3,732 research outputs found
Rick Strelan, Luke the Priest. The Autority of the Author of the Third Gospel, Aldershot, Ashgate, 2008
No full textGerber Daniel. Rick Strelan, Luke the Priest. The Autority of the Author of the Third Gospel, Aldershot, Ashgate, 2008. In: Revue d'histoire et de philosophie religieuses, 89e année n°3, Juillet-Septembre 2009. pp. 405-406
Hsp90-Mediated Maturation of Kinases and Nuclear Steroid Hormone Receptors: A Dissertation
No full textAmong heat shock proteins, Hsp90 is unusual because it is not required for the proper folding of most cellular proteins but rather is disproportionally linked to the activation of signal transduction proteins including over forty kinases and many steroid hormone receptors. Mutated forms of many Hsp90 clients are causative agents in cancer, making Hsp90 a promising pharmacological target. Many small molecular inhibitors have been identified that competitively bind to the ATP binding site of Hsp90, some of which are in clinical trials as anticancer agents. Although the activation of kinase and hormone receptor clients by Hsp90 and its co-chaperones has been extensively studied, the molecular mechanism of client protein activation is poorly understood. Hsp90 is a dimeric chaperone containing three domains: the N-terminal (N) and middle (M) domains contribute directly to ATP binding and hydrolysis and the C-terminal (C) domain mediates dimerization. At physiological concentration, Hsp90 predominantly forms dimers, but the possibility that full-length monomers might also function in cells has not been tested. In Chapter 3, we used a single-chain strategy to design a full-length Hsp90 monomer (NMCC). The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. NMCC Hsp90 was also defective at ATP hydrolysis and the activation of kinase and steroid hormone receptor clients in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric. After finding that a full-length Hsp90 monomer containing only one ATPase site was unable to support yeast viability or activate Hsp90 clients, we set out to further explore the role of ATPase activity in client protein activation. Approximately 10 % of the yeast proteome binds to Hsp90 making it important to study Hsp90 function in the cellular environment where all binding partners are present. In Chapter 4, we observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered super-stabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. We utilized these super-stabilized dimers to analyze the ability of ATPase mutant homodimers to activate known Hsp90 client proteins in yeast cells. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of the glucocorticoid hormone receptor (GR) and v-src confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo. In addition to its role in the activation of signal transduction client proteins, Hsp90 has been shown to suppress the in vitro aggregation of numerous hard-to-fold proteins. In Chapter 5, we examine the role of charge in Hsp90 anti-aggregation activity. The charge on Hsp90 is largely concentrated in two highly acidic regions. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. Addition of an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct rescued the anti-aggregation activity of Hsp90 indicating that the net charge contributes to its anti-aggregation activity. The in vitro anti-aggregation activity of Hsp90 studied in Chapter 5 occurs in the absence of ATP. However, all of the biologically important functions of Hsp90 in cells identified to date, including the maturation of kinases and nuclear steroid hormone receptors, clearly require ATP hydrolysis. Why does Hsp90 robustly hinder the aggregation of hard-to-fold proteins without ATP in vitro, but in vivo uses ATP hydrolysis for all of its essential functions? By utilizing separation of function Hsp90 variants (that specifically lack in vitro anti-aggregation activity) we have begun to address this question. We find that anti-aggregation deficient Hsp90 is unable to support yeast growth under stressful conditions, potentially due to reduced cellular expression. Interestingly, the ATP-independent anti-aggregation activity of Hsp90 has no measureable impact on cellular function. Thus, hindering the aggregation of most hard-to- fold proteins by Hsp90 (independent of ATP hydrolysis) does not appear to be important for cell function. These results suggest a cellular model where the Hsp40/60/70 machinery is responsible for hindering the aggregation of most hard-to-fold proteins while Hsp90 assists in the maturation of a select set of clients in an ATP-dependent fashion, potentially aided by its inherent anti-aggregation properties.Biochemistry and Molecular Pharmacolog
Ethnic identity, political identity and ethnic conflict: simulating the effect of congruence between the two identities on ethnic violence and conflict
No full textThis thesis outlines and presents an alternative hypothetical process to the emergence of ethnic conflict. Ethnic conflicts, rather than being dependent upon pre-existing 'ancient hatreds', are instead the result of a congruence between ethnic and political identity which grants individuals the ability to use ethnicity to identify and eliminate political threats. This hypothesis is formed by the examination of three case studies of ethnic conflict: Lebanon, Northern Ireland and Croatia. This hypothesis is then formalised and tested using an agent based simulation in which agent interactions are dependent upon ethnic and political identity and the congruence between the two. As predicted there was a strong positive correlation between how accurately ethnic identity reflected political identity and the level of ethnically motivated violence in the simulation, although the relationship was not linear. Furthermore the effect of a shift in congruence was found to be roughly comparable to the effect of initialising agents with a moderate level of pre-existing ethnic antagonism
Analyses of All Possible Point Mutations within a Protein Reveals Relationships between Function and Experimental Fitness: A Dissertation
No full textThe primary amino acid sequence of a protein governs its specific cellular functions. Since the cracking of the genetic code in the late 1950’s, it has been possible to predict the amino acid sequence of a given protein from the DNA sequence of a gene. Nevertheless, the ability to predict a protein’s function from its primary sequence remains a great challenge in biology. In order to address this problem, we combined recent advances in next generation sequencing technologies with systematic mutagenesis strategies to assess the function of thousands of protein variants in a single experiment. Using this strategy, my dissertation describes the effects of most possible single point mutants in the multifunctional Ubiquitin protein in yeast. The effects of these mutants on the essential activation of ubiquitin by the ubiquitin activating protein (E1, Uba1p) as well as their effects on overall yeast growth were measured. Ubiquitin mutants defective for E1 activation were found to correlate with growth defects, although in a non-linear fashion. Further examination of select point mutants indicated that E1 activation deficiencies predict downstream defects in Ubiquitin function, resulting in the observed growth phenotypes. These results indicate that there may be selective pressure for the activity of the E1enzyme to selectively activate ubiquitin protein variants that do not result in functional downstream defects. Additionally, I will describe the use of similar techniques to discover drug resistant mutants of the oncogenic protein BRAFV600E in human melanoma cell lines as an example of the widespread applicability of our strategy for addressing the relationship between protein function and biological fitness.Biochemistry and Molecular Pharmacolog
Experimental Illumination of Comprehensive Fitness Landscapes: A Dissertation
No full textEvolution is the single cohesive logical framework in which all biological processes may exist simultaneously. Incremental changes in phenotype over imperceptibly large timescales have given rise to the enormous diversity of life we witness on earth both presently and through the natural record. The basic unit of evolution is mutation, and by perturbing biological processes, mutations may alter the fitness of an individual. However, the fitness effect of a mutation is difficult to infer from historical record, and complex to obtain experimentally in an efficient and accurate manner. We have recently developed a high throughput method to iteratively mutagenize regions of essential genes in yeast and subsequently analyze individual mutant fitness termed Exceedingly Methodical and Parallel Investigation of Randomized Individual Codons (EMPIRIC). Utilizing this technique as exemplified in Chapters II and III, it is possible to determine the fitness effects of all possible point mutations in parallel through growth competition followed by a high throughput sequencing readout. We have employed this technique to determine the distribution of fitness effects in a nine amino acid region of the Hsp90 gene of S. cerevisiae under elevated temperature, and found the bimodal distribution of fitness effects to be remarkably consistent with near-neutral theory. Comparing the measured fitness effects of mutants to the natural record, phylogenetic alignments appear to be a poor predictor of experimental fitness. In Chapter IV, to further interrogate the properties of this region, library competition under conditions of elevated temperature and salinity were performed to study the potential of protein adaptation. Strikingly, whereas both optimal and elevated temperatures produced no statistically significant beneficial mutations, under conditions of elevated salinity, adaptive mutations appear with fitness advantages up to 8% greater than wild type. Of particular interest, mutations conferring fitness benefits under conditions of elevated salinity almost always experience a fitness defect in other experimental conditions, indicating these mutations are environmentally specialized. Applying the experimental fitness measurements to long standing theoretical predictions of adaptation, our results are remarkably consistent with Fisher’s Geometric Model of protein evolution. Epistasis between mutations can have profound effects on evolutionary trajectories. Although the importance of epistasis has been realized since the early 1900s, the interdependence of mutations is difficult to study in vivo due to the stochastic and constant nature of background mutations. In Chapter V, utilizing the EMPIRIC methodology allows us to study the distribution of fitness effects in the context of mutant genetic backgrounds with minimal influence from unintended background mutations. By analyzing intragenic epistatic interactions, we uncovered a complex interplay between solvent shielded structural residues and solvent exposed hydrophobic surface in the amino acid 582-590 region of Hsp90. Additionally, negative epistasis appears to be negatively correlated with mutational promiscuity while additive interactions are positively correlated, indicating potential avenues for proteins to navigate fitness ‘valleys’. In summary, the work presented in this dissertation is focused on applying experimental context to the theory-rich field of evolutionary biology. The development and implementation of a novel methodology for the rapid and accurate assessment of organismal fitness has allowed us to address some of the most basic processes of evolution including adaptation and protein expression level. Through the work presented here and by investigators across the world, the application of experimental data to evolutionary theory has the potential to improve drug design and human health in general, as well as allow for predictive medicine in the coming era of personalized medicine.Biochemistry and Molecular Pharmacolog
Enforced N-domain Proximity Stimulates Hsp90 ATPase Activity and Is Compatible with Function in Vivo
No full textThe Oxford Handbook of Daniel Defoe (Ed. by N Seager)
No full textThe Oxford Handbook of Daniel Defoe is the most comprehensive overview available of the author's life, times, writings, and reception. Daniel Defoe (1660-1731) is a major author in world literature, renowned for a succession of novels including Robinson Crusoe, Moll Flanders, and A Journal of the Plague Year, but more famous in his lifetime as a poet, journalist, and political agent. Across his vast oeuvre, which includes books, pamphlets, and periodicals, Defoe commented on virtually every development and issue of his lifetime, a turbulent and transformative period in British and global history. Defoe has proven challenging to position—in some respects he is a traditional and conservative thinker, but in other ways he is a progressive and innovative writer. He therefore benefits from the range of critical appraisals offered in this Handbook.The Handbook ranges from concerns of gender, class, and race to those of politics, religion, and economics. In accessible but learned chapters, contributors explore salient contexts in ways that show how they overlap and intersect, such as in chapters on science, environment, and empire. The Handbook provides both a thorough introduction to Defoe and to early eighteenth-century society, culture, and literature more broadly. Thirty-six chapters by leading literary scholars and historians explore the various genres in which Defoe wrote; the sociocultural contexts that inform his works; his writings on different locales, from the local to the global; and the posthumous reception and creative responses to his works
Homage to N. (after Chekhov)
No full textDaniel Halpern is the author of seven collections, most recently Foreign Neon (Knopf, 1991) and Tango (Viking Penguin, 1987). He has edited numerous volumes, including The Art of the Tale: An International Anthology of Short Stories. Knopf will release his Selected Poems in April 1994. Publisher of the Ecco Press and Antaeus, he lives in Princeton, New Jersey
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The Works of Daniel Defoe
No full textDaniel Defoe is now mainly thought of as a novelist - the author of such famous works as Robinson Crusoe and Moll Flanders - but this is not how his contemporaries regarded him. His reputation in his lifetime was as a poet, a journalist, and a polemical writer on political, economic and social affairs. The quantity and range of his writings is unparalleled in English Literature, and few writers have made such a lasting impact in so many literary genres.
This is the most comprehensive collection of the writings of this extraordinary man ever attempted. Grouped under five thematic headings, it aims, in 44 volumes, to give as extensive a representation as possible of Defoe’s work in all the literary genres to which he contributed
Guerres de mémoire en France
No full textMemory wars in France, Daniel Lindenberg.
The author proposes a transversal analysis of the periods which marked French national memory, from the Revolution to the Algerian war. He seeks to describe and explain the double movement of accumulation and selection characteristic of the link between French identity and the past.Lindenberg Daniel. Guerres de mémoire en France. In: Vingtième Siècle, revue d'histoire, n°42, avril-juin 1994. pp. 77-96
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