121 |
Isolation and Characterisation of the 5'-Nucleotidase from Escherichia coliMcMillen, Lyle, l.mcmillen@sct.gu.edu.au January 2001 (has links)
Escherichia coli 5'-nucleotidase is a periplasmically localised enzyme capable of hydrolysing a broad range of substrates, including all 5'-ribo- and 5'-deoxyribonucleotides, uridine diphosphate sugars, and a number of synthetic substrates such as bis (r-nitrophenyl) phosphate. The enzyme has been shown to contain at least one zinc ion following purification, and to have two metal binding sites in the catalytic cleft. 5'-Nucleotidase activity is significantly stimulated by the addition of particular divalent metal ions, most notably cobalt which results in a 30-50 fold increase in activity. Significant sequence homology between the E. coli 5'-nucleotidase and members of the Ser/Thr protein phosphatase family in the catalytic site has lead to 5'-nucleotidase being included in this protein family. This thesis describes the development of a rapid purification methodology for milligram quantities of 5'-nucleotidase, and the investigation of a number of physical and biochemical properties of the enzyme with the aim of comparing these properties to those of certain catalytic site mutants. The molecular weight of the mature protein was estimated as 58219 daltons, with a specific activity for 5'-AMP, in the presence of 4 mM Co2+ and 13 mM Ca2+ at pH 6.0, of 730 mmol/min/mg. The presence of up to two zinc ions associated with the purified enzyme was observed using ICP-ES analysis, suggesting both metal ion binding sites are occupied by zinc in vivo, and some degree of displacement of zinc by cobalt could be observed. Mass spectrometry data, gathered at 60 and 70 mS orifice potential, suggested the presence of a small proportion of material with a mass 118 to 130 daltons greater than the main 5'-nucleotidase mass estimation. This study suggests that this mass difference, only evident at the lower orificepotential, is due to the presence of two zinc ions closely associated with 5'-nucleotidase. To account for the observed high level of activation of 5'-nucleotidase activity by particular divalent metal ions, this thesis describes a proposed model in which these divalent ions may displace the zinc ion at one of the metal ion binding sites. This displacement only occurs at one of the two metal ion binding sites, with the other metal binding site retaining the zinc ion already present. Studies with purified enzyme, each with a single amino acid substitution, lend support to this hypothesis and suggest the identity of the metal ion binding site at which displacement occurs. Seven key catalytic site residues (Asp-41, His-43, Asp-84, His-117, Glu-118, His-217 and His-252) were selected on the basis of sequence conservation within the Ser/Thr protein phosphatases and 5'-nucleotidases. X-ray crystallographic data published by others during this study implicated five of the selected residues (Asp-41, His-43, Asp-84, His-217 and His-252) directly in metal ion binding, including two residues from each metal ion binding site and one directly involved in both sites (Asp-84). The remaining two residues (His-117 and Glu-118) are highly conserved but were not thought to play direct roles in metal ion binding. The seven selected residues were modified by site-directed mutagenesis, and the effect of the amino acid substitutions upon the kinetic properties of 5'-nucleotidase activity was determined. Residues hypothesised to be involved in metal ion displacement, and subsequent activation of 5'-nucleotidase activity, were identified by reductions in metal ion affinity and increased levels of activation by cobalt compared to the wild type 5'-nucleotidase. This study suggests that the metal binding site, M2, that includes residues Asp-84, His-217 and His-252, is involved in metal ion displacement, while the other metal binding site, M1, is not. This, in turn, suggests the metal binding sites are functionally non-equivalent and kinetically distinct. No residues were identified in this study as playing significant roles in substrate binding, as there was no significant reduction observed in affinity for 5'-AMP observed in any of the catalytic site mutants.
|
122 |
Expression et composition des motifs de conception avec les aspectsDenier, Simon 09 July 2007 (has links) (PDF)
Les patrons de conception répertorient les bonnes pratiques de la programmation par ob- jets. Les solutions des patrons, appelées motifs, apparaissent avec une densité croissante dans les bibliothèques et cadriciels. Les effets de cette densité sur la modularité, l'adaptation et la réutilisation des programmes sont mal connus. Or la dispersion et le mélange du code lié à l'im- plémentation des motifs rendent difficile l'étude de ces effets. La programmation par aspects est une technique nouvelle dédiée au traitement de ces deux symptômes. En modularisant les motifs dans des aspects, nous pouvons analyser de manière plus fine les problèmes d'implémentation et de composition des motifs liés à leur densité. Cette thèse aborde les problèmes de la densité, de l'implémentation et de la composition des motifs avec AspectJ, une extension de Java pour les aspects. À partir du cas concret du cadri- ciel JHotDraw, nous montrons qu'une forte densité est un facteur de risque sur la modularité et l'adaptation d'un programme objet. Nous présentons la transformation des motifs à l'aide des aspects et nous décrivons les idiomes d'AspectJ supportant leur modularisation. Nous exami- nons la modularité et la réutilisation des compositions de motifs définies avec les aspects. Nous proposons la résolution des interactions entre motifs à l'aide du langage de coupe des aspects. Enfin nous développons une méthode de programmation avec AspectJ basée sur l'usage conjoint des classes et des aspects. Ces travaux nous permettent de conclure sur l'intérêt des aspects comme moyen d'étude et de traitement de la densité des motifs. Ils ouvrent également des pistes pour l'amélioration des langages d'aspects.
|
123 |
Dictes hardiement, bons motz n'espargnent personne : approche typologique, esthétique et historique du comique dans PerceforestDelamaire, Anne 11 December 2010 (has links) (PDF)
Malgré l'attention que lui porte un nombre croissant de chercheurs, Perceforest, qui fait actuellement l'objet d'un travail d'édition intégrale mené par M. Gilles Roussineau, reste encore un domaine à défricher. Cette recherche se propose de l'aborder selon un angle lui aussi partiellement ignoré par les études de médiévistique consacrées aux romans arthuriens : le comique. L'objectif est d'offrir un inventaire fouillé du comique dans Perceforest. Ainsi qu'une étude du fonctionnement contextuel des « motifs » comiques et de leurs enjeux au niveau globale de l'oeuvre
|
124 |
Les alliances traditionnelles et modernes négro-africaines et la vie nouvelle en Jésus-Christ. Perspectives africaines pour une théologie morale de l'alliance.Kenkfuni Oblipe, Albert 14 May 2003 (has links)
Toute alliance donne à penser que les êtres humains sont capables de se lier par un engagement réciproque. Ce faisant, ils sont appelés à témoigner d'une solidarité vouée à se déployer dans le temps de l'alliance.
Le chrétien négro-africain est appelé à assumer ses engagements dans ses alliances traditionnelles et modernes aussi bien que dans l'Alliance christique. De là, la nécessité d'une réflexion éthique susceptible d'articuler ces alliances et d'éclairer le Négro-africain. Une réflexion éthico-théologique peut contribuer à relever ce défi en Afrique noire subsaharienne. Ce problème n'a pas échappé aux préoccupations théologiques dans cette partie du continent. Quelques monographies théologiques en témoignent. Mais ces études, à notre connaissance, se sont cantonnées aux alliances ancestrales. Or, il importe de prendre en compte aussi les alliances modernes dans lesquelles est engagé le chrétien négro-africain.
Pour interpeller ce dernier et éclairer sa conscience afin qu'il assume sa vie en alliance, nous procédons au discernement de ses alliances humaines à la lumière de la théologie morale de l'Alliance. A cette fin, notre recherche s'articule sur la triple démarche herméneutique d'une théologie contextuelle. La première partie qui analyse le contexte de l'étude, esquisse les alliances négro-africaines. Le premier chapitre porte sur les alliances socio-culturelles traditionnelles : les alliances matrimoniales, les alliances amicales et les alliances anthropocosmiques. Au second chapitre, il est question des alliances modernes tant politico-économiques qu'humanitaires. Dans la deuxième partie qui décontextualise la question de l'étude, nous analysons l'Alliance biblique et ses enjeux éthiques respectivement dans le Premier Testament au chapitre premier et le Nouveau Testament au second chapitre. Notre approche éthico-théologique s'appuie surtout sur J. L'Hour, A. Wénin dans le cadre vétérotestamentaire et sur H. Wattiaux et P. Beauchamp dans la perspective néotestamentaire. La troisième partie qui recontextualise l'étude entend placer les alliances négro-africaines dans la lumière de la morale biblique de l'Alliance. Le chapitre premier est consacré à l'évaluation théologique des alliances ancestrales à partir des études théologiques déjà réalisées dans la perspective négro-africaine. Le dernier chapitre commence par la lecture éthico-théologique des alliances modernes et se termine par une description du profil éthique du partenaire négro-africain du Dieu de Jésus-Christ.
En entrant dans l'Alliance résultant de l'initiative d'amour de Dieu pour l'humanité, le Négro-africain effectue et met au jour ce qu'il est et est appelé à devenir : un être qui n'est pas libre d'aimer ou de ne pas aimer, mais un être libre pour aimer. Le projet d'agapè de l'Alliance met les projets de solidarité des alliances dans une perspective telle que le service de la dignité humaine devient la pierre de touche de l'humanisation. Si la quête de l'humanisation est l'horizon de l'agir humain, l'identité et la spécificité de l'allié du Dieu de Jésus-Christ consiste à aimer, grâce à l'agapè de l'Alliance baptismale, à la manière de Jésus-Christ. En effet la manière d'être et d'aimer de celui-ci marque de son empreinte le chemin de l'humanisation : se servir de ses capacités pour humaniser l'homme et la société. L'éthique de l'Alliance aide le Négro-africain à prendre conscience de la limite et des faiblesses de son humanité. Ainsi l'amène-t-elle à apprécier et à vivre autrement les valeurs de solidarité, à contribuer au renouvellement des alliances d'Afrique aussi bien qu'à la reconstruction d'une humanité authentique de paix, de justice, d'égalité, de fraternité…dans le respect de la dignité humaine. Aimer, c'est participer de la nature théandrique du Christ. C'est à ce prix que le chrétien négro-africain assume au cœur de ses alliances humaines l'intuition célèbre de saint Augustin : " Dilige et quod vis fac " (Aime et fais ce que tu veux ).
|
125 |
Life Change Narratives: When The Road DivergesRyan, Bernadene J. 01 May 2013 (has links)
Transformation events can be a change in a person's work, a change in philosophy, a sudden insight, or a break in a relationship. According to David Hufford and Marilyn Motz, narrating these experiences are ways in which people perform, construct, and communicate belief systems. The narrators within the context of this thesis experience their transformation through a career transformation. The narrators rediscover their initial passion and transform that desire into actions that results in a shift of career. Sometimes seen as inexplicable, nevertheless the narrators provide analysis and reflection on the influences that led to their change. Some of the actions or thoughts that the narrators incorporate in their stories demonstrate not only the progression of events but also the alterations narrators experience in their worldviews. The context in which these changes occur is essential to interpreting and understanding the experience. Narrative components are filtered through an interpretive process that includes personal meaning, contrast with social norms and cultural beliefs and the impact on the receiver to enable narrators to justify their experience. It is the reflection on these experiences through which people gain insight and establish relevance to events that seem sometimes beyond their control. Stories from pop culture to ordinary citizens who change their lives daily demonstrate the pervasiveness of the transformational effect of states of crisis through which people journey. People's lives are turned upside-down through these experiences which place the narrator out of their normal element. There are two levels to these story types: external and internal transformation. At a superficial level there is the journey to change careers but at another level there is a relationship to opening up cultural expectations or acting generatively, as role-models. Narrators are effecting change through their positive attitudes and acceptance of the trials they encounter during their transitions. Narrators discuss specific actions that create transformative life changes or philosophical shifts. My investigation studies how individuals are involved in transitional events in which they experience a disengagement from a previous life, spending some time in liminal space where they transition or regenerate into a new place in society. Part of my approach to this subject matter used theories introduced by Victor Turner (pilgrimages) and by Arnold Van Gennep (rites of passage). Regina Holloman proposes that rites of passage can occur not just as physical/material transformation but can occur psychically as well. Some of the narrative patterns that narrators use to construct these tales are identified within the context of folk belief and folklore scholarship.
|
126 |
Structural and Biophysical Studies of the Role of Stromal Interaction Molecules STIM1 and STIM2 in Initiating Store-operated Calcium EntryZheng, Le 29 July 2010 (has links)
Store-operated calcium entry (SOCE) is the major Ca2+ entry pathway in most non-excitable cells maintaining prolonged elevation of cytosolic Ca2+ levels required for gene transcription. SOCE is activated by the loss of endoplasmic reticulum (ER) Ca2+ through stromal interaction molecules (STIM), ER-membrane associated Ca2+ sensors. In humans, STIM1 and STIM2 share 65% sequence similarity but differentially regulate SOCE. Biophysical studies on the luminal Ca2+-binding region suggests that STIM2 EF-SAM is more stable than STIM1. The NMR structure of Ca2+-loaded STIM2 EF-SAM determined in this work suggests a more stable SAM and a tighter EF-hand and SAM interaction in STIM2 may be account for its higher stability. Chimeric swapping of the EF-hand and SAM domains generates an unstable ES211. Introducing ES211 into cherryFP-STIM1 shows constitutive puncta which activate SOCE independent of ER depletion. The current work demonstrates that the instability of the EF-SAM plays an important role in regulating SOCE initiation.
|
127 |
Structural and Biophysical Studies of the Role of Stromal Interaction Molecules STIM1 and STIM2 in Initiating Store-operated Calcium EntryZheng, Le 29 July 2010 (has links)
Store-operated calcium entry (SOCE) is the major Ca2+ entry pathway in most non-excitable cells maintaining prolonged elevation of cytosolic Ca2+ levels required for gene transcription. SOCE is activated by the loss of endoplasmic reticulum (ER) Ca2+ through stromal interaction molecules (STIM), ER-membrane associated Ca2+ sensors. In humans, STIM1 and STIM2 share 65% sequence similarity but differentially regulate SOCE. Biophysical studies on the luminal Ca2+-binding region suggests that STIM2 EF-SAM is more stable than STIM1. The NMR structure of Ca2+-loaded STIM2 EF-SAM determined in this work suggests a more stable SAM and a tighter EF-hand and SAM interaction in STIM2 may be account for its higher stability. Chimeric swapping of the EF-hand and SAM domains generates an unstable ES211. Introducing ES211 into cherryFP-STIM1 shows constitutive puncta which activate SOCE independent of ER depletion. The current work demonstrates that the instability of the EF-SAM plays an important role in regulating SOCE initiation.
|
128 |
COPIA: A New Software for Finding Consensus Patterns in Unaligned Protein SequencesLiang, Chengzhi January 2001 (has links)
Consensus pattern problem (CPP) aims at finding conserved regions, or motifs, in unaligned sequences. This problem is NP-hard under various scoring schemes. To solve this problem for protein sequences more efficiently,a new scoring scheme and a randomized algorithm based on substitution matrix are proposed here. Any practical solutions to a bioinformatics problem must observe twoprinciples: (1) the problem that it solves accurately describes the real problem; in CPP, this requires the scoring scheme be able to distinguisha real motif from background; (2) it provides an efficient algorithmto solve the mathematical problem. A key question in protein motif-finding is how to determine the motif length. One problem in EM algorithms to solve CPP is how to find good startingpoints to reach the global optimum. These two questions were both well addressed under this scoring scheme,which made the randomized algorithm both fast and accurate in practice. A software, COPIA (COnsensus Pattern Identification and Analysis),has been developed implementing this algorithm. Experiments using sequences from the von Willebrand factor (vWF)familyshowed that it worked well on finding multiple motifs and repeats. COPIA's ability to find repeats makes it also useful in illustrating the internal structures of multidomain proteins. Comparative studies using several groups of protein sequences demonstrated that COPIA performed better than the commonly used motif-finding programs.
|
129 |
Innovative Algorithms and Evaluation Methods for Biological Motif FindingKim, Wooyoung 05 May 2012 (has links)
Biological motifs are defined as overly recurring sub-patterns in biological systems. Sequence motifs and network motifs are the examples of biological motifs. Due to the wide range of applications, many algorithms and computational tools have been developed for efficient search for biological motifs. Therefore, there are more computationally derived motifs than experimentally validated motifs, and how to validate the biological significance of the ‘candidate motifs’ becomes an important question. Some of sequence motifs are verified by their structural similarities or their functional roles in DNA or protein sequences, and stored in databases. However, biological role of
network motifs is still invalidated and currently no databases exist for this purpose.
In this thesis, we focus not only on the computational efficiency but also on the biological meanings of the motifs. We provide an efficient way to incorporate biological information with clustering analysis methods: For example, a sparse nonnegative matrix factorization (SNMF) method is used with Chou-Fasman parameters for the protein motif finding. Biological network motifs are searched by various clustering algorithms with Gene ontology (GO) information. Experimental results show that the algorithms perform better than existing algorithms by producing a larger number of high-quality of biological motifs.
In addition, we apply biological network motifs for the discovery of essential proteins. Essential proteins are defined as a minimum set of proteins which are vital for development to a fertile adult and in a cellular life in an organism. We design a new centrality algorithm with biological network motifs, named MCGO, and score proteins in a protein-protein interaction (PPI) network to find essential proteins. MCGO is also combined with other centrality measures to predict essential proteins using machine learning techniques.
We have three contributions to the study of biological motifs through this thesis; 1) Clustering analysis is efficiently used in this work and biological information is easily integrated with the analysis; 2) We focus more on the biological meanings of motifs by adding biological knowledge in the algorithms and by suggesting biologically related evaluation methods. 3) Biological network motifs are successfully applied to a practical application of prediction of essential proteins.
|
130 |
A Bioinformatics Study of Human Transcriptional RegulationAmeur, Adam January 2008 (has links)
Regulation of transcription is a central mechanism in all living cells that now can be investigated with high-throughput technologies. Data produced from such experiments give new insights to how transcription factors (TFs) coordinate the gene transcription and thereby regulate the amounts of proteins produced. These studies are also important from a medical perspective since TF proteins are often involved in disease. To learn more about transcriptional regulation, we have developed strategies for analysis of data from microarray and massively parallel sequencing (MPS) experiments. Our computational results consist of methods to handle the steadily increasing amount of data from high-throughput technologies. Microarray data analysis tools have been assembled in the LCB-Data Warehouse (LCB-DWH) (paper I), and other analysis strategies have been developed for MPS data (paper V). We have also developed a de novo motif search algorithm called BCRANK (paper IV). The analysis has lead to interesting biological findings in human liver cells (papers II-V). The investigated TFs appeared to bind at several thousand sites in the genome, that we have identified at base pair resolution. The investigated histone modifications are mainly found downstream of transcription start sites, and correlated to transcriptional activity. These histone marks are frequently found for pairs of genes in a bidirectional conformation. Our results suggest that a TF can bind in the shared promoter of two genes and regulate both of them. From a medical perspective, the genes bound by the investigated TFs are candidates to be involved in metabolic disorders. Moreover, we have developed a new strategy to detect single nucleotide polymorphisms (SNPs) that disrupt the binding of a TF (paper IV). We further demonstrated that SNPs can affect transcription in the immediate vicinity. Ultimately, our method may prove helpful to find disease-causing regulatory SNPs.
|
Page generated in 0.0321 seconds