Comparison of the transcriptomic "stress response" evoked by antimycin A and oxygen deprivation in saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Acute changes in environmental parameters (e.g., O₂, pH, UV, osmolarity, nutrients, etc.) evoke a common transcriptomic response in yeast referred to as the "environmental stress response" (ESR) or "common environmental response" (CER). Why such a diverse array of insults should elicit a common transcriptional response remains enigmatic. Previous functional analyses of the networks involved have found that, in addition to up-regulating those for mitigating the specific stressor, the majority appear to be involved in balancing energetic supply and demand and modulating progression through the cell cycle. Here we compared functional and regulatory aspects of the stress responses elicited by the acute inhibition of respiration with antimycin A and oxygen deprivation under catabolite non-repressed (galactose) conditions.</p> <p>Results</p> <p>Gene network analyses of the transcriptomic responses revealed both treatments result in the transient (10 – 60 min) down-regulation of MBF- and SBF-regulated networks involved in the G1/S transition of the cell cycle as well as Fhl1 and PAC/RRPE-associated networks involved in energetically costly programs of ribosomal biogenesis and protein synthesis. Simultaneously, Msn2/4 networks involved in hexose import/dissimilation, reserve energy regulation, and autophagy were transiently up-regulated. Interestingly, when cells were treated with antimycin A well before experiencing anaerobiosis these networks subsequently failed to respond to oxygen deprivation. These results suggest the transient stress response is elicited by the acute inhibition of respiration and, we postulate, changes in cellular energetics and/or the instantaneous growth rate, not oxygen deprivation <it>per se</it>. After a considerable delay (≥ 1 generation) under anoxia, predictable changes in heme-regulated gene networks (e.g., Hap1, Hap2/3/4/5, Mot3, Rox1 and Upc2) were observed both in the presence and absence of antimycin A.</p> <p>Conclusion</p> <p>This study not only differentiates between the gene networks that respond to respiratory inhibition and those that respond to oxygen deprivation but suggests the function of the ESR or CER is to balance energetic supply/demand and coordinate growth with the cell cycle, whether in response to perturbations that disrupt catabolic pathways or those that require rapidly up-regulating energetically costly programs for combating specific stressors.</p

Use of neural networks to model molecular structure and function

This thesis is a study of some applications of neural networks - a recent computer algorithm - to modelling the structure and function of biologically important molecules. In Chapter 1, an introduction to neural networks is given. An overview of quantitative structure activity relationships (QSARs) is presented. The applications of neural networks to QSAR and to the prediction of structural and functional features of protein and nucleic acid sequences are reviewed. The neural network algorithms used are discussed in Chapter 2. In Chapter 3, a two-layer feed-forward neural network has been trained to recognise an ATP/GTP-binding local sequence motif. A comparably sophisticated statistical method was developed, which performed marginally better than the neural network. In a second study, described in Chapters 4 and 5, one of the largest data sets available for developing a quantitative structure activity relationship - the inhibition of dihydrofolate reductase by 2,4-diamino-6,6-dimethyl-5-phenyldihydrotriazine derivatives has been used to benchmark several computational methods. A hidden-layer neural network, a decision tree and inductive logic programming have been compared with the more established methods of linear regression and nearest neighbour. The data were represented in two ways: by the traditional Hansch parameters and by a new set of descriptors designed to allow the formulation of rules relating the activity of the inhibitors to their chemical structure. The performance of neural networks has been assessed rigourously in two distinct areas of biomolecular modelling; sequence analysis and drug design. The conclusions of these studies are presented in Chapter 6

Structural and functional studies of mucin-interacting adhesion domains from Candida glabrata and Helicobacter pylori

Epithelial adhesins from Candida glabrata Epithelial adhesins (Epa) are crucial proteins in the colonization, pathogenesis and virulence of Candida glabrata. These adhesins have a similar modular structure to Saccharomyces cerevisiae flocculins, with an N-terminal adhesive A domain, a central neck-like B domain, and an anchorage C-terminal C domain. A hallmark of many fungal adhesins is the presence of a calcium binding PA14 domain within the A domain. The PA14 domain is responsible for carbohydrate binding in a calcium dependent manner, which allows for the classification of these proteins as C-type lectins[1]. In this study, it was possible to elucidate the crystal structures of Epa1A and three variants at resolutions from 1.4 to 2.0 Å. The latter were meant to emulate the specificities of Epa2, Epa3 and Epa6 adhesive domains. The results yielded a profound knowledge of the binding pocket of Epa1A and the mechanisms through which specificity is controlled in the Epa A domain. Especially surprising was the fact that, even though the proteins were crystallized in the presence of lactose, the protein co-crystals never showed the aforementioned sugar. Instead, a galactoseβ1-3glucose disaccharide unit could be modeled into the electron density. The disaccharide is commonly found on cell surfaces and milk derivates, from which the employed lactose was obtained[2]. Epa1A , Epa1→2A, Epa1→3A and Epa1→6A were also functionally characterized by semi- quantitative, high-throughput methodologies. In collaboration with the consortium for functional glycomics, fluorescently labeled proteins were set in contact with large-scale glycan arrays. The results showed a marked preference for galactoseβ1-3 terminal oligosaccharides in the case of Epa1A. For the other proteins, varying degrees of promiscuity were noted. Epa1→6A presented a very similar binding profile to the one presented by Zupancic et. al. in 2008 for Epa6A, demonstrating the validity of the method. Epa1→2A and Epa1→3A were much less active, and presented a preference for sulfated glycans, along with terminal galactose. Fluorescence titrations showed for Epa1A a ~20 time stronger affinity for the T antigen (galactoseβ1-3N-acetyl- galactosamine) than for the milk-derived lactose, showing how marked the adhesin preference for β1-3 linkages is, as compared to β1-4 glycosidic bonds. Adhesins of Helicobacter pylori The adhesins of H. pylori have been shown to be critical for the colonization and immune recognition of the bacterium during gastric invasion and disease development[3]. BabA and SabA figure prominently, as the former is the primary adhesin during early stage colonization, while the latter binds strongly to inflamed tissue[4]. Both of them are autotransporters, with a C-terminal, membrane bound translocation unit and an N-terminal passenger domain which contains the adhesive portion of the protein[5]. Pure, soluble passenger domains of BabA and SabA were successfully overproduced by recombinant expression in Escherichia coli. BabA could be functionally characterized by the same method as the Epa proteins. The results showed that BabA activity was strongly pH dependent, with a ~100 times stronger activity at pH 5.8 than at pH 2.5. This behavior could be further characterized through circular dichroism spectroscopy and size exclusion chromatography, which showed that BabA is in a reversible molten globule-like, aggregation-prone and relaxed conformation at pH 2.5. At pH 5.8, on the other hand, the protein is in a much more compact, defined conformation with a strong tendency to precipitate. H. pylori has been shown to present many pH dependent virulence factors, like the urea transporter, but up to now no direct biochemical data had been presented supporting pH dependent conformational changes in its adhesins

Investigation Of Oxidative Stress Response In Yeast And Rat

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2009Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2009Bu projede, oksidatif stresin çeşitli ön koşullamalara bağlı olarak oluşan etkilerini iki model organizma olan sıçan ve maya da incelenmesi amaçlanmıştır. Projenin sıçan tabanlı olan kısmında, erken iskemiyi reperfüzyona bağlı olarak işleyen önemli bir mekanizma olduğunu tanımlamakla birlikte iskemi ve ısı ön koşullamasının global gen ekspresyonu düzeyinde mikroışın analizine bağlı olarak sıçan böbrek dokusundaki değişiklikler incelenmesi amaç edinilmiştir ve bu yüzden çeşitli istatistiksel analizler yapılmıştır. Farklı eksprese olmuş gen grupları ile ileri analizler yapılmıştır. Projenin maya kısmında ise hidrojen peroksit oksidatif strese sebep olan ajan olarak kullanılırken, Saccharomyces cerevisiae ise basit ökaryotik organizma olduğu için kullanılmıştır. Sıçanda kullanılan benzer önkoşullamala koşulları bu çalışmada da kullanılmak amaçlanmış olup evrimsel mühendisliği yöntemiyle elde edilmeye çalışılan oksidatif strese dirençli mutant maya hücreleri üzerinde önkoşullamaların etkisi araştırılmıştır. Bunun yanı sıra, bu mutant hücrelerin çeşitli stres koşullarına çapraz direnci de incelenmiştir.In this project, the effect of oxidative stress along with different preconditioning in two different model organisms, yeast and rat, was investigated. In the rat part of the study, it was about to characterize the early ischemia as a function of reperfusion in rat kidneys and to investigate the effects of ischemia-reperfusion along with ischemic or heat preconditioning on global gene expression profile of rat kidney tissues by cDNA microarray analysis and hence, some statistical analysis were done. Differentially expressed genes were further characterized in silico. In yeast part of the study, hydrogen peroxide was used as oxidative stress causing agent and Saccharomyces cerevisiae was used a simple eukaryotic model organism. The same preconditioning conditions that were used in rat studies were aimed to be used in the yeast model organism to see the effect of preconditioning on obtaining oxidative stress resistant mutant yeast cells via evolutionary engineering methodology. Moreover, the cross resistance of oxidative stress resistant mutant yeast cells to other stress conditions was observed.Yüksek LisansM.Sc