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1
Tokarew, Jacqueline M. « A Novel Role for the Parkinson Disease-Linked and Neuromelanin-Associated Parkin Protein as a Cysteine-Dependent Redox-State Regulator ». Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42389.
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Parkinson disease (PD) is an incurable disease, second only to Alzheimer’s disease as the most common neurodegenerative disease in adults. Unfortunately, the course of disease is significantly longer for individuals diagnosed at an early age (20-40 years of age). Although early-onset, recessively inherited cases represent a small subset of individuals with PD (~5- 10%), their clinical presentation is unique, with symptoms being almost exclusively motor-related. The expressivity of early-onset PD is partially explained by post-mortem neuropathological findings, which demonstrate a specific loss of dopamine synthesizing cells in brainstem nuclei that also produce neuromelanin (i.e. Substantia nigra and Locus coeruleus). With the majority of early-onset PD cases being caused by homozygous and biallelic heterozygous mutations in the PRKN gene, its gene product, parkin, has been extensively studied. It is generally accepted that loss of its E3 ligase function leads to neurodegeneration by either one of the following two mechanisms: i) toxic substrate accumulation from the loss of target protein ubiquitination (and related degradation), or ii) accumulation of dysfunctional mitochondria due to impaired mitophagy initiation. However, whether these mechanisms lead to selective neuronal loss within the human brain remains unknown. This thesis represents a body of work that supports a novel role for parkin as a thiol-based anti-oxidant and redox homeostasis regulator, which helps explain the cell-specificity observed in recessive, PRKN-linked PD. These findings include: i) evidence that human brain parkin uniquely and natively undergoes age-associated aggregation beginning at 40 years of age (Chapter 2); ii) identification of multiple, reversible and irreversible oxidative modifications of parkin cysteines, both in cells and tissues, including dopamine-adduct conjugation on primate sequence-specific cysteine 95 (Chapter 2); iii) the demonstration that irreversible oxidation of parkin cysteines causes aggregate formation ii in cells and mice exposed to exogenous and/or endogenous sources of oxidative and dopamine stress (Chapter 2 and 3); iv) evidence that parkin functions as a thiol-dependent anti-oxidant similar to glutathione (Chapter 2), which lowers oxidation state in cells and tissues under native and stress conditions (Chapter 2 and 3); v) the demonstration that parkin cysteines, notably C95, directly bind glutathione and regulate glutathione redox homeostasis in cells and tissues in a dynamic fashion (Chapter 3); and vi) the development of novel, human-specific, anti-parkin monoclonal antibodies that preferentially detect oxidized and aggregated forms of parkin found associated with neuromelanin and lysosomal storage vesicles within neurons of human Substantia nigra (Chapter 2 and 4). Future studies focusing on further validation of in situ oxidative modifications of parkin cysteines and their effect on protein structure, notably the poorly studied linker region that contains C95, will provide insight into how these oxidative modifications affect the function of parkin in vivo, including in adult human brain. Also, identifying the bona fide intracellular redox partners of parkin will be crucial to understanding how this protein regulates cellular redox state. Of clinical importance, the findings presented here indicate a potential, human-specific link between parkin and neuromelanin formation, which deserves to be further explored, such as with parkin mouse models engineered to produce neuromelanin. Finally, designing clinical trials using anti-oxidants specifically in individuals affected by PRKN-associated PD represents a logical, translational treatment approach to explore.
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Lui, James Kwok Ching. « A fluorescent labelling technique to detect changes in the thiol redox state of proteins following mild oxidative stress ». University of Western Australia. School of Biomedical, Biomolecular and Chemical Sciences, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0056.
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There is increasing evidence that hydrogen peroxide (H2O2) can act as a signalling molecule capable of modulating a variety of biochemical and genetic systems. Using Jurkat T-lymphocytes, this study initially investigated the involvement of H2O2 in the activation of a specific signalling protein extracellular signal-regulated protein kinase (ERK). It was found that as a result of H2O2 treatment, mitochondrial complex activities decreased which led to subsequent increase of mitochondrial reactive oxygen species (ROS) production. The increase of ROS resulted in higher cellular H2O2 as well as increased ERK activation. This study demonstrated that in an oxidative stress setting, H2O2 production from the mitochondria was an essential component in maintaining the activation of a signalling protein. One way in which H2O2 could influence protein function is by the oxidation of susceptible thiol groups of cysteine residues. To further understand the variety of signalling pathways that H2O2 may be involved in, an improved proteomics technique was developed to globally identify proteins with susceptible thiol groups. The
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Guo, Liang. « Structural and functional studies of mitochondrial small Tim proteins ». Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/structural-and-functional-studies-of-mitochondrial-small-tim-proteins(03dde6fd-6692-4af5-9023-b85a33803fcd).html.
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Most mitochondrial proteins are encoded by nuclear DNA, and synthesised in the cytosol, then imported into the different mitochondrial subcompartments. To reach their destination, mitochondrial inner membrane proteins require import across the outer mitochondrial membrane, and through the intermembrane space. This passage through the IMS is assisted by the small Tim proteins. This family is characterised by conserved cysteine residues arranged in a twin CX3C motif. They can form Tim9-Tim10 and Tim8-Tim13 complexes, while Tim12 appears to form part of a Tim9-Tim10-Tim12 complex that is associated with the inner membrane translocase TIM22 complex. Current models suggest that the biogenesis of small Tim proteins and their assembly into complexes is dependent on the redox states of the proteins. However, the role of the conserved cysteine residues, and the disulphide bonds formed by them, in small Tim biogenesis and complex formation is not clear. As there is no research about the structural characterisation of Tim12 and double cysteine mutants of Tim9, purification of these proteins was attempted using different methods. To investigate how cysteine mutants affect complex formation, the purified double cysteine mutants of Tim9 were studied using in vitro methods. It showed that the double cysteine mutants were partially folded, and they can form complexes with Tim10 with low affinities, suggesting disulphide bonds are important for the structures and complex formation of small Tim proteins. The effect of cysteine mutants on mitochondrial function was addressed using in vivo methods. It showed that cysteines of small Tim proteins were not equally essential for cell viability, and growth defect of the lethal cysteine mutant was caused by low level of protein. Thus, the conclusion of this study is that disulphide bond formation is highly important for correct Tim9- Tim10 complex formation, and yeast can survive with low levels of complex, but it results in instability of the individual proteins.
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Maxwell, Brian Andrew. « Multi-disciplinary Investigation of the Kinetics and Protein Conformational Dynamics of DNA Replication and Oxidative DNA Damage Bypass and Repair ». The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1405961617.
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Mensah, Eric. « Creation of a Site-Directed Mutant of Hen Egg White Lysozyme Working Toward Site-Specific Oxidation as it Relates to Protein Structure ». Connect to resource online, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1251756763.
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Bilous, I. I. « The dynamics of the parameters of lipid peroxidation, the oxidative modification of proteins and the state of the blood antioxidant system 3 and 6 months after treating diabetic polyneuropathy ». Thesis, БДМУ, 2017. http://dspace.bsmu.edu.ua:8080/xmlui/handle/123456789/17030.
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Bilous, Iryna Ivanivna. « The dynamics of the parameters of lipid peroxidation, the oxidative modification of proteins and the state of the blood antioxidant system 3 and 6 months after treating diabetic polyneuropathy ». Thesis, ВДНЗ України "Буковинський державний медичний уніврситет", 2017. http://dspace.bsmu.edu.ua:8080/xmlui/handle/123456789/14083.
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Tuckey, Nicholas Pierre Lemieux. « Technologies for tissue preservation : the role of endogenous and exogenous antioxidants in preserving tissue function in chinook salmon, Oncorhynchus tshawytscha ». Thesis, University of Canterbury. Biological Sciences, 2008. http://hdl.handle.net/10092/1510.
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The seafood industry is of considerable importance to both the New Zealand and global economies and therefore tissue preservation technologies that increase product quality and/or prolong shelf life have the potential to add significant value. Technologies for maintaining the viability of isolated tissues also have a wide range of other medical and industrial applications. This thesis examines the relationship between metabolic function, oxidation and cell death and the resulting stability of the non-viable tissues during long term storage in chinook salmon (Oncorhynchus tshawytscha) red and white skeletal muscle tissue. This research also looks at the role of the aquatic anaesthetic AQUI-S™, in which the active ingredient is isoeugenol (a lipid soluble antioxidant), and other antioxidant compounds in preserving metabolic function in viable tissues and tissue stability in nonviable tissues. Perfusion of salmon tails at 15℃ over 5 or 10 hours with oxygen saturated saline resulted in significant increases in protein and lipid oxidation (protein carbonyl and TBARS concentrations respectively) in the red muscle, but not the white muscle. The introduction of ascorbic acid and uric acid into the saline did not reduce the oxidation in the red muscle despite significantly increasing their respective concentrations in the tissue. This indicates the difficulties associated with attempting to extend tissue viability by delivering free oxygen to the tissue and also highlights the difference in susceptibility of the two muscle types to oxidation. Tail fillets from salmon harvested in both rested and exhausted physiological states using AQUI-S™, and fillets from exhausted salmon harvested without AQUI-S™, were exposed to air at 15℃ for up to 96 hours. Protein carbonyls increased in a roughly linear fashion over the entire 96 hours in all three groups. Both lipid peroxides (TBARS) and uric acid concentrations began to increase in the exhausted group after 30 hours. In contrast, no significant increases in lipid peroxides or uric acid was seen in the fillets from either group harvested using AQUI-S™. Vitamin E concentrations reduced slowly but did not change significantly despite the oxidation that was evident in the tissue. These processes also occurred in salmon tail fillets during storage at 6℃. The measurement of ATP related compounds provides an effective indicator of both the metabolic state of the tissue post-harvest and the quality. The breakdown of these compounds is also associated with the production of ammonia and hydrogen peroxide. Fresh rested salmon fillets had high concentrations of ATP and creatine phosphate, which were both depleted after 12 hours storage at 15℃. This indicates that cell viability lasted a number of hours following harvesting. These metabolites were depleted in exhausted fillets and metabolic potential appeared to be immediately compromised. The concentration of the taste enhancing compound IMP was significantly reduced in fresh rested tissue, but increased during storage, and was significantly higher than in exhausted tissues following 12 hours of storage at 15℃. This indicates that some properties of rested tissues may improve with limited storage times. The accumulation of uric acid - the metabolic end point for ATP related compounds - was also significantly reduced in rested tissue and increases in K-value were slowed. AQUI-S™ showed an ability to preserve tissue function through its anaesthetic action allowing tissue to be harvested in a rested state, and to reduce late stage lipid oxidation in stored salmon tail fillets. The antioxidant action of isoeugenol in salmon fillets may be mediated through its ability to chelate transition metals released during tissue degradation. This research shows that during reperfusion and during fillet storage there is a significant level of oxidative stress, which needs to be minimized while maintaining basic tissue metabolism to prolong tissue and cellular viability. The development of future technologies to preserve tissue viability may depend on the development of a synthetic oxygen carrying compound with properties similar to red blood cells. This may allow more control over oxygen delivery, potentially reducing the oxidative stress associated with high concentrations of free oxygen in solution. However, preserving cell viability will also require the maintenance of endogenous antioxidant function and there is also the potential to use iron chelating compounds including plant derived flavonoids to preserve non-viable tissues. Future research in these areas is necessary.
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Du, Aiguo. « Prediction of oxidation states of cysteines and disulphide bridges in proteins ». unrestricted, 2007. http://etd.gsu.edu/theses/available/etd-11272007-024411/.
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Thesis (Ph. D.)--Georgia State University, 2007.Title from file title page. Y. Pan, committee chair; G. Qin, A. Bourgeois, A. Zelikovski, committee members. Electronic text (124 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed June 3, 2008. Includes bibliographical references (p. 111-124).
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Harvey, Anna Ross. « Oxidative protein folding in Aspergillus niger ». Thesis, University of Nottingham, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.523081.
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Xiao, Ruoyu. « Protein disulfide isomerase : function and mechanism in oxidative protein folding / ». Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-238-1/.
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Du, Aiguo. « Prediction of Oxidation States of Cysteines and Disulphide Connectivity ». Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/cs_diss/28.
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Knowledge on cysteine oxidation state and disulfide bond connectivity is of great importance to protein chemistry and 3-D structures. This research is aimed at finding the most relevant features in prediction of cysteines oxidation states and the disulfide bonds connectivity of proteins. Models predicting the oxidation states of cysteines are developed with machine learning techniques such as Support Vector Machines (SVMs) and Associative Neural Networks (ASNNs). A record high prediction accuracy of oxidation state, 95%, is achieved by incorporating the oxidation states of N-terminus cysteines, flanking sequences of cysteines and global information on the protein chain (number of cysteines, length of the chain and amino acids composition of the chain etc.) into the SVM encoding. This is 5% higher than the current methods. This indicates to us that the oxidation states of amino terminal cysteines infer the oxidation states of other cysteines in the same protein chain. Satisfactory prediction results are also obtained with the newer and more inclusive SPX dataset, especially for chains with higher number of cysteines. Compared to literature methods, our approach is a one-step prediction system, which is easier to implement and use. A side by side comparison of SVM and ASNN is conducted. Results indicated that SVM outperform ASNN on this particular problem. For the prediction of correct pairings of cysteines to form disulfide bonds, we first study disulfide connectivity by calculating the local interaction potentials between the flanking sequences of the cysteine pairs. The obtained interaction potential is further adjusted by the coefficients related to the binding motif of enzymes during disulfide formation and also by the linear distance between the cysteine pairs. Finally, maximized weight matching algorithm is applied and performance of the interaction potentials evaluated. Overall prediction accuracy is unsatisfactory compared with the literature. SVM is used to predict the disulfide connectivity with the assumption that oxidation states of cysteines on the protein are known. Information on binding region during disulfide formation, distance between cysteine pairs, global information of the protein chain and the flanking sequences around the cysteine pairs are included in the SVM encoding. Prediction results illustrate the advantage of using possible anchor region information.
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Hammerman, Malin. « Oxidative Stress and Protein Acetylation in Adipocytes ». Thesis, Linköpings universitet, Proteinkemi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-75785.
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Obesity is an increasing health problem which is causally associated with insulin resistance and type 2 diabetes. Oxidative stress, i.e. overproduction of reactive oxygen species, is associated with insulin resistance and obesity and may be a major risk factor in the onset and progression of diabetes. Bernlohr Lab at University of Minnesota have study oxidative stress in adipocytes by silencing the enzyme glutathione S-transferase A-4 (GSTA4), an enzyme detoxifying 4-hydroxynonenal formed during oxidative stress. Their results indicate that lysine acetylation, an important post-translational modification, may be involved during oxidative stress. In this study lysine acetylation has been investigated in condition of oxidative stress in 3T3-L1 adipocytes and subcutaneous adipose tissue from mice using SDS-PAGE gel electrophoresis and western blot. Lysine acetylation was analyzed in different compartments of the cell such as in cytoplasm, mitochondria as well as in whole cell extracts. Silencing of GSTA4 and stimulation by TNF-α in 3T3-L1 adipocytes resulted in an increase of lysine acetylation in cytoplasm. Furthermore, stimulation by IL-6 did not have any effect on lysine acetylation. Surprisingly, subcutaneous adipose tissue from mice fed on a high-fat diet showed a decrease of lysine acetylation in cytoplasm compare to mice fed on a chow diet. In conclusion, lysine acetylation seems to change during oxidative stress and may be an important factor during insulin resistance, type 2 diabetes and obesity. Therefore, studying lysine acetylation and enzymes modulating acetylation may potentially increase our understanding of insulin resistance, type 2 diabetes and obesity and could lead to new therapies.
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Costa, N. J. « Mitochondrial protein thiol modifications during oxidative stress ». Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598052.
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The focus of this thesis is to investigate the interactions of mitochondrial protein thiols with ROS and examine the oxidation of these thiols in response to oxidative stress. Among these protein thiol modifications, an area of great interest is the interactions of mitochondrial protein thiols with glutathione, therefore possible connections between the modifications and cell death were investigated. I explored the possibility that staurosporine (STS) induces apoptosis via the mitochondrial pathway, causing early changes in the mitochondrial membrane potential (Δψ), by changing the thiol redox status of the cell. The hypothesis tested was that there may be a common link between oxidative stress and thiol changes of mitochondrial proteins and oxidation of the cellular glutathione pool which then initiates the critical mitochondrial events that lead to apoptosis. STS caused apoptosis after 2-4 hours of treatment and caused a decrease in total glutathione measured in cells, with the depletion of cellular glutathione occurring after the induction of apoptosis. There were no changes in glutathione (GSH)/glutathione disulphide (GSSG) redox state up to one hour of STS treatment, and thus no association of cellular GSH oxidation with the early mitochondrial Δψ changes observed. However, the GSH pool was significantly oxidised after 2 hours. Even so, no significant changes in protein glutathionylation by STS were observed at 2 hours. I next explored the possibility that protein thiol modifications might respond to oxidative stress for the purpose of redox signalling, whereby redox-sensitive modifications might contribute to biological regulation. Therefore, I went on to quantify total and exposed protein thiols in subcellular fractions including mitochondria. This analysis showed that 74% of total liver cell lysate protein thiols are exposed compared to 57% of mitochondrial proteins, 67% of cytosolic proteins and 62% of soluble proteins. The amount of exposed thiols as a percentage of total thiols in each of the four fractions did not differ when comparing rat liver and rat heart tissue. I then further characterised the distribution of protein thiols in the mitochondria and found that approximately two-thirds of protein thiols present in the mitochondrial membrane fraction were exposed compared to 78% in the mitochondrial matrix fraction. In addition, by using two membrane-impermeant thiol-alkylating agents, I was able to show that approximately a third of all the exposed protein thiols in the mitochondrial membrane fraction were either on the outer membrane, facing outward into the cytosol or facing inward into the intermembrane space, within the intermembrane space itself or on the outer leaflet of the inner membrane. The possibility that protein thiols might have a protective role acting as a redox buffer was also explored, and loss of exposed protein thiols during oxidative stress was investigated.
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CÌŒemazar, Masa. « Oxidative folding of a cystine knot protein ». Thesis, Open University, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.275108.
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Annangudi, Palani Suresh Babu. « Lipid-based Oxidative Protein Modifications in Glaucoma ». Case Western Reserve University School of Graduate Studies / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=case1129558048.
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Grundy, Nicholas Matthew. « Protein S-thiolation and oxidative stress in plants ». Thesis, Durham University, 2002. http://etheses.dur.ac.uk/3950/.
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The tripeptides glutathione (GSH; γglutamyl-cysteinyl-glycine) and homoglutathione (hGSH; γglutamyl-cysteinyl-β-alanine) are abundant cytosolic tripeptides in legumes. The reactive cysteinyl sulphydryl group enables GSH or hGSH to act as the major cellular redox buffer through the formation of disulphides with other GSH/hGSH molecules. GSH can also form disulphides with cysteinyl groups within proteins, which is termed 5-thiolation, a reversible modification, protecting proteins from irreversible inactivation of thiol residues, as well as being important in regulating protein activity. Following treatment with fungal cell wall elicitors, plant cells produce reactive oxygen species (ROS) which results in cellular oxidative stress. In animal cells ROS generation induces antioxidant defences which include the accumulation of glutathione (GSH) and the formation of mixed disulphides between proteins and GSH. It was hypothesised that following treatment with a fringal elicitor, plant cells also thiolate proteins. It was of interest to determine how protein thiolation changed in response to changes in thiol metabolism known to occur during elicitation, as well as identifying proteins which underwent this modification. Using cell cultures of alfalfa (Medicago saliva L.), a leguminous plant containing both GSH and hGSH, changes in thiol content upon treatment with a fungal cell wall preparation elicitor were determined. By inhibiting protein synthesis and labelling the thiol pools with L-[(^35)S]cysteine, the degree and rate of protein mixed disulphide formation could be monitored in-vivo. To induce the elicitation response, alfalfa cell cultures were treated with a fungal cell-wall elicitor. Following elicitor treatment GSH, but not hGSH, was found to accumulate, with an associated increase in GSH, but not hGSH, forming mixed disulphide with protein. In order to use proteomic tools to identify thiolated proteins, the oxidative stress response in cell cultures of Arabidopsis, a GSH containing species, was then characterised. The level of protein-bound GSH was found to increase following treatment of cell cultures with the oxidant tert-hutyl hydroperoxide and this was associated with changes in cellular thiols. When proteins S-thiolated either in-vivo, or in-vitro, with [(^35)S]-GSH were resolved by SDS-PAGE under non-reducing conditions, a large number of radiolabelled polypeptides were identified in oxidatively stressed preparations. Testing the hypothesis that GSH-dependent enzymes may undergo S-thiolation, proteins which bound GSH were isolated from Arabidopsis using GSH-afFinity chromatography. A number of 30 kDa polypeptides were isolated and found to be S-thiolated under oxidative conditions in-vitro. Several of these were subsequently identified, notably members of the glutathione transferase (GST) superfamily. Representative recombinant GSTs from Arabidopsis, maize and soybean were expressed, Violated in-vitro and the effect on activity determined. Several thiolatable GSTs were identified from Arabidopsis, notably the members of the family of dehydroascorbate reductases (DHAR I, 11, III) and lambda GSTs. Further analysis by elecfrospray mass-spectroscopy confirmed the covalent binding of GSH to DHAR isoenzymes during in-vitro thiolation. It was concluded that S-thiolation of proteins is a commonly observed reversible modification of proteins in plants exposed to oxidative stress with potentially important consequences in cytoprotection and regulation.
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Carr, M. D. « NMR studies of oxidative phosphorylation ». Thesis, University of Oxford, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382584.
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Tan, Yew-Foon. « Metal-protein interactome in plant mitochondria ». University of Western Australia. School of Biomedical, Biomolecular and Chemical Sciences, 2009. http://theses.library.uwa.edu.au/adt-WU2009.0162.
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[Truncated abstract] Transition metals in the plant mitochondrion have dual roles in regulating the function of the organelle. While metals participate in mitochondrial respiratory metabolism as ligands in bioenergetic, detoxifying, and various other metabolic enzymes, a breakdown in metal homeostasis during oxidative stress can perpetuate the cycling of ROS by redox active metal ions. Large-scale studies into the duplicitous roles of metal ions in biological systems has been lacking and in this thesis, a combination of metallomics, database annotations, membrane proteomics, metal-protein interactomics, structural biology, functional assays and mass spectrometry were all used to gain a clearer insight into the involvement of metal ions in affecting plant mitochondrial function. The Arabidopsis mitochondrion was shown to contain the transition metals cobalt, copper, iron, manganese, molybdenum, and zinc. Interestingly, the redox active copper and iron represented 75% of the mitochondrial metallome and these metal species were revealed to be highly labile during oxidative stress suggesting a possible contribution of metal-catalysed oxidation (MCO) in the damage of biological macromolecules. Bioinformatic analysis of metalloproteins predicted and experimentally determined to be mitochondrially localised revealed that metal ion transporters are poorly characterised. An in-depth proteomic analysis of the membrane proteome was conducted on mitochondria isolated from unstressed and stressed cell cultures resulted in the identification of stress-responsive as well as potential metal ion transporters. Also, many of the annotated metalloproteins predicted to be mitochondrial lack experimental evidence for subcellular localisation. ... However, based on evidence in the literature, it was hypothesised that metal-interacting sites may be the targets for MCO due to their affinity for metal ions. Attempts were made to identify the site specificity of MCO on mitochondrial proteins but no carbonyl sites could be found owing to technical problems associated with non-specific binding of proteins to the enrichment resin and low abundance of the labelled protein carbonyls. The use of the model protein BSA showed that protein oxidation occurs in clusters and the use of model peptides demonstrated that the ability of amino acid residues to complex metal ions is important in dictating susceptibility to MCO. Further experimental verification for the site specificity of MCO is required to determine the consequences of MCO on mitochondrial protein function. Overall, this thesis provided a large-scale analysis of the contributions of metal ions to mitochondrial respiratory metabolism with an emphasis on metal ion induced toxicity. Using multi-facetted approaches, an insight into the dynamic nature of mitochondrial metal homeostasis, stress responsive transporters, the interactions of metal ions with mitochondrial proteins and the possible mechanism in which proteins are specifically oxidised by MCO has been uncovered paving the way for future focused studies characterising the consequences of oxidative stress on specific proteins and their function.
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Luca, Corneliu Constantin. « MTERFD3 is a Mitochondrial Protein that Modulates Oxidative Phosphorylation ». Scholarly Repository, 2008. http://scholarlyrepository.miami.edu/oa_dissertations/132.
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Mitochondrial function is critical for the survival of eukaryotes. Hence, mitochondrial dysfunctions are involved in numerous human diseases. An essential process for a normal mitochondrial function is mitochondrial gene expression which is tightly regulated in response to various physiological changes. The accurate control of mitochondrial gene expression is essential in order to provide the appropriate oxidative phosphorylation capacity for diverse metabolic demands. Recent findings in the basic mitochondrial replication and transcription regulation helped advance our understanding of organelle function and basic pathogenetic mechanisms of mitochondrial DNA mutations associated with oxidative phosphorylation defects. Mitochondrial transcription is regulated by the mitochondrial transcription termination factor (mTERF) both at the initiation and termination levels. A protein family containing highly conserved mTERF motifs has been identified recently and its members named generically as "terfins." In this work, one of these factors, mTERFD3, has been characterized in vitro and in vivo. The mTERFD3 protein is highly conserved throughout evolution. It is a mitochondrial protein localized to the matrix and is abundantly expressed in high energy demand tissues. We found that it contains 4 putative leucine zippers and is able to form dimers in vitro. We showed that mTERFD3 binds mtDNA at the transcription initiation site in the mtDNA regulatory region. These findings suggest that mTERFD3 may be involved in regulating mitochondrial gene expression at the transcriptional initiation level. In order to study the functional significance of mTERFD3 in vivo we developed a mouse deficient in mTERFD3 using a gene trapping strategy. The KO mice had a normal lifespan but showed decreased weight gain and decreased fat content in females. Fibroblasts isolated from KO mice displayed decreased growth rate when compared with WT in respiratory media, and had decreased complex IV activity. Consistent with the above findings, we found that muscle, one of the tissues with high energy demands, showed abnormal mitochondrial function, displaying features characteristic of mitochondrial myopathy such as decreased muscle strength and endurance. Muscle mitochondria of the KO mice showed a significant decrease in the complex II +III and complex IV activity. The decrease in OXPHOS complexes activity was associated with increased citrate synthase activity, suggesting mitochondrial proliferation, a feature typical for mitochondrial disorders. Another important finding was a decrease in the muscle mitochondrial transcripts in the KO animals associated with decreased steady state levels of OXPHOS subunits. Together these data suggest that mTERFD3 is a mitochondrial protein involved in the regulation of mtDNA transcription. mTERFD3 KO is not embryonic lethal suggesting that it is involved in the fine tuning of mitochondrial transcription. We conclude that mTERFD3 is a mitochondrial protein that modulates oxidative phosphorylation function, probably by directed interactions with the mtDNA regulatory region. This work shows the importance of mTERFD3, an mTERF family member, in the mitochondrial gene expression regulation.
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Clements, Casey M. Ting Jenny P. Y. « Functional characterization of DJ-1 an oxidative response protein / ». Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1218.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.Title from electronic title page (viewed Mar. 26, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology." Discipline: Microbiology and Immunology; Department/School: Medicine.
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Matsusaki, Motonori. « Molecular Mechanism of Oxidative Protein Folding by Soybean Protein Thiol Disulfide Oxidoreductases/ERO1 Pathway ». Doctoral thesis, Kyoto University, 2016. http://hdl.handle.net/2433/217183.
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京都大学0048
新制・課程博士
博士(農学)
甲第20008号
農博第2192号
新制||農||1045(附属図書館)
学位論文||H28||N5017(農学部図書室)
33104
京都大学大学院農学研究科農学専攻
(主査)教授 裏出 令子, 教授 松村 康生, 教授 三上 文三
学位規則第4条第1項該当
Doctor of Agricultural Science
Kyoto University
DFAM
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Matsusaki, Motonori. « Molecular Mechanism of Oxidative Protein Folding by Soybean Protein Thiol Disulfide Oxidoreductases / ERO1 Pathway ». Kyoto University, 2009. http://hdl.handle.net/2433/217183.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第20008号
農博第2192号
新制||農||1045(附属図書館)
学位論文||H28||N5017(農学部図書室)
33104
京都大学大学院農学研究科農学専攻
(主査)教授 裏出 令子, 教授 松村 康生, 教授 三上 文三
学位規則第4条第1項該当
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Gregory, Mary Sarah-Jane, et 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.
Texte intégralRésumé :
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.
25
Gregory, Mary Sarah-Jane. « Thioredoxin and Oxidative Stress ». Thesis, Griffith University, 2004. http://hdl.handle.net/10072/367183.
Texte intégralRésumé :
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
Full Text
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Karamullaoglu, Gulsun. « Dynamic And Steady-state Analysis Of Oxidative Dehydrogenation Of Ethane ». Phd thesis, METU, 2005. http://etd.lib.metu.edu.tr/upload/12606269/index.pdf.
Texte intégralRésumé :
In this research, oxidative dehydrogenation of ethane to ethylene was studied over Cr-O and Cr-V-O mixed oxide catalysts through steady-state and dynamic experiments. The catalysts were prepared by the complexation method. By XRD, presence of Cr2O3 phase in Cr-Oand the small Cr2O3 and V2O4 phases of Cr-V-O were revealed. In H2-TPR, both catalysts showed reduction behaviour. From XPS the likely presence of Cr+6 on fresh Cr-O was found. On Cr-V-O, the possible reduction of V+5 and Cr+6 forms of the fresh sample to V+4, V+3 and Cr+3 states by TPR was discovered through XPS. With an O2/C2H6 feed ratio of 0.17, Cr-O exhibited the highest total conversion value of about 0.20 at 447°
C with an ethylene selectivity of 0.82. Maximum ethylene selectivity with Cr-O was obtained as 0.91 at 250°
C. An ethylene selectivity of 0.93 was reached with the Cr-V-O at 400°
C. In the experiments performed by using CO2 as the mild oxidant, a yield value of 0.15 was achieved at 449°
C on Cr-O catalyst. In dynamic experiments performed over Cr-O, with C2H6 pulses injected into O2-He flow, the possible occurrence of two reaction sites for the formation of CO2 and H2O was detected. By Gaussian fits to H2O curves, the presence of at least three production ways was thought to be probable. Different from Cr-O, no CO2 formation was observed on Cr-V-O during pulsing C2H6 to O2-He flow. In the runs performed by O2 pulses into C2H6-He flow over Cr-V-O, formation of CO rather than C2H4 was favored.
27
Gonzalez, Veronica. « The role of protein disulfide isomerase (PDI) in oxidative folding ». To access this resource online via ProQuest Dissertations and Theses @ UTEP, 2008. http://0-proquest.umi.com.lib.utep.edu/login?COPT=REJTPTU0YmImSU5UPTAmVkVSPTI=&clientId=2515.
Texte intégral
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McKenna, Tomás. « Oxidative stress on mammalian cell cultures during recombinant protein expression ». Licentiate thesis, Linköping University, Linköping University, Biotechnology, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-51823.
Texte intégralRésumé :
When the cell is under stress arising from oxidation, heat, infection, toxic contamination or any other stressful condition, proteins may unfold and expose residues in their structure that under normal physiological conditions are hidden and shielded from chemical reactions.
In this licentiate thesis the effects of general oxidative stress on the production of recombinant protein by mammalian cells are considered.
The work consisted of a broad literary review focused on oxidative stress and cellular response, cross-protection, gene regulation in response to oxidative stress and the relevance of this to pharmaceutical industry. A series of oxidative stressors is described and examined for experimental use. Experimental cultivation and maintenance of several mammalian cell lines was performed and several candidate stressing agents were proven on these cell lines. Menadione was selected as a powerful and consistent stressing agent, and so several experiments were performed where batches of cells were exposed to varying degrees of stress.
The performance of the cells in regard to production of recombinant protein was then examined by ELISA, showing a strong downregulation of production under stressful conditions. Recombinant protein taken from stressed and control cultures is then isolated, purified and examined with MALDI-TOF spectrometry. Finally mRNA from the cells is isolated and examined by means of microarray. Genes that are significantly regulated are examined, and those genes that may have significance in the area of stress regulation and reaction are listed.
The results of the study show that mitomycin C exerts oxidative stress on the industrial protein expressing mammalian cell lines tested.
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Basoah, Afua. « The effect of oxidative stress on protein modification and degradation ». Thesis, University of Oxford, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400552.
Texte intégral
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Scheinost, Johanna C. « A cholesterol oxidative metabolite and its role in protein misfolding ». Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.504523.
Texte intégral
31
Auciello, Francesca Romana. « Canonical and non-canonical regulation of AMP-activated protein kinase ». Thesis, University of Dundee, 2015. https://discovery.dundee.ac.uk/en/studentTheses/2720a2b7-3f1e-445c-b008-c5c235f35395.
Texte intégralRésumé :
The AMP-activated protein kinase (AMPK) is a sensor of cellular energy stress that, once activated, promotes ATP-producing process while it switches off ATP-consuming pathways, in order to restore the cellular energetic balance under conditions of stress. Activation of AMPK is dependent on the phosphorylation of the residue Thr172 in its α subunit. This phosphorylation is generally mediated by the known tumour suppressor LKB1, but also CaMKKβ has been shown to phosphorylate AMPK. As its name suggests, AMPK is also activated by the binding of AMP to its γ subunit. This binding causes a >10 fold allosteric stimulation, promotes phosphorylation of Thr172 by upstream kinases and protects AMPK from dephosphorylation of Thr172 by protein phosphatase(s). In 2010 it was reported that oxidative stress mediated by H2O2 activated AMPK by increasing the cellular AMP:ATP and ADP:ATP ratios (Hawley et al, 2010). However, the same year another work suggested that the mechanism of activation of AMPK by H2O2 was direct, independent of AMP and involved the oxidation of two cysteine residues in the α subunit of AMPK (Zmijewski et al, 2010). Given this discrepancy, here we provided evidence that H2O2, generated by addition of glucose oxidase in the cell medium, activates AMPK mostly through an increase of AMP:ATP and ADP:ATP ratios, as previously suggested in our laboratory. However, it seems that there might be a second, minor mechanism of activation that is independent of the changes in cellular nucleotides. This second mechanism was not identified in our previous work because we were not aware of how rapidly a single bolus of H2O2 can be metabolized by the antioxidant defences of the cell. We could not identify the alternative mechanism of activation by H2O2 but showed that H2O2 could protect Thr172 from dephosphorylation, which might suggest a direct effect of H2O2 on the phosphatase(s) dephosphorylating AMPK. However, since the identity of this phosphatase(s) remains unclear, we could not rule out the possibility that the protection from dephosphorylation that we observed could still be mediated by the increase in AMP:ATP and ADP:ATP ratios. Moreover, it remains still possible that a direct effect of H2O2 on AMPK might be responsible for the small but significant activation we detected in cell expressing a nucleotides-insensitive mutant of AMPK. Recently, a new crystal structure of AMPK obtained by Xiao et al (2013) provided new insights about AMPK structure and regulation. In particular, the authors identified a new binding pocket located at the interface between the N-lobe of the α-kinase domain and the β-CBM of AMPK, which appeared to be the binding site for two direct activators of AMPK: A769662 and 991. Here we confirm that this novel binding pocket is indeed the binding site for both A769662 and 991, and provide evidence that another direct activator of AMPK, MT63-78, also binds at the same site. Mutation of two important residues in this pocket (Lys29 and Lys31 of the α2 subunit) abolished the allosteric stimulation of AMPK by A769662, 991 and MT63-78 while it had no effect on allosteric stimulation by AMP. However, we also showed that the same mutation abolished protection against Thr172 dephosphorylation not only by A769662, 991 and MT63-78, but also by phenformin and H2O2, which are known to activate AMPK by increasing the AMP:ATP and ADP:ATP ratios. These data show that the integrity of this pocket is important for the effect of AMP to protect against Thr172 dephosphorylation, but not for its ability to cause allosteric stimulation. Moreover, in HEK-293 cell stably expressing an α2 subunit carrying the mutation of both Lys29 and Lys31, the basal activity of AMPK due to Thr172 phosphorylation was almost 6-fold less than in cells expressing wild-type α2. This result pointed out for the first time that there might be a natural ligand binding in the newly discovered binding pocket that is not able to bind to the double mutant, explaining the difference in activity observed. However the identity of this possible natural ligand remains unclear and more studies will be necessary to uncover it.
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Dunner, Emily M. « Defective Signal Transduction and Oxidative Stress in Ataxia-Telangiectasia ». Thesis, Griffith University, 2007. http://hdl.handle.net/10072/367278.
Texte intégralRésumé :
Ataxia-telangiectasia (A-T) is a debilitating disease caused by the functional loss of the ataxia-telangiectasia mutated (ATM) protein. A-T is characterised by immunodeficiency, reduced growth, insulin resistance and a predisposition to cancer. However, the hallmark symptom of ataxia, which presents within the first years of life, is caused by degeneration of cerebellar Purkinje and granule cells. At the cellular level, the loss of ATM leads to increased oxidative stress. The major function of ATM in normal cells is to activate DNA repair pathways in response to double strand breaks. However, the mechanisms leading to neurodegeneration of cerebellar Purkinje cells are still being elucidated. The focus of this study was to examine how oxidative stress contributes to defective cell signalling and the A-T phenotype. Examination of the insulin and epidermal (EGF) receptor signalling pathways by Western blotting indicated abnormal signalling in fibroblasts from A-T patients. Reduced expression of the membrane receptors and some effector proteins were a likely cause of the observed signalling defects. Fluorescence studies on transferrin receptor recycling identified delayed intracellular trafficking as a novel characteristic of the cellular A-T phenotype, and a contributing factor to altered signalling...Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Science, Environment, Engineering and Technology
Full Text
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Zhou, Deyu. « Learning the hidden vector state model for extracting protein-protein interactions ». Thesis, University of Reading, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.515744.
Texte intégral
34
Wang, Wenzhong. « Mechanistic studies of flavoenzymes in fatty acid oxidation and oxidative protein folding ». Access to citation, abstract and download form provided by ProQuest Information and Learning Company ; downloadable PDF file, 233 p, 2007. http://proquest.umi.com/pqdweb?did=1362529911&sid=3&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Lee, Julie 1983. « Role of oxidative stress in the regulation of iron regulatory protein 2 ». Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116073.
Texte intégralRésumé :
Iron homeostasis is regulated by iron regulatory proteins, IRP1 and IRP2, which bind to iron responsive elements (IRE) in the mRNA of proteins of iron metabolism such as ferritin (iron storage). IRP2 undergoes iron-mediated degradation, and this pathway shares notable similarities with that of hypoxia-inducible factor 1 (HIF-1). It has been reported that oxidative stress marked by increased reactive oxygen species (ROS) signal HIF-1 stabilization in hypoxia. The role of ROS in IRP2 regulation is not well-established. We show that the degree of hypoxia induces differential effects on iron-mediated degradation of IRP2, such that IRP2 levels are 3-fold higher when exposed to 0.1% O 2 compared to 3% O2 after 4 hours of iron treatment. Hydrogen peroxide (H2O2) affects IRP2 by inducing IRE-binding activity after 12 hours, which is accompanied by decreased ferritin levels. Furthermore, the ability of H2O2 to protect IRP2 against iron-dependent degradation is similar to that of hypoxia. Finally, both intracellular and extracellular sources of oxidative stress protect IRP2 from ascorbate-mediated degradation. Taken together, these results support a role of ROS in protecting IRP2 against iron-mediated degradation and indicate that oxidative stress modulates downstream effects of IRP2.
36
Frand, Alison R. (Alison Renee) 1971. « The role of ERO1 in oxidative protein folding in the endoplasmic reticulum ». Thesis, Massachusetts Institute of Technology, 1999. http://hdl.handle.net/1721.1/9361.
Texte intégralRésumé :
Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1999.Includes bibliographical references.
The formation of native disulfide bonds is critical for the folding and stability of many secreted proteins. We describe an essential S. cerevisiae gene, ER01, which encodes a conserved ER membrane protein required for disulfide bond formation in the er .doplasmic reticulum (ER). In a conditional ero 1-1 mutant, secretory proteins that would normally contain disulfide bonds, such as carboxypeptidase Y (CPY), are retained in the ER in reduced form, as shown by thiol modification with AMS. ER01 levels determine cellular oxidizing capacity, since mutation of ER01 causes hypersensitivil/ to the reductant OTT, whereas overexpression of ER01 confers resistance to OTT. Moreover, the thiol oxidant diamide can restore growth and secretion to ero1 mutants. These results suggest that Ero1p provides the oxidizing equivalents utilized for disulfide bond formation in the ER. Oxidizing equivalents are transferred directly from Ero1p to the abundant ER oxidoreductase PDI (protein disulfide isomerase) and its homolog Mpd2p. PDI is oxidized in wild-type cells, but reduced in the ero 1-1 mutant. Thiol-disulfide exchange between POI and Ero1p is indicated by the capture of PD1-Ero1p mixed-disulfides. PDI oxidizes secretory proteins, since newly-synthesized CPY remains fully reduced in POI-depleted cells. Mixed-disulfides between PDI and p1 CPY are also detected, indicating that PDI engages directly in thiol-disulfide exchange with this substrate. Together, these results define a pathway for protein disulfide bond formation in the ER wherein oxidizing equivalents flow from Ero1p to POI (and Mpd2p) and then to substrate proteins through direct thiol-disulfide exchange reactions. Oxidized glutathione (GSSG) does not serve as an obligate intermediate In this pathway, since oxidative protein folding proceeds normally in a gsh 1.1 mutant devoid of intracellular glutathione. Mutational analysis of ER01 identifies two pairs of conserved, vlclnal cystelnes essential for Ero1p function. Mutation of Cys100, Cys105, Cys352, or Cys355 of Ero1 p disrupts cell viability, CPY folding, and thiol-disulfide exchange between POI and Ero1p. Cys100 of Ero1p may be preferentially attacked by POI, while the Cys352- Cys355 disulfide may re-oxidize the Cys 100-Cys 105 cystelne pair. The properties of yeast Ero1 p resemble those of E. coli DsbB.
by Alison R. Frand.
Ph.D.
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Shute, Max. « Effect of Whey Protein Isolate on Oxidative Stress, Exercise Performance, and Immunity ». Diss., Virginia Tech, 2004. http://hdl.handle.net/10919/11113.
Texte intégralRésumé :
The purpose of this study was to evaluate the effectiveness of a whey protein isolate (WPI), a reported glutathione (GSH) booster, on exercise performance, immune function, and antioxidant status during weight maintenance and energy restriction in humans. Twenty well-trained, college age, male cyclists performed a cycling exercise test for 45 min, the first 7 min at 70% of VO2peak and the remaining 38 min at 55% VO2peak immediately followed by a performance test set at 90% VO2peak until exhaustion. Blood samples were collected prior to the exercise test, after 45 min of exercise, within 5 min of exhaustion, and 1 h after exercise. Blood samples were analyzed for GSH, GSH/GSSG ratio, glutathione peroxidase (GPx), lipid hydroperoxides (LPO), phagocytosis, oxidative burst, peripheral blood mononuclear cell (PBMC) proliferation, and PBMC phenotyping. Subjects consumed 40g/day of WPI or casein placebo (P) along with their normal diet for 2 wk, repeated the exercise test, and then began a low energy period continuing the same supplementation for 4 d before the final exercise test. WPI was not associated with superior exercise performance or antioxidant status following exercise or weight loss. WPI supplementation did result in 33% greater lymphocyte proliferation capacity following exercise. Following exhaustive exercise for all trials, tGSH and GPx increased 7% and 11%, respectively, while WBCGSH decreased 13%. For WPI, GPx activity was 10% lower than P following exhaustive exercise for all trials combined. Weight loss (2.67 ± 0.26 kg) resulted in increases in phagocytosis (65%), white blood cell (WBC) GSH (40%), and GPx (35%) while decreasing the GSH/GSSG ratio (55%) and LPO (16%). Exhaustive exercise caused a 28% increase in CD8+ PBMCs and decreased CD4+ (34%), CD3+ (15%), the CD4+/8+ ratio (45%), and phagocytosis (8%) with all values returning to baseline after 1 h recovery. Supplementation with WPI did not enhance GSH status or exercise performance in trained cyclists, during weight maintenance or energy restriction. Following exercise, WPI is associated with greater lymphocyte proliferation of PBMCs which may help maintain an athleteâ s health during heavy training or competition.Ph. D.
38
Weids, Alan. « Protein aggregation, oxidative stress and the role of the yeast peroxiredoxin Tsa1 ». Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/protein-aggregation-oxidative-stress-and-the-role-of-the-yeast-peroxiredoxin-tsa1(742029c5-9b7e-47f6-b0e9-2f396b4af52a).html.
Texte intégralRésumé :
Peroxiredoxins are ubiquitous, thiol-specific proteins that have multiple functions in stress protection, including oxidative stress. Tsa1 is the major yeast peroxiredoxin and we show that it functions as a specific antioxidant to protect against oxidative stress caused by nascent protein misfolding and aggregation. Yeast mutants lacking TSA1 are sensitive to misfolding caused by exposure to the proline analogue azetidine-2-carboxylic acid (AZC). AZC promotes protein aggregation and its toxicity to a tsa1 mutant is caused by reactive oxygen species (ROS). Generation of [rho0] cells lacking mitochondrial DNA rescues the tsa1 mutant AZC sensitivity indicating that mitochondria are the source of ROS. Inhibition of nascent protein synthesis with cycloheximide prevents AZC-induced protein aggregation and abrogates ROS generation confirming that aggregate formation causes ROS production. Protein aggregation is accompanied by mitochondrial fragmentation and we show that Tsa1 localizes to the sites of protein aggregation, which are formed adjacent to mitochondria. Further investigation reveals that AZC-induced protein aggregation leads to an inhibition of mitochondrial respiration and the depolarisation of the mitochondrial membrane. Remarkably, this was entirely dependent on the presence of Tsa1. We show that the effects of protein aggregation on mitochondrial function are mediated by the Ras/PKA pathway and that Tsa1 appears to influence the activity of this pathway through its effects on the yeast phosphodiesterase, Pde2. Together, these data indicate a new role for peroxiredoxins in the response to ROS, generated as a result of protein misfolding and aggregate formation. Finally, we analysed the characteristics of proteins found within protein aggregates, isolated from different conditions during the course of the study. Our results highlight the differences between proteins that aggregate under normal, mid-exponential growth conditions (physiological aggregates) and those which aggregate during cellular stress. We were able to establish the characteristics of an archetypical physiological aggregate, through an assessment of a range of properties, identifying factors that significantly differed from genomic expectations. Furthermore, our observations indicate that, in general, cellular stress reduces the threshold of metrics associated with protein aggregation propensity. We also found that different stresses result in the aggregation of proteins that are, largely, physicochemically indistinct from one another, regardless of the mode of toxicity. Finally we show that a significant number of proteins, identified in our protein aggregates, were also present in protein aggregates isolated from aged C. elegans. This suggests that the factors and components of protein aggregates are conserved.
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Bader, Martin. « Elucidation of electron transfer pathways during oxidative protein folding in Escherichia coli / ». [S.l. : s.n.], 2001. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB9167722.
Texte intégral
40
Liu, Quan. « PHOSPHORYLATION AND SEQUENCE DEPENDENCY OF NEUROFILAMENT PROTEIN OXIDATIVE MODIFICATION IN ALZHEIMER DISEASE ». Connect to text online, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1102024839.
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41
Zhu, Donghui. « Effects of oxidative stress and Alzheimer's amyloid-beta peptide on astrocytes ». Diss., Columbia, Mo. : University of Missouri-Columbia, 2006. http://hdl.handle.net/10355/5900.
Texte intégralRésumé :
Thesis (Ph. D.)--University of Missouri-Columbia, 2006.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (March 3, 2007) Vita. Includes bibliographical references.
42
Liu, Fenglong. « Calcium-dependent protein kinase regulates soybean serine acetyltransferase in response to oxidative stress ». [Gainesville, Fla.] : University of Florida, 2002. http://purl.fcla.edu/fcla/etd/UFE0000561.
Texte intégral
43
Yi, Dong-Hui Chemistry Faculty of Science UNSW. « The Study of Biomarkers of Protein Oxidative Damage and Aging by Mass Spectrometry ». Awarded by:University of New South Wales. School of Chemistry, 1999. http://handle.unsw.edu.au/1959.4/17636.
Texte intégralRésumé :
The physiologically important free radicals, nitrogen monoxide and superoxide, can combine to form the reactive intermediate peroxynitrite. Peroxynitrite can react with proteins and their constituent amino acids, such as tyrosine, resulting in protein peroxidation, oxidation and nitration. The nitration of proteins, assessed by the analysis of 3-nitrotyrosine, is a proposed index of pathophysiological activity of peroxynitrite. The aim of the work was to investigate the reaction products between peroxynitrite and protein, develop an assay for 3-nitrotyrosine and measure its levels in biological samples. To study the amino acid products arising from the reaction of peroxynitrite and protein, both liquid chromatography (LC) and gas chromatography (GC) combined with mass spectrometry (MS) were adopted. Approaches to 3-nitrotyrosine assay development were first, to take advantage of the intrinsic sensitivity of electron capture negative ionization GC-MS. Secondly, to avoid possible artefactual problems associated with the derivatisation step in GC-MS, an assay for 3-nitrotyrosine based on combined LC-MS-MS was developed. When a selection of peptides was exposed to peroxynitrite under physiological conditions in vitro, the hydrolysis products showed that 3-nitrotyrosine was the major product. Detectable minor products were 3,5-dinitrotyrosine and DOPA. The GC-MS assay was found to be fraught with difficulty due to artefactual formation of 3-nitrotyrosine. In order to quantify and correct for artefact formation, this complication was approached by incorporating a second isotopomer. This method, however, was confounded by large errors that reduced the overall sensitivity. Either negative or zero levels of endogenous 3-nitrotyrosine were found in tested samples after correction for artefact formation. The LC-MS-MS assay was then used to analyse 3-nitrotyrosine levels in a range of biological samples, including human plasma from healthy volunteers, synovial fluid samples from arthritis patients and tissue extracts from a mouse model of amyotropic lateral sclerosis. In contrast to published data, 3-nitrotyrosine levels were found to be below the limit of detection (1 pg/????L, 10 pg o/c) for all samples - a result somewhat consistent with the negative GC-MS data. It is suggested that the high 3-nitrotyrosine levels previously reported in the literature might reflect artefactual generation of 3-nitrotyrosine and that other approaches to assessing pathophysiological nitration should be sought in future.
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Li, Shengchun. « Antioxidant systems and protein phosphatases in metabolic and signaling responses to oxidative stress ». Thesis, Paris 11, 2013. http://www.theses.fr/2013PA112086.
Texte intégralRésumé :
Le stress oxydant est un acteur clé dans les réponses des plantes à des conditions contraignantes. En raison de la complexité de la régulation de l’état redox cellulaire, il reste beaucoup à élucider concernant les interactions entre différentes composantes dans ces conditions. Grâce à une approche de génétique inverse basée sur un mutant d’Arabidopsis déficient en catalase (cat2) qui présente des modifications d’état redox prévisibles et bien définies, cette étude a exploré les interactions entre le stress oxydant et (1) un gène spécifique impliqué dans la déphosphorylation des protéines, (2) des enzymes spécifiques impliquées dans les systèmes antioxydants réducteurs. Les résultats obtenus révèlent que la sous-unité B'γ de la protéine phosphatase de type 2A (PP2A-B'γ) est importante dans la détermination des phénotypes et des réponses de défense photopériode-dépendantes chez cat2. En conditions de jours courts (SD), un double cat2 pp2a-b'γ mutant montrait une gamme de réponses qui n’étaient pas observées chez cat2. Ces effets comprenaient l’apparition de lésions ainsi que l’accumulation de l’acide salicylique et d’autres composés de défense. Des analyses métabolomiques et protéomiques ont permis de démontrer que ces effets étaient accompagnés de modifications de l’abondance de métabolites et protéines spécifiques, ainsi que des changements dans le statut de phosphorylation de certains polypeptides. Dans un deuxième volet du travail, l’importance d’une enzyme productrice du NADPH a été évaluée en produisant des doubles cat2 nadp-me2 mutants chez lesquels l’isoforme majeure de l’enzyme malique cytosolique n’est plus exprimée. Malgré une induction de cette enzyme par le stress oxydant aux niveaux de transcrits et d’activité, et une diminution importante de l’activité foliaire associée aux mutations nadp-me2, peu de différence a été observée entre les lignées cat2 et cat2 nadp-me2. De même, la mutation nadp-me2 n’a pas affecté la réponse phénotypique de plantes exposées à l’ozone. Dans la troisième partie du travail, le couplage entre les pools ascorbate et glutathion lors du stress oxydant a été exploré par l’introduction de mutations pour la déshydroascorbate réductase (DHAR) dans le fond génétique cat2. L’activité extractible de cette enzyme a été diminuée à des niveaux très faibles chez des lignées portant à la fois les mutations dhar1 et dhar3. Cependant, peu de différence a été observée dans les phénotypes et les statuts d’ascorbate et de glutathion chez un triple mutant cat2 dhar1 dhar3 par rapport à cat2. Des analyses préliminaires d’un quadruple cat2 dhar1 dhar2 dhar3 mutant semblent pourtant indiquer que les trois DHARs jouent des rôles fonctionnellement redondants dans le stress oxydant. Dans son ensemble, ces travaux apportent des données nouvelles sur les enzymes qui régulent les réponses aux stress oxydants et ont généré des outils intéressants pour des études ultérieuresOxidative stress is a key player in plant responses to challenging environmental conditions. The intricate nature of the regulation of cellular redox state means that much remains to be elucidated on interactions between different components in these conditions. By using a genetic approach based on a catalase-deficient Arabidopsis mutant (cat2) that presents well-defined, predictable changes in redox state, this study explored interactions between oxidative stress and (1) a specific gene involved in protein dephosphorylation, and (2) specific enzymes involved in the antioxidative/reducing system. The results showed that protein phosphatase 2 subunit B'γ (PP2A-B'γ) is involved in determining day length-dependent phenotypes and related defense responses in cat2. A cat2 pp2A-B'γ double mutant showed a range of responses that were not observed in cat2 grown in short days, including lesion formation and accumulation of salicylic acid (SA) and related metabolites. Metabolomics and proteomics analyses showed that these effects were associated with altered abundance of specific metabolites and proteins, as well as changes in protein phosphorylation status. A second part of the study investigated the importance of NADP-generating enzymes in oxidative stress by production of cat2 nadp-me2 double mutants, in which the cytosolic isoform of NADP-malic enzyme is knocked out. Although NADP-ME2 was shown to be induced by oxidative stress, and mutants for this gene had much decreased leaf NADP-malic enzyme activity, no effects on cat2 phenotypes or redox profiles were apparent. Similarly, phenotypic responses to ozone were not affected in an nadp-me2 single mutant. In the third part, coupling between ascorbate and glutathione pools during oxidative stress was investigated by introduction of loss of function mutations for dehydroascorbate reductase (DHAR) into the cat2 background. In lines carrying a combination of dhar1 and dhar3 mutations, extractable leaf activity was decreased to very low levels. Despite this, cat2 dhar1 dhar3 and cat2 phenotypes and ascorbate and glutathione pools were similar. However, preliminary functional analysis of a cat2 dhar1 dhar2 dhar3 quadruple mutant suggested that the three DHARs play functionally redundant roles in oxidative stress. Overall, the work provides new data on enzymes that regulate responses to oxidative stress and has produced interesting genetic tools for further study
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Okuda, Aya. « Novel Soybean Enzymes Involved in the Oxidative Protein Folding in the Endoplasmic Reticulum ». 京都大学 (Kyoto University), 2017. http://hdl.handle.net/2433/225656.
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Drießen, Marc D. [Verfasser]. « Investigation of nanoparticle toxicity : Characterization of protein corona and evaluation of oxidative stress by protein carbonylation / Marc D. Drießen ». Berlin : Freie Universität Berlin, 2016. http://d-nb.info/1111558787/34.
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Lohman, Jeremy R. « Two-state conformational behavior in protein active centers / ». Connect to title online (ProQuest) Connect to title online (Scholars' Bank), 2007. http://hdl.handle.net/1794/6198.
Texte intégralRésumé :
Thesis (Ph. D.)--University of Oregon, 2007.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 74-82). Also available online in Scholars' Bank; and in ProQuest, free to University of Oregon users. Also available online.
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Lohman, Jeremy R. 1981. « Two-state conformational behavior in protein active centers ». Thesis, University of Oregon, 2007. http://hdl.handle.net/1794/6198.
Texte intégralRésumé :
xiv, 82 p., ill. (some col.)Cellular processes are carried out by proteins, which often utilize conformational changes for function. In theory, conformational changes can be harnessed to promote, prevent or monitor cellular processes. Such changes in protein active centers require perturbations through interactions with other proteins, small molecules or through energy input into the system, for example light. The work presented incorporates rational design and crystallographic elucidation of two-state conformational changes in two proteins, green fluorescent protein (GFP) and malate synthase (MS). GFP indicators were previously developed to quantitate the thiol/disulfide redox status within cells. Cysteine residues were introduced in close proximity on the surface of GFP and allow the formation of a disulfide bond. The indicators provide a fluorescent readout of the ambient thiol/disulfide equilibrium, however thermodynamic studies showed the resulting thiol/disulfide to be unusually stable (-287 mV) in comparison to the cellular redox buffer glutathione (-240 mV). In order to produce a family of redox indicators suitable for use in less reducing environments, amino acids were inserted near the introduced cysteine pair in order to destabilize the disulfide. The resulting family of redox indicators, termed roGFP-iX, exhibit midpoint potentials in the more desirable range of -229 to -246 mV. Crystallographic analysis indicates that roGFP-iX indicators undergo much larger two-state conformational changes than the original indicators. Surprisingly, a cis-peptide was discovered between the cysteine and the inserted residue which in combination with the conformational changes helps to explain the reduced stability of the disulfide. Malate synthase is an important virulence factor for certain microbes and carries out the Claisen condensation between glyoxylate and acctyl-CoA to produce malate. Crystal structures of Mycobacterium tuberculosis and Escherichia coli malate synthase isoform G had previously been determined with substrates or products bound. To determine the conformational changes necessary for substrate binding and product release, crystal structures of Escherichia coli malate synthase isoform A were determined in both the apo and acetyl-CoA/inhibitor bound forms. The crystallographic models revealed two-state conformational changes in the part of the active-site loop necessary for substrate binding, which has important implications for drug design. This dissertation includes my unpublished co-authored materials.
Adviser: S. James Remington
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Milner, Steven John. « The oxidative folding of insulin-like growth factor-I analogues / ». Title page, table of contents and summary only, 1996. http://web4.library.adelaide.edu.au/theses/09PH/09phm65945.pdf.
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Ugur, Zafer. « Mass Spectroscopic Identification and Quantification of Protein Carbonyls ». VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/386.
Texte intégralRésumé :
It is well established that free radical mediated oxidative stress plays a critical role in aging and age-related diseases. Among the post-translational protein modifications, carbonylation has attracted a great deal of attention due to its irreversible and irreparable nature. Despite the fact that protein carbonylation is associated with a series of physiological and pathological processes, there are still issues to be clarified such as why certain proteins are more vulnerable to modification, what are the locations of the protein modifications, and how does the nature of the oxidant affect the preferred site of modification. In this study, we will seek an answer to these questions and examine the global effect of oxidative stress on protein abundance. The study embraces three distinct specific aims. In the first, methods are developed for identifying sites of protein carbonylation. In the second specific aim, these methods are used to identify carbonylaytion sites in model proteins subjected to chemical oxidants. In the third aim, the focus is on a model organism, C. elegans, subjected to paraquat-induced oxidative stress. This is exploratory work and mass spectrometry is used to assess the impact of oxidative stress on the mitochondrial proteome.