To see the other types of publications on this topic, follow the link: Post translational modification (PTM).
Dissertations / Theses on the topic 'Post translational modification (PTM)'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Post translational modification (PTM).'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Dumont, Quentin. "Applications of Ion Mobility Mass Spectrometry - Screening for SUMOylation and Other Post-Translational Modifications." University of Toledo / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1345130293.
Full text
2
Matsumiya, Nozomi. "Optimization of disulfide mapping using mass spectrometry." Thesis, Kansas State University, 2009. http://hdl.handle.net/2097/1358.
Full textAbstract:
Master of ScienceBiochemistry
John Tomich
One of the important keys to characterize the biological function of a protein is the study of post-translational modification (PTM). Formation of disulfide bond linkages between cysteine residues within a protein is a common PTM which not only contributes to folding and stabilizing the protein structure, but also to accomplishing its native function. Therefore, the study and discovery of structural-functional relationships of expressed proteins using an isolated proteomics approach has been one of the biggest advances within the field of structural biology in recent years. In this study, rapid disulfide bond mapping of freshly obtained equine serum albumin (ESA) was performed using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Highly sensitive MALDI-TOF MS is commonly used for the investigation of disulfide bond linkages in the proteomics field. However, it has also been known that the presence of disulfide bond linkages absorbs the energy which is created by the cysteine-cysteine kinetic vibration, resulting in a decrease of the instrumental sensitivity. To overcome this problem, the disulfide bond mapping method was optimized by applying a combination of chemical labeling, proteolytic enzymes, and matrices. With the optimized method, we were also able to achieve high protein sequence coverage. Obtaining higher sequence coverage of a protein provides more information about a protein which helps to identify the protein by peptide mass fingerprint (PMF) technique. These analyses eventually contribute to the estimation of the possible PTM sites.
3
Diallo, Issa. "Nouvelle méthode en protéomique pour améliorer l'identification et la quantification des protéines acétylées." Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAS035.
Full textAbstract:
L'acétylation des protéines constitue l’une des plus importantes modifications post-traductionnelles (PTMs). Elle intervient dans de multiples processus bologiques et physiopathologiques tels que, l’activité transcriptionnelle, l'apoptose, la régulation des voies métaboliques, les cancers, les maladies inflammatoires et cardiovasculaires. Face à l’importance de l’acétylation des protéines, il apparaît donc indispensable de bien comprendre les mécanismes qui y sont associés, et donc, de pouvoir identifier et quantifier les protéines acétylées à partir du protéome complet d’échantillons complexes tels que des extraits cellulaires ou tissulaires. La spectrométrie de masse est une technique de choix pour de telles études, car elle permet d’identifier les protéines et les sites d’acétylation, mais aussi de les quantifier en l’associant à des techniques de quantification (label free, SILAC, iTRAQ/TMT, AQUA). Malheureusement, ces méthodes ne ciblent pas particulièrement les acétylations et requièrent l’utilisation de techniques d’enrichissement ou de fractionnement qui ne sont dédiées qu’à certains types d’acetylation : les N-ter et K-acetylation. Aucun enrichissement n’est disponible pour les O- acétylations et ces méthodes d’enrichissement ne sont pas toujours compatibles avec les techniques de quantification citées ci-dessus. Pour améliorer la détection et la quantification des acétylations, nous proposons la méthode RAQIAT (Relatif Absolute Quantification Isobaric Affinity Tag) qui se résume en trois grandes étapes: i) Le blocage des fonctions amines libres à l'aide de la di-méthylation réductrice, ceci empêchera ces dernières de réagir avec le réactif RAQIAT, ii) La désacétylation des lysines acétylées pour permettre une quantification sélective des acétylations, iii) Le marquage des amines primaires précédemment désacétylées dans l’étape 2 par le réactif RAQIAT pour permettre leurs identifications et quantifications. Ce manuscrit a porté en partie sur les deux premières étapes de la méthode RAQIAT.Dans la première étape, les échantillons de protéines de levure ont été digérés puis di-méthylés et fractionnés par OFFGEL en 24 fractions. Ensuite, chacune de ces 24 fractions OFFGEL a été soumise à un fractionnement nano-RPLC et analysée par MALDI TOF/TOF (4800 MALDI-TOF/TOF, Sciex). En parallèle, la même expérience a été réalisée, cette fois-ci sans di-méthylation. L'analyse des données a été réalisée en utilisant le logiciel Mascot comme moteur de recherche.L’efficacité de la réaction de di-méthylation démontrée, nous avons montré que sans réaliser la di-méthylation réductrice 164 sites acétylés ont pu été identifiés alors que 385 sites acétylés distincts ont été identifiés avec la di-méthylation réductrice. De plus, l'amélioration de la détection de l'acétylation en utilisant la méthode de di-méthylation a été observée pour chacune des différents types acétylations: N-ter, K- et O-acétylation.Dans la deuxième étape, nous avons présenté des résultats préliminaires de déacétylation par la sirtuine 1 en présence du peptide de la p53 (Ac-Arg-His-Lys-Lys-(Ac)-AMC) connu comme étant un substrat de cette enzyme. Nous avons observé la formation d’un peptide non acétylé, suggérant une déacétylation de ce peptide acétylé de p53. Cependant, la formation de cet ion étant très faible et l’ion acétylé étant fortement préservé, nous en avons conclu que l’efficacité de la déacétylation du peptide de p53 n’était pas suffisante pour l’intégrer à la méthode RAQIATProtein acetylation is one of the most widespread post-translational modifications which is involved in many cellular physiologies and pathologies such as cancers. Regarding the important biological effect of protein acetylation and a non-negligible number of proteins bearing this PTM, several methods emerged last decade to investigate such PTM. But the detection of acetylations and their quantification are still limited and enrichment method allowing a better detection of acetylation target mostly one kind of acetylation (K-acetylation). To improve the detection of the three kind of acetylation (N-ter, K, and O-) and their quantification, we propose the RAQIAT method (Relative Absolute Quantification Isobaric Affinity Tag), based on protein digestion followed by 3 steps : i) a protection of free primary amines at N-ter, lysine (i.e. primary amine not bearing PTM) based on a reductive di-methylation strategy ii) a deacetylation of acetylated residues to obtain free primary amine corresponding to peptides previously acetylated iii) a RAQIAT labeling on the free primary amine obtained in the previous step to allow the enrichment of peptides previously acetylated and their quantifications. Herein, we present the investigation of the two first steps of RAQIAT method.In the first step, we evidenced that the reductive di-methylation strategy improved the detection of the three kind of acetylation: N-ter, K- and O- acetylations. Yeast protein samples were digested with trypsin prior di-methylation of resulting peptide mixture. Then, di-methylated peptide mixtures were fractionated by OFFGEL and reverse phase liquid chromatography followed by MALDI-TOF/TOF mass spectrometry analysis. Data analysis was performed by using Mascot as search engines.Our results showed that OFFGEL fractionation is a useful step to increase detection of acetylations. Moreover, we showed that our di-methylation treatment improved significantly detection of acetylation. Indeed, after di-methylation treatment, 385 unique acetylated sites were identified while 164 unique acetylated peptides were detected without di-methylation treatment. The improvement of acetylation detection using our di-methylation strategy is observed for each of acetylations: N-ter, K- and O-acetylations. Thus, this new proteomic method is promising to enhance N-ter, K- and O-acetylation detection.In the second step, we presented preliminary results of deacetylation by sirtuin 1 in the presence of p53 peptide (Ac-Arg-His-Lys-Lys- (Ac) –AMC. However, the low deacetylation efficiency of the p53 peptide observed, conclude that is not suitable to applicate into RAQIAT Method
4
Schiller, Rachel Shamo. "Investigating the inhibitor and substrate diversity of the JmjC histone demethylases." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:1e7fd2a1-a9c3-48f7-8fa7-a041299d42f9.
Full textAbstract:
Epigenetic control of gene expression by histone post-translational modifications (PTMs) is a complex process regulated by proteins that can 'read', 'write' or 'erase' these PTMs. The histone lysine demethylase (KDM) family of epigenetic enzymes remove methyl modifications from lysines on histone tails. The Jumonji C domain (JmjC) family is the largest family of KDMs. Investigating the scope and mechanisms of the JmjC KDMs is of interest for understanding the diverse functions of the JmjC KDMs in vivo, as well as for the application of the basic science to medicinal chemistry design. The work described in this thesis aimed to biochemically investigate the inhibitor and substrate diversity of the JmjC KDMs, it led to the identification of new inhibitors and substrates and revealed a potential combinatorial dependence between adjacent histone PTMs. Structure-activity relationship studies gave rise to an n-octyl ester form of IOX1 with improved cellular potency and selectivity towards the KDM4 subfamily. This compound should find utility as a basis for the development of JmjC inhibitors and as a tool compound for biological studies. The rest of this thesis focused on the biochemical investigations of potential substrates and inhibitors for KDM3A, a JmjC demethylase with varied physiological functions. Kinetic characterisation of reported KDM3A substrates was used as the basis for evaluations of novel substrates and inhibitors. Further studies found TCA cycle intermediates to be moderate co-substrate competitive inhibitors of KDM3A. Biochemical investigations were carried out to study potential protein-protein interactions of KDM3A with intraflagellar transport proteins (IFTs), non-histone proteins involved in the formation of sperm flagellum. Work then addressed the exploration of novel in vitro substrates for KDM3 (KDM3A and JMJD1C) mediated catalysis, including: methylated arginines, lysine analogues, acetylated and formylated lysines. KDM3A, and other JmjC KDMs, were found to catalyse novel arginine demethylation reaction in vitro. Knowledge gained from studies with unnatural lysine analogues was utilised to search for additional novel PTM substrates for KDM3A. These results constitute the first evidence of JmjC KDM catalysed hydroxylation of an Nε-acetyllysine residue. The H3 K4me3 position seems to be required for acetyllysine substrate recognition, implying a combinatorial effect between PTMs. Preliminary results provide evidence that JMJD1C, a KDM3 protein previously reported to be inactive, may catalyse deacetylation in vitro. An additional novel reaction, observed with both KDM3A and JMJD1C, is deformylation of Nε-formyllysine residues on histone H3 fragment peptides. Interestingly, H3 K4 methylation was also observed to enhance the apparent deformylation of both KDM3A and JMJD1C catalysed reactions. Overall, findings in this thesis suggest that the catalytic activity of JmjC KDMs extends beyond lysine demethylation. In a cellular context, members of the KDM3 subfamily might provide a regulatory link between methylation and acylation marks. Such a link will further highlight the complex relationships between histone PTMs and the epigenetic enzymes that regulate them. The observed dependency of H3 K9 catalysis on H3 K4 methylation adds another layer of complexity to the epigenetic regulation by histone PTMs.
5
Gopalswamy, Mohanraj [Verfasser]. "Aggregation and post-translational modification of the parathyroid hormone and its agonistic activity towards the G-protein coupled PTH receptors / Mohanraj Gopalswamy." Halle, 2017. http://d-nb.info/1141177951/34.
Full text
6
Dedieu, Alain. "Exploration des modifications post-traductionnelles des protéines : nouvelles approches et nouveaux modèles biologiques." Thesis, Montpellier 1, 2014. http://www.theses.fr/2014MON13516/document.
Full textAbstract:
L'étude des modifications post-traductionnelles a connu au cours des dernières années un regain d'intérêt notable. Tout d'abord car elle s'effectue aujourd'hui au travers d'approches basées sur la spectrométrie de masse, technique qui pendant cette période a connu de profonds bouleversements, conduisant à des études plus aisées et systématiques.Mais aussi car tant par leur variété que par le rôle qu'elles jouent dans la vie et la régulation cellulaire, ces modifications ne peuvent plus être négligées. Par ailleurs au cours de ces quinze dernières années, nous avons assisté concernant les procaryotes à un changement total de paradigme. En effet à la fin des années 90, l'idée dominante était que ces modifications pouvaient exister chez ceux-ci mais de façon très partielle et/ou très particulière.Dans ce travail, les divers degrés d'iodation de la tyrosine ont été sondés par une approche de type «shotgun » sur un organe entier, la thyroïde de souris. L'efficacité de ce type d'approche démontrée, les modifications post-traductionnelles potentiellement présentes dans des organismes modèles radiorésistants, la bactérie Deinococcus deserti et l'archée Thermococcus gammatolerans ont été analysées. Dans le premier cas, les données de protéomique montrent que de nombreuses acétylations N-terminales portent sur un motif spécifique (essentiellement des thréonines et sérines), cas très atypique pour une bactérie. Chez Thermococcus gammatolerans les acétylations N-terminales sont rares, mais la présence d'acétylations sur les chaînes latérales des lysines est notable. La présence de phosphorylations sur ces mêmes protéines, laisse entrevoir un possible phénomène de « cross talk » entre les lysines acétylées et les sérines et/ou thréonines phosphorylées.Ici, nous démontrons que la complexité du protéome chez les procaryotes par le biais des MPT est bien réelle et que de possibles interdépendances entre MPT mériteraient un regard nouveauRecently, the study of post-translational modifications has greatly evolved, mainly because of crucial progresses in mass spectrometry methodology which have allowed high-throughput, high resolution analysis. Their variety and their role in the regulation of key molecular mechanisms are increasingly documented. In this work, the different degrees of iodination of tyrosine were probed with a "shotgun" approach carried out from an entire organ, the mice thyroid. Post-translational modifications present in two radioresistant organism models, the bacterium Deinococcus deserti and the archaeon Thermococcus gammatolerans, were analyzed. The large scale exploration of N-terminal acetylation in D. deserti indicates a specific pattern of this modification on serine and threonine, as well as an atypical, high propension to acetylation with 50% of modified N-termini. In T. gammatolerans, N-terminal acetylation is rare, but the presence of acetylation on lysine side chains is significant. The presence of phosphorylation on these proteins suggests a potential "cross talk" between the acetylated lysine and phosphorylated serine or threonine residues. This work demonstrates that the complexity of the proteome in prokaryotes through post-translational modifications is higher than expected when extremophiles are scrutinized compared to classical prokaryote models. Interdependencies between post-translational modifications definitively deserve a fresher look
7
Gibson, Matthew D. "Reading the Epigenetic State of Chromatin Alters its Accessibility." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480534756664384.
Full text
8
Silverman, H. S. "Post-translational modification of mucins." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365785.
Full text
9
Inche, Adam. "The post translational modification of the retinoblastoma protein." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491620.
Full textAbstract:
The retinoblastoma protein (pRb) is a central figure in the control of not only the cell cycle, but also other cellular functions such as differentiation. The regulation of pRb function is through a variety of post translational modifications, either on pRb itself, or by the controlling influence of pRb on the post translational modification of the histone proteins. Phosphorylation of pRb is a key mechanism in the regulation of the cell cycle. pRb is also involved in the recruitment of histone methyltransferase (HMT) and acetyltransferase (HAT) to the chromatin to modify histones. Previous work within the group showed that pRb was subjected to acetylation by the HAT p300. The acetylation of pRb was shown to negatively influence phosphorylation by interfering with a proposed kinase binding site. Another group showed that acetylation of pRb may also be involved in the differentiation of myoblast cells. The work presented here characterises, for the first time, the methylation of pRb by the HMT Set7/9, previously shown to methylate p53. This post translational modification was identified by a combination of ill vitro mutational analysis and mass spectroscopy. The site of methylation was shown to be at lysine 873 (K873), the same region that was subjected to acetylation by the HAT p300. It was also shown that methylation at K873 occurs il1 vivo and is up regulated in response to myoblast differentiation. The ability for these cells to differentiate is compromised when a mutant of pRb is used that cannot be methylated at position 873. . The methylation ofpRb also seems to exert a regulatory control over other post translational modifications that occur on pRb. It was shown that pRb methylated by Set7/9 was able to reduce -acetylation at positions 873/4. The over expression of Set7/9 in cells was also shown to specifically negatively affect the phosphorylation of S811. This site has been implicated in the regulation cell cycle exit, and therefore supports the theory that methylation of pRb is required for the progression of differentiation. It is unclear at this time if the regulation of phosphorylation at S811 is as a consequence of methylation at K873, or at a secondary site that was identified by in vitro mass spectroscopy at K810.
10
Strong, Emily. "Post translational modification of Exo1 in Saccharomyces cerevisiae." Thesis, University of Sheffield, 2017. http://etheses.whiterose.ac.uk/19163/.
Full textAbstract:
Meiosis is the cell division that produces haploid gametes for the purpose of sexual reproduction. During this division it is essential for homologous chromosomes to be securely paired and segregated in order for the gametes to receive a single copy of each chromosome. An important protein in this process is the exonuclease Exo1. Exo1 has two important and distinct roles during meiosis: resection of DNA at double-strand breaks (DSBs) exposing single stranded DNA suitable for strand invasion, and resolution of double-Holliday junctions (dHJs) as cross-overs. Exo1 also acts as a nuclease during DSB repair during mitosis. Previous studies have shown that Exo1 is phosphorylated in response to DNA damage in mitotically cycling cells. The role of this phosphorylation is yet to be definitively determined. This study aimed to test the hypothesis that Exo1 might also be phosphorylated in response to meiotic DSBs. It was confirmed that Exo1 was phosphorylated in response to mitotic DNA damage by designing a tagged version of Exo1 in the Saccharomyces cerevisiae background SK1, a strain commonly used for meiotic experiments. Meiotic progression and spore viability appeared to be normal in cells with exo1 mutated at the phosphorylation sites reported as active in mitosis. In meiotic studies Exo1 was found to be phosphorylated during meiosis, and this phosphorylation was different to that seen in previous mitotic studies. It was found that this phosphorylation was transient during meiosis and that it reflected the presence of Spo11-DSBs and their repair.
11
Svensson, Nilsson Caroline. "Post-translational modification of protein kinase RNA-activated." Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-355076.
Full text
12
Wright, Tom. "Post-translational mutagenesis : radical methods for protein modification." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:774aa93a-489e-4051-aa01-b33781215968.
Full textAbstract:
The naturally occurring post-translational modification of proteins expands the chemical and structural diversity available for protein function. Enzymatic processes are known to modify proteins after translation, and in many cases these modifications have been demonstrated to be critical for biological function. However, the paucity of methods for introduction of site-specific modifications to proteins remains a key obstacle to their biochemical study. Access to these naturally modified proteins has long been complicated by their low abundance, difficulties in purifying homogeneous samples from cellular samples and the intractability of bacterial expression systems towards post-translational modification. In this thesis, we report an important step towards circumventing these issues by using a novel 'tag and modify' chemical strategy, termed 'post-translational mutagenesis'. We describe the development and application of C(sp3)-C(sp3) bond-forming reactions on proteins under biocompatible conditions, exploiting unusual free-radical chemistry to form a wide diversity of protein side-chains. Natural, unnatural, posttranslationally-modified (methylated, glycosylated, phosphorylated, hydroxylated) and labeled (fluorinated, isotopically-labeled) side-chains have been installed on a number of proteins. Extensive characterisation of modification site, connectivity and modified protein structure and function confirm the benign nature of our protocols. The modified proteins thus produced have been used to investigate novel aspects of epigenetic regulation of histone proteins, in the context of free histones and in nucleosome particles, and as novel 'isotope PTMs' for protein characterisation by NMR. We believe this approach, a form of 'protein editing', will have wide application in biochemistry and synthetic biology for accessing diverse modified proteins for research.
13
Boulanger, Marie-Chloé. "Arginine methylation, the characterization of a post-translational modification." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85889.
Full textAbstract:
Proteins are known to be post-translationally modified. This thesis will discuss arginine methylation, one of the many post-translational modifications that occur within the cell. The enzymes that catalyze this post-translational modification are called arginine methyltransferases. The three main types of methylated arginines include monomethylated arginine (MMA), asymmetric dimethylated arginine (aDMA) and symmetric dimethylated arginines (sDMA). Type I arginine methyltransferases catalyze the formation of MMA and aDMA; Type II enzyme catalyze the formation of MMA and sDMA. Protein arginine methylation has been implicated in the regulation of many different cellular processes, including transcription, cellular localization, protein-protein interaction and signal transduction.The purpose of this work was to further characterize arginine methylation by identifying new members of the arginine methyltransferase enzyme family in Drosophila melanogaster and to study the effects of protein arginine methylation on novel substrates. I identified and characterized nine homologues of arginine methyltransferases in Drosophila that were named DART1 to DART9, for drosophila arginine methyltransferases 1-9. All nine enzymes are expressed at various developmental stages. I discovered that a substrate of mammalian enzyme protein arginine methyltransferase 1 (PRMT1) can also be methylated by PRMT5. I also identified HIV-1 Tat protein as the first substrate of the novel enzyme PRMT6.
14
Lupi, Rosita. "Characterization of post translational modification of heterotrimeric G proteins." Thesis, Open University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343748.
Full text
15
Chapman, Tajekesa Kudzaishe Pamacheche. "Regulation of PABP1 function by differential post-translational modification." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25875.
Full textAbstract:
Post-transcriptional control of gene expression is critical for normal cellular function and viability. Poly(A)-binding protein (PABP) 1 is the prototypical member of a family of RNA-binding proteins which are key post-transcriptional regulators. PABP1 is multifunctional, acting as a primary determinant of translation efficiency and mRNA stability, regulating the fate of specific mRNAs, and participating in microRNAmediated regulation and nonsense-mediated mRNA decay. As well as binding various mRNAs, PABP1 achieves its multifunctionality through protein-protein interactions with numerous PABP-interacting motif (PAM)-2 motif-containing protein partners. These have been identified to bind the same site within the C-terminal PABC domain, therefore it is unclear how different PABP1 functions are coordinated. Recently, PABP1 was found to exhibit extensive post-translational modification (PTM), including putative lysine acetylation/methylation switches, which were suggested as a potential mechanism by which interactions with different PAM2 motifcontaining proteins may be regulated. In particular, in silico molecular modelling of the acetylation or dimethylation of the position 606 lysine residue (Lys606) within the PABC domain, using available structures of PABC in complex with PAM2 peptides of eukaryotic release factor (eRF)-3a and PABP-interacting protein (Paip)-2, suggested that modification of this residue, which is critical in PABC-PAM2 interactions, may differentially affect these PABP1 interactions. To examine the role of the Lys606 modification as a molecular switch to dictate PABC-mediated protein-protein interactions, site-specifically acetylated recombinant PABC domain was generated using cutting–edge amber codon suppression recoding technology. Following sequential purification by affinity, ion exchange and size exclusion chromatography, recombinant PABC protein quality was analysed by biophysical approaches such as thermal denaturation assay (TDA), dynamic light scattering (DLS), circular dichroism (CD) and liquid chromatography mass spectrometry (LCMS). Biochemical and biophysical analysis of PABC-PAM2 interactions was subsequently undertaken using GST-pulldown analysis, with the well characterised Paip2 protein, and Surface Plasmon Resonance (SPR) using PAM2 peptides of eRF3, Paip2 and trinucleotide repeat-containing (Tnrc) 6C (or GW182) proteins. These revealed that PABC Lys606 acetylation significantly increased the affinity and increased the association rate for eRF3 peptide. In contrast, effects on Paip2 peptide binding were less suggestive. Furthermore, although approaches to decipher the biological relevance of Lys606 and its modifications within cells are in their infancy, they reflect the complexity of studying PTM function in vitro. Overall, these data provide support for the hypothesis that Lys606 modification status confers selectivity between PABP1 protein partners suggesting a potential mechanism for how its multi-functionality may be coordinated.
16
Perez, Morales Tiara G. "SdpAB are required for post-translational modification of SdpC." Thesis, University of Iowa, 2010. https://ir.uiowa.edu/etd/577.
Full textAbstract:
Bacillus subtilis is a Gram-positive spore-forming soil bacterium. Under environmental stress conditions such as starvation, B. subtilis enters the pathway of sporulation. Earlier work demonstrated that B. subtilis can delay sporulation by undergoing cannibalism. Sporulating cells secrete toxins that kill nearby siblings, thus allowing cells to feed on the released contents. One of these toxins, SdpC, is encoded by the sdpABC operon. To uncover the requirements for SdpC toxic activity during cannibalism, all proteins in the sdpABC operon were analyzed. We report that mutations of SdpC which block signal peptidase cleavage also block toxin production. In addition, production and secretion of SdpC do not require SdpA and SdpB. Our results indicate that SdpC secretion is indispensable for induction of the immunity operon sdpRI . Furthermore, SdpC secreted from a Δ sdpAB strain does not fully induce sdpRI expression and has decreased toxicity to cells that are sensitive to wild type SdpC. Lastly, differences in SdpC mobility are observed in the presence of SdpA and SdpB. Thus, we propose that SdpA and SdpB may function by post-translationally modifying SdpC into the active form of the toxin.
17
Leocadio-Victoria, Daniel. "Post-translational modification and nuclear targeting of beta-dystroglycan." Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/9316/.
Full textAbstract:
Cellular communication and the link between the extracellular and intracellular environments are in part mediated by signalling events from plasma membrane proteins. Their tight regulation by a diverse array of post-translational modifications (PTM) is essential to maintain the cellular integrity. Understanding these modifications would help with understanding cancer cell progression. Dystroglycan is one of the plasma membrane proteins with both structural and signalling properties, and is a core component of the dystrophin associated protein complex (DAPC). The nuclear localisation of dystroglycan and some DAPC members suggests similar roles to those observed in the plasma membrane. Dystroglycan is composed of an alpha and beta subunits that are modified by different PTM. The altered glycosylation of alpha and the phosphorylation of Y890 in beta, are triggering factors for the instability of dystroglycan leading to the disruption of the DAP complex. In cancer studies, it has recently been indicated that in addition to the above-mentioned PTM, other signalling events such as additional phosphorylation sites and ubiquitination, could mediate the rapid turnover of dystroglycan from the plasma membrane and from the nucleus. Here we show that beta-dystroglycan is susceptible to additional phosphorylation, and we were able to demonstrate its multiple ubiquitination. Further experiments revealed that beta-dystroglycan is subject to intramembrane proteolytic events induced by increased cellular density and PDBu treatment. Interestingly, we observed the translocation of beta- dystroglycan to the nucleus due to high cell density growth. By enzymatic inhibition studies we were able to identify gamma-secretase and furin as the enzymes responsible for the shedding of beta-dystroglycan from both the plasma membrane and nuclear envelope. Interestingly, we observed the preferential nuclear translocation and degradation by the proteasome of the cytoplasmic fragment of beta-dystroglycan. An interactome analysis by mass spectrometry techniques revealed that beta-dystroglycan interacts with components of the ubiquitin-proteasome system, the cell-cycle, and the nucleus. This all together suggests that, the regulated intramembrane proteolysis mediated by ubiquitination and phosphorylation PTM of beta-dystroglycan triggers downstream nuclear signalling events. These findings provide more ideas of the mechanisms implicated in the regulation of beta-dystroglycan and importantly, of some nuclear processes wherein beta-dystroglycan is involved. These insights may have further implications in the understanding of the progression of cancer and the development of useful therapies.
18
Filipe, Soares Renata. "Proteomic analysis of post-translational modification of streptococcal proteins." Thesis, King's College London (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.618344.
Full text
19
Pasquel, Danielle R. "Regulation of Pregnane X Receptor by Post-translational Modification." Thesis, Yeshiva University, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10019210.
Full textAbstract:
Pregnane X receptor (PXR) is a major transcriptional regulator of xenobiotic metabolism and transport pathways in the liver and intestines, which are critical for protecting organisms against potentially harmful xenobiotic and endobiotic compounds. Inadvertent activation of drug metabolism pathways through PXR is known to contribute to drug resistance, adverse drug-drug interactions, and drug toxicity in humans. In both humans and rodents, PXR has been implicated in non-alcoholic fatty liver disease, diabetes, obesity, inflammatory bowel disease, and cancer. Because of PXR's important functions, it has been a therapeutic target of interest for a long time.
Recent mechanistic studies have shown that PXR is modulated by multiple PTMs. In this thesis work, we conducted the first detailed examination of PXR regulation by acetylation. We found that PXR is efficiently acetylated in vitro and in vivo in multiple cell lines (293T, HepG2, LS174T). Acetylation and deacetylation are mediated by p300 and SIRT1, respectively. We found that PXR is directly acetylated by p300 at K109 by LC-MS/MS analysis. The K109Q acetylation mimicking mutant displayed reduced transcriptional activity and reduced ability to induce cyp3A4 target gene mRNA and protein compared to the WT and the K109R acetylation-defective mutant. The diminished activity of the K109Q mutant appears to be due to impaired heterodimerization with RXRa and impaired binding of the PXR-RXRa heterodimer to DNA response elements. Furthermore, PXR acetylation appears to have an effect at the phenotypic level, in that pharmacological modulation of PXR acetylation levels can modulate its lipogenic function in mouse primary hepatocytes independent of a ligand. Moreover, the K109Q mutant displays impaired chemoprotective function based on morphological assessment of cells overexpressing K109Q and challenged with indomethacin, suggesting that K109 acetylation downregulates PXR's chemoprotective and perhaps anti-apoptotic functions, although this must be explored further. Notably, the K109R mutant displayed the WT phenotype, further supporting that acetylation itself, not just any arbitrary mutation confers the effect. Altogether, the data suggests that acetylation at K109 represents an overall "loss of function" effect on PXR activity. Implications of our findings are discussed in the context of known roles for PXR in transcription, health, and disease.
20
Beswick, H. T. "Post-translational modification of lens proteins in relation to cataract." Thesis, University of Oxford, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.354818.
Full text
21
Alsaadi, Reham. "Characterization of Post-Translational Modification of ATG16L1 in Antibacterial Autophagy." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39151.
Full textAbstract:
Autophagy is a highly regulated catabolic pathway that is potently induced by stressors including starvation and infection. An essential component of the autophagy pathway is an ATG16L1-containing E3-like enzyme, which is responsible for lipidating LC3B and driving autophagosome formation. ATG16L1 polymorphisms have been linked to the development of Crohn’s disease (CD) and phosphorylation of CD-associated ATG16L1 (caATG16L1) has been hypothesized to contribute to cleavage and autophagy dysfunction. Here we show that ULK1 kinase directly phosphorylates ATG16L1 in response to infection and starvation. Moreover, we show that ULK1-mediated phosphorylation drives the destabilization of caATG16L1 in response to stress. Additionally, we found that phosphorylated ATG16L1 was specifically localized to the site of internalized bacteria indicating a role for ATG16L1 in the promotion of anti-bacterial autophagy. Lastly, we show that stable cell lines harbouring a phospho-dead mutant of ATG16L1 have impaired xenophagy. In summary, our results show that ATG16L1 is a novel target of ULK1 kinase and that ULK1-signalling to ATG16L1 is a double-edged sword, enhancing function of the wildtype ATG16L1, but promoting degradation of caATG16L1.
22
Bentley-DeSousa, Amanda. "Investigating Polyphosphate Biology: From Post-Translational Modification to Rare Disease." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42225.
Full textAbstract:
The first report of polyphosphates (polyP) was in 1890 by L. Liberman and since then, polyP’s role in biology has been explored. PolyPs are chains of phosphoanhydride-linked inorganic phosphates ranging from 3-1000s of units in length. These chains are implicated in many cellular pathways including blood clotting, bacterial virulence, and neuroproteotoxic disease. Given the diversity of polyP, they make an excellent candidate in the development of novel therapeutics. In yeast, polyP is synthesized by the vacuolar transporter chaperone (VTC) complex as a translocation event into the vacuole lumen. In 2015, polyP chains were found to act as a post-translational modification termed polyphosphorylation on yeast proteins (Nsr1 and Top1). This modification occurs non-enzymatically on lysine residues within poly-acidic, serine, and lysine (PASK) motifs and can only be detected via electrophoretic mobility shift on NuPAGE gels. We have since expanded the pool of yeast polyphosphorylated substrates to 25, with an enrichment of proteins with roles related to RNA biology. Additionally, we were the first group to demonstrate polyphosphorylation of 6 human proteins by expressing E. coli PPK1 in HEK293T cells. We next focused on elaborating how polyP is being regulated via the VTC complex by assessing which protein trafficking pathways are critical for VTC localization at the vacuole membrane. We found the adaptor protein 3 (AP-3) complex is responsible for localizing Vtc5 subunit to the vacuole membrane and in AP-3 mutants, Vtc5 becomes mislocalized to the vacuole lumen and degraded. Vtc5 degradation, upon AP-3 mutation, is mediated by the endosomal sorting complex required for transport (ESCRT) complex. The loss of polyP in AP-3 mutants is imparted by Vtc5 mislocalization. In humans, mutations in AP-3 cause a rare genetic disorder termed Hermansky-Pudlak Syndrome (HPS) which has a wide range of symptoms. These include defects in polyP accumulation in platelets, likely related to a loss of polyP. We expect that our work using yeast will provide a framework for understanding fundamental aspects of polyP biology related to HPS and other health conditions.
23
Charvet, Casey Douglas. "Post-Translational Modification By Isolevuglandins: Retinal Detection, Effects, and Prevention." Case Western Reserve University School of Graduate Studies / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=case1366296569.
Full text
24
Abraham, Jacinth. "Post-translational modification of p53 protein in response to DNA damage." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0027/NQ49932.pdf.
Full text
25
Murnan, Eric Joseph. "Post-translational modification and regulation of the chaperone Hsp27 during apoptosis." Connect to resource, 2009. http://hdl.handle.net/1811/37289.
Full text
26
Hurley, Christopher James. "Regulation of the transcription factor Neurogenin 3 by post-translational modification." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607900.
Full text
27
Yang, Yujie. "POST-TRANSLATIONAL MODIFICATION AND DEGRADATION MECHANISMS OF THE ARYL HYDROCARBON RECEPTOR." Scholarly Commons, 2021. https://scholarlycommons.pacific.edu/uop_etds/3753.
Full textAbstract:
The aryl hydrocarbon receptor (AHR) is a transcription factor first discovered to be activated by exogenous ligands, such as dioxins, and helps promote downstream gene (e.g. CYP1A1) transcription to metabolize the toxicants. With the reports of various AHR targets genes, the expression levels and activities of AHR have been implicated in many physiological and pathological situations. Understanding how AHR protein level is regulated would provide more information to target AHR. AHR stays in the cytosol in the absence of ligand in a complex with HSP90, p23 and XAP2. After ligand activation, AHR translocates into the nucleus, fulfilling its transactivation function and then is finally degraded by proteasomes. Here, we discovered a new mechanism that controls basal AHR protein level: the selective autophagy. Loss of AHR co-chaperone p23 leads to increased protein degradation of AHR through autophagy in HeLa cells. Inhibition of autophagy using several inhibitors (chloroquine, bafilomycin A1 or 3-methyladenine) increased AHR protein levels. Knocking down of key macroautophagy protein LC3B increases AHR protein levels and decreases the responsiveness of AHR to CQ treatment. The interaction between AHR and LC3B as well as AHR and autophagy receptor p62 were confirmed in vitro and in situ. AHR is found to be lysine (K) 63-ubiquitinated in HeLa cells, which is a common signal for the autophagy-lysosomal degradation.6 We also discovered that AHR is controlled by glycogen synthase kinase 3β (GSK3β) phosphorylation. Inhibition of GSK3β activity or its expression level increased AHR protein levels while expression of HA tagged-GSK3β lowers AHR protein levels. AHR protein level is regulated through autophagy. We confirmed the GSK3β-mediated phosphorylation of AHR by phos-tag gel electrophoresis couples with Western blot analysis and identified three putative phosphorylation sites of AHR in the C-terminal half of AHR sequence. Moreover, phosphorylated AHR constitutes the active pool for transactivation and phosphorylation tagged AHR for the autophagy-lysosomal degradation, which may act as way to limit its function.
28
Freschi, Luca. "Post-translational modifications regulatory networks : evolution, mechanisms et implications." Doctoral thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/25812.
Full textAbstract:
Les modifications post-traductionnelles (PTM) sont des modifications chimiques des protéines qui permettent à la cellule de réguler finement ses fonctions ainsi que de coder et d’intégrer des signaux environnementaux. Les progrès récents en ce qui a trait aux techniques expérimentales et bioinformatiques nous ont permis de determiner les profils de PTM pour des protéomes entiers ainsi que d’identifier les molécules qui sont responsables d’ « écrire » ou d’« effacer » ces PTM. Avec ces donnés, il a été possible de commencer à definir des réseaux de régulation cellulaire par PTM. Ici, nous avons étudié l’évolution de ces réseaux pour mieux comprendre comment ils peuvent contribuer à expliquer la complexité et la diversité des organismes ainsi que pour mieux comprendre leurs mecanismes d’action. Avant tout, nous avons abordé la question de comment les réseaux de régulation des PTM peuvent être recablés après un évenement de duplication des gènes en étudiant comment le réseau de phosphorégulation de la levure bourgeonnante a été récablé après un évenement de duplication complète du génome qui a eu lieu il y a 100 milions d’années. Nos résultats mettent en évidence le rôle de la duplication des gènes comme mécanisme clé pour l’innovation et la complexification des réseaux de régulation par PTM. Par la suite, nous avons abordé la question de comment les PTM peuvent contribuer à la diversité des organismes en comparant les profils de phosphorylation de l’homme et de la souris. Nous avons trouvé des différences substantielles dans les profils de PTM de ces deux espèces qui ont le potentiel d’expliquer, au moins en partie, les différences phénotypiques observées entre eux. Nous avons aussi trouvé des évidences qui supportent l’idée que les PTM peuvent « sauter » vers des nouvelles localisations et quand même réguler les mêmes fonctions biologiques. Ce phénomène doit être pris en considération dans les comparaisons des profils de PTM qui appartiennent à des espèces différentes, pour éviter de surestimer la divergence causée par la régulation par les PTM. Enfin, nous avons investigué comment plusieures PTM alternatives pour un même residu pouvent interagir pour réguler des fonctions cellulaires. Nous avons examiné deux des PTM les plus connus, la phosphorylation et la O-GlcNAcylation, qui modifient les sérines et les thréonines, et nous avons étudié les mécanismes potentiels d’interaction entre ces deux PTM. Nos résultats supportent l’hypothèse que ces deux PTM contrôlent plusieurs fonctions biologiques plutôt qu’une seule fonction. Globalement, les résultats présentés dans cette thèse permettent d’élucider les dynamiques évolutives, les mécanismes de fonctionnement et les implications biologiques des PTM.Post-translational modifications (PTMs) are chemical modification of proteins that allow the cell to finely tune its functions as well as to encode and integrate environmental signals. The recent advancements in the experimental and bioinformatic techniques have allowed us to determine the PTM profiles of entire proteomes as well as to identify the molecules that write or erase PTMs to/from each protein. This data have made possible to define cellular PTM regulatory networks. Here, we study the evolution of these networks to get new insights about how they may contribute to increase organismal complexity and diversity and to better understand their molecular mechanisms of functioning. We first address the question of how and to which extent a PTM network can be rewired after a gene duplication event, by studying how the budding yeast phosphoregulatory network was rewired after a whole genome duplication event that occurred 100 million years ago. Our results highlight the role of gene duplication as a key mechanism to innovate and complexify PTM regulatory networks. Then, we address the question of how PTM networks may contribute to organismal diversity by comparing the human and mouse phosphorylation profiles. We find that there are substantial differences in the PTM profiles of these two species that have the potential to explain, at least in part, the phenotypic differences observed between them. Moreover, we find evidence supporting the idea that PTMs can jump to new positions during evolution and still regulate the same biological functions. This phenomenon should be taken into account when comparing the PTM profiles of different species, in order to avoid overestimating the divergence in PTM regulation. Finally, we investigate how multiple and alternative PTMs that affect the same residues interact with each other to control proteins functions. We focus on two of the most studied PTMs, protein phosphorylation and O-GlcNAcylation, that affect serine and threonine residues and we study their potential mechanisms of interactions in human and mouse. Our results support the hypothesis that these two PTMs control multiple biological functions rather than a single one. Globally this work provides new findings that elucidate the evolutionary dynamics, the functional mechanisms and the biological implications of PTMs.
29
Hertlein, Erin K. "Post-translational modification of NF-kappaB regulation of stability and gene expression /." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1167336380.
Full text
30
Abdelghany, Magdy Korashy. "Profiling post translational modification of histone and p53 in human breast carcinomas." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12234/.
Full textAbstract:
Breast cancer is one of the most common cancers in females in the western world, and despite the advances in diagnosis and treatment it is still associated with significant morbidity. Thus, improvements to existing treatment modalities remain a priority. Understanding the molecular mechanisms controlling tumour growth and its modulation will be key to developing new therapies. In recent years it has been shown that posttranslational modifications (PTMs) of histones and p53 are functionally important in the regulation of cellular processes such as proliferation, differentiation and DNA damage repair. Thus, this study assessed the incidence of histone and p53 PTMs in breast tumours, and investigated how small molecule inhibitors of acetyltransferases can manipulate the levels of these PTMs in tumour cells. Our initial study demonstrated that hypoacetylation of H4K16 is associated with higher grade breast tumours (Elsheikh et al., 2009). Therefore, the expression levels of enzymes that are known to modulate H4K16 acetylation in vivo was assessed using immunohistochemical staining of 880 human breast tumour tissue microarrays. This led to the identification of a cluster of biomarkers (hMOF, H4K16ac, H3K9me3 and SUV39H1) which are significantly associated with patient outcome. We also assessed in tumours the incidence of other potential biomarkers including selected p53 PTMs such as p53K373ac and p53 K386ac. These were also found to be associated with favourable patient outcome in Kaplan-Meier survival analysis. Other potential biomarkers were also assessed such as the histone variant H2A.Z and its hyperacetylated form. H2A.Z correlated with Estrogen Receptor status of the tumours, consistent with a report that the gene encoding this histone variant is estrogen-regulated. In summary, this study has revealed that histone and p53 PTMs in breast tumours are potentially useful biomarkers for the classification of tumour type and as prognostic indicators, for use in conjunction with other clinicopathological indicators, and other well established biomarkers such as estrogen receptor and HER2. In a second aspect of the study, we investigated the effects of the acetyltransferase inhibitors curcumin and garcinol on a breast cancer cell model (MCF-7 cells). Garcinol blocked transcription-related PTMs such as H3K18ac, but surprisingly induced hyperacetylation of H4K16. This was found to be correlated with increased TIP60 expression, and correlated with increased incidence of DNA damage and cell cycle arrest. Other changes in cancer -associated PTMs were also observed, including increased H4K20 trimethylation. Garcinol compounds also reduced colony formation by MCF-7 cells and augmented sensitivity to etoposide. In summary, the data shows that histone and p53 PTMs constitute novel biological prognostic markers in breast cancer, and that targeting the enzymes that regulate these events may provide new avenues to drug therapies. This version does not contain the previously published journal article reproduced in the print thesis.
31
Hertlein, Erin K. "Post-translational modification of NF-kB: regulation of stability and gene expression." The Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=osu1167336380.
Full text
32
Hozoji, Masako. "Post-translational modification and regulation of ABCA1 involved in cellular cholesterol homeostasis." Kyoto University, 2009. http://hdl.handle.net/2433/126528.
Full textAbstract:
Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第14842号
農博第1782号
新制||農||974(附属図書館)
学位論文||H21||N4482(農学部図書室)
27248
UT51-2009-F484
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 和光, 教授 阪井 康能, 教授 三芳 秀人
学位規則第4条第1項該当
33
Brewster, Richard Christian. "Development of Halomethyl-Triazole reagents for installation of protein post-translational modification mimics." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31116.
Full textAbstract:
Triazoles have been widely used as amide bond isosteres in chemical biology as linkers and to enhance proteolytic stability. The use of triazoles has grown exponentially since the discovery of the copper (I) catalysed alkyne azide cycloaddition reaction in 2002 as the reaction is solvent and functional group tolerant, and usually high yielding. The reaction is also orthogonal to reactions used in nature, meaning it has become a powerful coupling tool. In post-translational modification (PTM), proteins are modified by covalent attachment of functional groups to amino acid side chains. These PTM processes are generally thought to be dynamic and highly regulated by cell machinery, controlling protein function in response to stimuli. The ability to control function post protein synthesis allows organisms to have a smaller genome, which is advantageous as it reduces the energy required for DNA replication and repair. Research into the function of PTMs has been limited by the difficulty in generating recombinant proteins that bear a single PTM in a specific location. Although many elegant methods have been proposed that solve this problem, to date cysteine alkylation is one of the most successful techniques. For lysine PTMs, thia-lysine II (sLys) derivatives have been shown to be excellent mimics of lysine, where the only perturbation between the native lysine-containing analogue is the switch of a CH2 for S in the side chain. Biotin is a well-known PTM in biotin dependent carboxylases, where biotin is involved in CO2 transfer. Recently biotinylation has also been shown to be a PTM on many other proteins, however the role of biotinylation is not well understood. Biotin triazole III has been shown to be a good mimic of the biotin amide bond and retains excellent affinity to Avidin (Av). In Chapter 1 the effects of modification to the valeryl side chain, and orientation of the biotin triazole bond affect affinity to Av using ITC are investigated. Compounds III, V and VI are shown to have a KD < 120 pM, but further information on the binding affinity of these compounds could not be assessed by ITC. Biotin triazoles III-VI were also shown to be resistant to hydrolysis in serum, unlike the native biotin amide bond, which is hydrolysed by the enzyme biotinidase (BTD). Generation of amide sLys derivatives has been shown to be synthetically challenging. In Chapter 2, the synthesis and applications of chloromethyl-triazole biotin as a sulfhydryl selective alkylation reagent are investigated. The electron withdrawing nature of the triazole was proposed to give a ‘pseudo-benzylic’ halide α to the triazole, thus increasing reactivity. The controlled alkylation of peptides and proteins has shown that chloromethyl-triazole biotin shows enhanced reactivity over many commercial alkylation reagents and also gives good selectivity for cysteine. Alkylation of histone H4K12C gave the singly alkylated product, accompanied by low amounts of double alkylation. Biotinylation was confirmed by Western blot with anti-biotin. Due to the wide range of readily available functional azides, it was envisaged that halomethyltriazoles could be incorporated into other PTM mimics. In Chapter 3, efforts to expand the range of PTMs accessible using halomethyl-triazoles and further enhance the reactivity of chloromethyl triazoles by preparation of bromo- and iodomethyl triazoles are detailed. Synthesis of reagents to mimic malonylation, succinylation and GlcNAcylation PTMs is described and the reactivity of these halomethyl-triazole reagents is assessed. An alternate approach to the development of PTM mimics through cysteine propargylation and subsequent CuAAC coupling is also described in chapter 3. In conclusion, a series of new reagents have been developed to mimic protein PTMs through alkylation of cysteine. The reagents, which include biotin, GlcNAc, succinyl and malonyl mimics, are based on a halomethyl-triazole scaffold and have been successfully reacted with cysteine containing peptides and proteins.
34
Wingrove, Callum Scott. "Post-translational modifications of recombinant human interferon-#gamma# in the CHO 320 cell line." Thesis, University of Kent, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.357687.
Full text
35
Keller, Patrick. "Developmental regulation and post-translational modification of lactase-phlorizin hydrolase and sucrase-isomaltase /." [S.l.] : [s.n.], 1994. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10881.
Full text
36
Humbard, Matthew Adam. "Post-translational modification and the 20S proteasome system of the haloarchaeon Haloferax volcanii." [Gainesville, Fla.] : University of Florida, 2009. http://purl.fcla.edu/fcla/etd/UFE0024797.
Full text
37
Liu, Qingbin. "Post-translational modification on arginine and function of CCAAT/enhancer binding protein alpha." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16620.
Full textAbstract:
Der Transkriptionsfaktor CCAAT/enhancer-binding protein α (C/EBPα) kontrolliert Zellzyklusarrest und terminale Differenzierung von neutrophilen Granulozyten und Adipozyten. Mutationen von C/EBPα treten häufig im Zusammenhang mit akuter myeloischer Leukämie auf. Massenspektrometrische Untersuchungen zeigten, dass C/EBPα an mehreren konservierten Argininen citrunilliert ist, einschließlich R297 in der C-terminalen basischen Region von C/EBPα. Mutationen von C/EBPα R297 wurden bereits beschrieben, weshalb der Schwerpunkt dieser Arbeit auf die Analyse der Modifikation dieses Aminosäurerestes gelegt wurde. Die Ergebnisse zeigen, dass die Peptidyl-Arginin-Deaminase (PADI4) mit C/EBPα interagiert und an mehreren Aminosäureresten citrunilliert. Citrunillierung oder Mutation von R297 beeinflusst die Aktivität von C/EBPα, einschließlich DNA-Bindung und Interaktion mit Partnerproteinen. Mutationsanalysen legen nahe, dass die positive Ladung des Aminosäurerestes R297 für die Bindung an cis-regulatorische DNA-Elemente, Protein Interaktionen, Genaktivierung, Fettzelldifferenzierung und Zellzyklusarrest ausschlaggebend ist. Knock-down von PADI4 in der myeloischen Vorläufer-Zelllinie 32D oder in der leukämischen U937 Zelllinie induziert Granulozyten-Differenzierung, möglicherweise durch Blockierung der PADI4-vermittelten Citrunillierung und Inaktivierung von C/EBPα. Zusammengefasst ergibt sich aus den Daten, dass PADI4 die positiv-geladene Seitenkette von C/EBPα R297 in eine ungeladene, citrunillierte Form umwandelt, die die Assoziation mit DNA destabilisiert und die C/EBPα-E2F-Interaktion beeinflusst, was wiederum das Gleichgewicht zwischen Proliferation und Differenzierung bestimmt.The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) coordinates cell cycle arrest and terminal differentiation of neutrophil granulocytes and adipocytes. Mutations in C/EBPα are frequently associated with acute myeloid leukemia. Mass spectrometric analysis revealed that citrullination occurred on multiple conserved C/EBPα arginine residues including R297 in the C/EBPα basic region. C/EBPα R297 was previously reported to be mutated in acute myeloid leukemia and we therefore focused on the modification this residue. Data presented here show that peptidylarginine deiminase 4 (PADI4) interacts with and citrullinates C/EBPα at several sites. Citrullination or mutation of R297 dramatically changed C/EBPα activities, including DNA binding and interaction with protein partners. Mutational analysis demonstrated that the positive charge of residue R297 was critical for binding to cis-regulatory sites on DNA, gene activation, adipocytic differentiation, and cell cycle arrest. Knock down of PADI4 in the myeloid precursor cell line 32D or U937 leukemia cells induced granulocyte differentiation, potentially through relieving PADI4 mediated citrullination and inactivation of C/EBPα. Taken together, the data suggest that PADI4 converts the positive C/EBPα R297 side chain to the non-charged citrulline side chain which destabilizes the association with DNA and affects C/EBPα - E2F interaction that determines the balance between proliferation and differentiation.
38
Thatte, Jayashree. "The role of post translational modification in regulating Human Papillomavirus (HPV) E6 functions." Thesis, Open University, 2018. http://oro.open.ac.uk/58133/.
Full textAbstract:
The Human Papillomavirus (HPV) E6 oncoprotein from cancer-causing HPV types is highly multi-functional, capable of targeting many different cellular partners and pathways. Integral to this multi-functionality is its regulation by phosphorylation. Here I describe studies to firstly investigate when E6 is phosphorylated within the PDZ binding motif (PBM), and demonstrate a complex pattern of phosphorylation events which take place upon the exposure of the cells to different forms of stress. Most important of which is phosphorylation by kinases belonging to the core of the DNA Damage Response (DDR) machinery. Functionally this redirects E6 from interaction with PDZ substrates to association with 14-3-3 proteins. This in turn appears to contribute towards the ability of E6 to inhibit p53 transcriptional activity on a subset of p53 responsive promoters, thereby linking DDR signaling to the function of the E6 PBM. Functionally I have also been able to precisely dissect the sequence constraints within the E6 PBM governing phosphorylation by different kinases, PDZ recognition and interaction with different 14-3-3 isoforms. I also show that phospho-regulation of the E6AP ubiquitin ligase can also play a role in these pathways, and can be utilized and redirected by different HPV E6 oncoproteins with varying degrees of efficiency. Depending upon the specific HPV E6 protein, I also show the first evidence for phosphorylation at a site outside of the PBM and provide insights into the potential functional consequences thereof. Taken together, these studies shed new light on the role of E6 phosphorylation in a range of different biological activities, and begin to explain the multi-functional nature of the high risk HPV E6 proteins.
39
Li, Xiang. "Post-translational modification and sequestration of cAMP-specific phosphodiesterase 4 in signalling complexes." Thesis, University of Glasgow, 2006. http://theses.gla.ac.uk/39014/.
Full textAbstract:
cAMP is a ubiquitous second messenger that is pivotal in controlling a wide array of cellular functions. The sole means to inactivate cAMP is to degrade it into 5'- AMP through the action of cyclic nucleotide phosphodiesterases (PDEs). It is now well appreciated that cAMP hydrolysis by PDEs is as important as its synthesis by adenylyl cyclases to achieve cAMP homeostasis in cells. The cAMP-specific phosphodiesterase-4 (PDE4) is encoded by four different genes (PDE4A, PDE4B, PDE4C and PDE4D), which generate over 16 different isoforms by alternative 5' mRNA splicing. This process gives rise to PDE4 isoforms with unique N-terminal region, complete or truncated UCR1/UCR2 modules, a central catalytic domain and an extreme C-terminal region. This plethora of PDE4 isoforms, complete with distinct N-terminal targeting domains and particular regulatory properties, confers compartmentalized action on cAMP signaling in various cell types. Understanding the distinct molecular mechanisms and functional outcomes of modification and sequestration of specific PDE4 isoforms can be expected to aid the development of more selective PDE4 inhibitor-based therapeutics. I have assessed the involvement of PDE4 isoenzymes in the β2-adrenergic receptor (β2AR) desensitization process in Chapter 3. It has been previously shown that agonist-activated P2ARS can recruit PDE4 isoforms in complex with β-arrestins and, in particular, PDE4D5 whereupon they undergo a dual desensitization. Using the PDE4-selective inhibitor rolipram or siRNA-mediated knockdown of PDE4B and PDE4D, I determined a new facet of PDE4-mediated process in the early stage of β2AR desensitization. In HEKB2 cells (stably overexpressing β2ARS) that have been pretreated with either rolipram or with siRNA to PDE4B and PDE4D, PKA phosphorylated GRK2 was accelerated in response to treatment with the β-agonist isoprenaline, as was isoprenaline-induced membrane translocation of GRK2, phosphorylation of the β2AR by GRK2, membrane translocation of β-arrestins/PDE4D complex and internalization of β2ARS, in comparison with the cells that were only challenged with isoprenaline. In the absence of isoprenaline, rolipram- induced inhibition of PDE4 activity in HEKB2 cells also stimulated the phosphorylation of GRK2 by PKA, with consequential effects on GRK2 membrane recruitment and GRK2 phosphorylation of β2AR. These results collectively suggest that PDE4 plays a fundamental role in influencing β2AR functioning by gating for the ability of PKA to phosphorylate GRK2 in resting cells and thereby regulating the degree of feedback through this system in stimulated cells. This provides a finely tuned adaptive response for preventing inappropriate activation of GRK through fluctuations in the basal levels of cAMP in resting/unstimulated cells. In Chapter 4 and 5 of this thesis, I analyzed the potential post-translational modification on one PDE4 isoform, namely PDE4D5. I have used site-directed mutagenesis to show that the ubiquitin-interacting motif (UIM) can confer the ability of PDE4D5 to undergo ubiquitination in response to extracellular stimuli. Furthermore, this ubiquitination was shown to be mediated by the E3 ligase Mdm2 that is brought in close proximity to PDE4D by the associated β-arrestin. My data shows that only a small subset of PDE4D5, namely that to which β-arrestins bind, is susceptible to ubiquitination.
40
Hoffman, Michael David. "Method development for the comprehensive analysis of post translational modifications by mass spectometry." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/1051.
Full textAbstract:
Signal Transduction is mediated by protein complexes whose spatial- and temporal-distribution, composition and function within cells are often regulated by different post-translational modifications (PTM). As PTMs add or subtract a specific mass difference to a protein, mass spectrometry becomes very amenable for modification analysis. These modifications have conventionally been monitored by fragmenting the modified protein or peptide by collision induced dissociation (CID) within the mass spectrometer, and then screening for the characteristic neutral fragment or fragment ion (marker ion), which is particular to the modification in question. Unfortunately, there are two major issues with respect to the traditional mass spectrometric analysis of PTMs: (1) as there are over 300 known types of modifications, the characteristic fragmentation of only a fraction of these modifications has been studied and (2) the traditional mass spectrometric approaches can only monitor these modifications sequentially, and thus comprehensive modification analysis would be unfeasible considering the breadth of PTMs. The following work aims to address these issues by (1) analyzing PTMs that have never been characterized mass spectrometrically and (2) developing a multiplexed technique for comprehensive PTM monitoring by simultaneously screening for all known characteristic fragments. With respect to the first issue, the characteristic fragmentation of lipid modifications and HNO-induced modifications was investigated. The most prevalent indicator(s) of the modification within the mass spectra are as follows: fragmentation of N-terminal myristoylated peptides produced marker ions at 240 and 268 Th, fragmentation of cysteine farnesylated peptides produced a marker ion at 205 Th and a neutral fragment of 204 Da, and fragmentation of cysteine palmitoylated peptides produced a neutral fragment of 272 Th. For HNO-induced modifications, fragmentation of the sulfinamide- and sulfinic acid-modified peptides produced a neutral fragment of 65 Da and 66 Da, respectively. With respect to the second issue, a multiplexed technique for monitoring modifications that fragment as neutral losses, termed Multiple Neutral Loss Monitoring (MNM), has been developed, successfully validated, and then shown to be the most sensitive approach for PTM analysis. MNM, combined with a second multiplexed approach, targeted Multiple Precursor Ion Monitoring, has been used to provide a comprehensive PTM analysis.
41
Liu, Ling, and 刘凌. "Regulation of post-translational modifications of the protein kinase LKB1: molecular mechanisms and physiologicalimplications." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47660831.
Full textAbstract:
Background and objectives:
Endothelial dysfunction and cancer are two of the important aspects of obesity-related medical complications, the prevalence of which is reaching an epidemic level worldwide. The protein kinase LKB1 has been shown to play opposite roles in these two metabolic diseases by promoting cellular senescence and inhibiting cell proliferation through regulating a series of its downstream targets. However, the molecular mechanisms wherebyLKB1 itself is regulated by its upstream molecules remains poorly understood.
The major objectives of this study are to identify novel upstream regulators of LKB1 and to investigate how these upstream regulators modulate the subcellular localization and physiological functions of LKB1 by post-translational modifications.
Key findings:
1. Our proteomic analysis demonstrated that LKB1 was modified by both acetylation and phosphorylation. The acetylation sites of mouseLKB1 include Lys48, Lys64and Lys312. The phosphorylation sites of mouseLKB1 include: Ser31, Thr32,Tyr36, Ser69, Thr71, Ser334and Thr336.
2.
In both human embryonic kidney 293 (HEK293)cells and primary porcine aortic endothelial cells (PAECs), the nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase SirT1 attenuated the acetylation levels of LKB1,which consequently resulted in enhancedLKB1ubiquitination, thereby leading to the proteasome-mediated degradation of LKB1.
3.
In primary PAECs, overexpression of SirT1 protected cells from cell cycle arrest and cellular senescence, whereas overexpression of LKB1 exhibited the opposite effects.SirT1 antagonizedLKB1-induced G1 phase arrest and cellular senescence by promoting the deacetylation and protein degradation of LKB1.
4. The in vitro phosphorylation assay and mass spectrometry analysis demonstrated
that LKB1 could be phosphorylated by the Akt kinase at Ser334which was critical for the interaction between LKB1 and 14-3-3. The enhanced association between LKB1 and 14-3-3 subsequently attenuated the interaction between LKB1 and Ste20 related adaptor α(STRADα), which further promoted the nuclear accumulation of LKB1.
5. The cell proliferation and cell cycle distribution analysis of the stably-transfected MDA-MB-231 breast cancer cells demonstrated that overexpression of the LKB1 mutant S334D, which mimicked Ser334 phosphorylation and localized exclusively in the nucleus, completely lost its anti-tumor activities. On the other hand, the S334A mutation enhanced the tumor suppressive functions of LKB1.
6. Nude mice inoculated with the LKB1 S334A stably-transfected MDA-MB-231 cells exhibited delayed tumor onset, decreased tumor growth rate and tumor weight. By contrast, inoculation of nude mice with the MDA-MB-231 cells overexpressing LKB1 S334D mutation showed the opposite effects on these parameters.
Conclusions:
These results collectively suggest that the deacetylase SirT1 and the protein kinase Aktare the two important upstream regulators of LKB1. SirT1 prevents LKB1-induced cellular senescence and protect endothelial ageing by promoting proteasome-mediated degradation of LKB1. Akt inhibits the tumor-suppressive activity of LKB1 by enhancing the phosphorylation-dependent nuclear translocation.
Further investigations on the precise mechanisms whereby SirT1 and Akt regulate LKB1 functions may help to design novel therapeutic strategies for treating obesity-related diseases, such as diabetes, cardiovascular disease and cancer.published_or_final_version
Medicine
Doctoral
Doctor of Philosophy
42
Gabant, Guillaume. "Spectrométrie de masse des modifications induites ou post-traductionnelles de protéines : méthodologie et application à des protéines d’intérêt thérapeutique." Thesis, Orléans, 2014. http://www.theses.fr/2014ORLE2061/document.
Full textAbstract:
Les modifications de protéines, qu’elles soient post-traductionnelles (PTMs) ou induites chimiquement, ont une influence considérable sur l'activité des protéines. Des méthodes de spectrométrie de masse (MS) HRMS, MS/MS CID et ETD, et de biochimie ont été développées pour la caractérisation structurale et cinétique de complexes protéine-ligand et de PTMs, dans le but de disséquer leur mécanisme et de concevoir des médicaments covalents contre des protéines liant des protéases, des kinases, ou l'ADN. La MS combinée avec des outils biochimiques a permis de séquencer l'inhibiteur de protéases grégline, et de détecter une PTM originale. De même, la transposase MOS1, modèle de l'intégrase du VIH pour la conception d'inhibiteurs, s'avère être à la fois acétylée et phosphorylée. Pour la lyase Abf2, une stratégie de piégeage, purification, protéolyse et hydrolyse ADN du complexe covalent Abf2-ADN, couplée à l’analyse MS, a été développée. Enfin, l’interaction entre le surpresseur de métastase hPEBP1 et la locostatine a été disséquée sur la protéine entière et par approche bottom-up. La locostatine s’hydrolyse en butyrate après fixation. Afin d’identifier le site ciblé par la locostatine, les conditions de réaction et de protéolyse ont été optimisées. La présence de réactions non spécifiques a conduit au développement 1) d'un modèle mathématique permettant de déterminer la fraction de liaison optimale pour discriminer le site spécifique des sites non-spécifiques, et 2) d'une méthode pour la quantification parallèle et exhaustive du degré de modification de tous les sites modifiés d'une protéine. Ces outils sont applicables aux ligands covalents de protéines et/ou à leurs PTMsProtein modifications, whether post-translational (PTMs) or chemically induced, play a crucial role on the activity of proteins. Mass spectrometry (MS) techniques such as HRMS, CID/ETD MS/MS, and biochemistrybased methods for structural and kinetic characterization of protein-ligand complexes and PTMs have been developed. MS combined with several biochemical tools has been used to sequence the proteinase inhibitor gregline and to detect a novel PTM. A similar approach shows that the transposase MOS1, a model for the design of HIV integrase inhibitors, is both phosphorylated and acetylated. For the lyase Abf2, a strategy of trapping, purification, proteolysis, and DNA hydrolysis of the Abf2-DNA covalent complex, coupled to MS analysis, has been developed. Finally, the interaction between the metastasis suppressor hPEBP1 and locostatin was dissected. Upon binding to hPEBP1, locostatin undergoes hydrolysis. To identify the site targeted by locostatin, the conditions of reaction and proteolysis were optimized. The qualitative approach reveals the presence of non-specific reactions, leading to the development of 1) a mathematical model to determine the optimum bound fraction for discriminating the specific site from non-specific sites, and 2) a method for the parallel and exhaustive quantification of the degree of modification of all modified sites of a protein. These tools are widely applicable to covalent protein ligands and/or PTMs
43
Lakshmi, Sreeja [Verfasser], and Eugen [Akademischer Betreuer] Kerkhoff. "Post-translational modification and regulation of human Spir protein / Sreeja Lakshmi. Betreuer: Eugen Kerkhoff." Regensburg : Universitätsbibliothek Regensburg, 2011. http://d-nb.info/1023276283/34.
Full text
44
Nixon, Benjamin R. "Effect of Post-translational Modification Crosstalk on Thin Filament Regulatory Function in Cardiac Muscle." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397558646.
Full text
45
Liu, Serena E. B. "Kinome-wide RNAi Screening to Identify Kinases Involved in Post-translational Modification of FUS." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/34199.
Full textAbstract:
Amyotrophic lateral sclerosis (ALS) is a devastating adult onset neurodegenerative
disorder characterized by the selective degeneration of upper and lower motor neurons.
Patients typically die from respiratory failures within 2-5 years after diagnosis. One of
the milestones in ALS research is the discovery Fused in Sarcoma (FUS), an ALS causative gene. FUS is an RNA/DNA-binding protein and predominantly resides in the nucleus.
Majority of the FUS mutations are located in the C-terminus and causing aberrant
misdistribution to the cytoplasm. Currently, only a few binding partners of FUS are
known, which makes it difficult to speculate on the function and interaction of the
protein. In this study, we conducted a kinome-wide RNAi screen to identify kinases that
affect the localization of FUS. A dual specificity protein kinase named CDC2-like kinase
(CLK1) from the screen was found to be responsible for in post-translational modification of
FUS and affects the localization of FUS in the nucleus. The identification of CLK1 as FUSmodifying kinase is consistent with roles ascribed to both in the binding and regulation of RNA.
46
Zhou, Shanggen. "Post-translational regulation of CCAAT/enhancer binding protein [delta] (C/EBP[delta]) by ubiquitin family proteins." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1195227986.
Full text
47
Kesic, Matthew J. "Identification and characterization of the post-translational modifications of the HTLV types 1 and 2 regulatory protein Rex." Columbus, Ohio : Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1241104210.
Full text
48
Liu, Li. "Purification and characterization of a protein palmitoyltransferase that acts on H-Ras protein and on a C-terminal N-Ras peptide /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/8664.
Full text
49
Lens, Zoé. "Sumo et le désordre structural font-ils bon ménage ?" Doctoral thesis, Universite Libre de Bruxelles, 2010. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210023.
Full textAbstract:
La sumoylation représente, après l’ubiquitination, l’exemple le plus étudié de modification post-traductionnelle impliquant la liaison d’une protéine à une autre. Cependant, alors que l’ubiquitination est impliquée principalement dans la dégradation des protéines par le protéasome, la sumoylation semble réguler les propriétés biochimiques de ses substrats (localisation cellulaire, interaction protéique, activité, …). Pour venir lier une protéine appelée Sumo (Small Ubiquitin-like Modifier) sur un substrat, la sumoylation emprunte une voie enzymatique analogue à celle de l’ubiquitination mais utilise des enzymes différentes. A ce jour, bien que plusieurs centaines de substrats de la sumoylation aient été identifiés, seules 5 structures de protéines sumoylées ont été résolues. Elles ne sont vraisemblablement pas représentatives de l’ensemble des substrats de la sumoylation et mon travail de thèse vise à élargir les connaissances structurales sur la sumoylation pour permettre de dégager des concepts généraux. Les études sur la sumoylation se heurtent généralement à la difficulté d’obtenir les substrats sumoylés. Ce projet a donc nécessité, au niveau technique, la mise au point d’un système de sumoylation in vivo en bactérie permettant de modifier des quantités importantes de protéines et de les purifier efficacement.
Des analyses bioinformatiques nous ont permis d’identifier des substrats de la sumoylation propices à une étude structurale de leur forme sumoylée. Au terme de ces analyses, nous avons retenu 3 protéines :DJ-1, PPARγ et IκBα. Bien que la complexité du sujet nous ait ensuite amené à écarter DJ-1 et PPARγ, nous sommes parvenus à purifier la forme sumoylée d’IκBα. Ce résultat nous a permis d’entreprendre une campagne de cristallogenèse d’IκBα complexé au facteur de transcription NF-κB. L’obtention d’IκBα sumoylé permettra également d’aborder des études fonctionnelles pour améliorer la compréhension du rôle de la sumoylation de ce substrat.
Nos analyses bioinformatiques ont également révélé que dans plus de 60% des cas, les sites de sumoylation des substrats se trouvent dans des zones prédites intrinsèquement désordonnées. L’importance du désordre dans le processus de sumoylation était jusqu’alors largement sous-estimée. A titre d’exemple, nous avons étudié par diffusion des rayons X aux petits angles la structure du domaine transactivateur du facteur de transcription ERM sous forme non modifiée et sous forme sumoylée. Cette étude indique que la sumoylation d’ERM n’induit pas le repliement de ce domaine transactivateur. De même, il apparait peu probable, au vu de la flexibilité de cette région, que la sumoylation empêche des interactions avec certains partenaires cellulaires. Dans ce contexte, la sumoylation semble servir de plateforme de recrutement de partenaires, reconnaissant de manière spécifique le Sumo. Ce mécanisme pourrait se généraliser à l’ensemble des sites de sumoylation prédits dans des zones intrinsèquement désordonnées.
Le système de sumoylation que nous avons développé permet de produire des protéines sumoylées pures en grande quantité et pourra également servir à identifier des protéines reconnaissant spécifiquement les substrats modifiés. Tous ces éléments devraient permettre de progresser dans la compréhension de cette modification post-traductionnelle impliquée dans de nombreux processus cellulaires fondamentaux.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished
50
Sefton, Mark. "The synthesis and post-translational modification of uvomorulin during compaction of the preimplantation mouse embryo." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309206.
Full text