza a più di 350 isolati di P. hordei provenienti da tutto il mondo (Weerasena et al. 2004). L’interazione pianta-patogeno attiva numerosi processi di signalling cellulare e, molto probabilmente, l’accumulo delle proteine e i cambiamenti nel pattern di fosforilazione delle proteine giocano un ruolo centrale nella risposta della pianta in seguito a stress biotico. In questo lavoro, un approccio di tipo proteomico è stato intrapreso per studiare i cambiamenti nei pattern proteici totali e delle proteine fosforilate in seguito a risposta alla ruggine fogliare in due linee quasi isogeniche di orzo, Bowman e la linea Rph15, che differiscono per l’ introgressione del gene Rph15. Due tempi di infezione, 24 ore e quattro giorni, sono stati presi in considerazione per le analisi. Nessuna differenza statisticamente significativa è stata individuate nel primo tempo di infezione precoce, a 24 ore dopo l’inoculo, sia per quanto riguarda le proteine totali che per le proteine fosforilate. A 4 giorni dall’infezione, l’ analisi delle proteine totali ha consentito di identificare ventuno spot proteici significativamente up o down regolati in risposta all’ infezione con un fold-change almeno di 2. La maggior parte delle proteine down-regolate sono state trovate nel campione infettato della linea isogenica contenente il gene di resistenza Rph15, mentre non è stata riscontrata alcuna differenza statisticamente significativa nel pattern proteico della linea isogenica suscettibile. Diciannove dei 21 spot proteici sono stati caratterizzati mediante analisi LC-MS/MS e identificati essere implicati in processi come fotosintesi, metabolismo degli zuccheri, bilancio energetico e risposte di difesa. L’analisi del fosfoproteoma è stata condotta a quattro giorni dopo l’inoculo. Una tecnica di arricchimento in fosfoproteine basata su MOAC (cromatografia di affinità mediante ossidi metallici) che è stata ottimizzata per la successiva analisi 2DE.
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Hamnell-Pamment, Ylva. "Novel methods for the identification of cellular S-glutathionylated proteins and sites of glutathionedependent modification using affinity chromatography and proteomic analyses /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-248-9/.
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Caviness, James A. "Stress biomarkers in a rat model of decompression sickness /." Download the thesis in PDF, 2005. http://www.lrc.usuhs.mil/dissertations/pdf/Caviness2005.pdf/.
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