Malaria is a treatable communicable disease yet remains a common cause of death and disease especially among pregnant women and children. Most of malaria's worldwide burden disproportionately lies in Southeast Asia and Sub-Saharan Africa. Western medicine's 100+ year history of combating Plasmodium falciparum has taught us that the global population of malaria parasites has a unique and dangerous ability to rapidly evolve and spread drug resistance. Recently it was documented that resistance to the first-line antimalarial artemisinin may be developing in Southeast Asia.

Στην παρούσα εργασία, εξετάσαμε λεπτομερώς την κινητική της ενεργοποίησης της δραστικότητας της RNase P του E. coli από τη σπιραμυκίνη. Τα αποτελέσματα δείχνουν ότι η σπιραμυκίνη δρα σαν μικτού τύπου «μη απαραίτητος» ενεργοποιητής . Η κινητική συμπεριφορά του ενεργοποιητή που εξετάστηκε, μπορεί να εξηγηθεί μ’ ένα κινητικό σχήμα ανάλογο αυτού που περιγράφεται στο Segel (1993). Σε κορεσμένη συγκέντρωση σπιραμυκίνης η τιμή της φαινομενικής Vmax (Vmax,app) για το ανασχηματισμένο ολοένζυμο αυξάνεται κατά 2,5 φορές και τιμή της φαινομενικής K(Ks s,app) μειώνεται κατά 7,1 φορές. Ομοίως, σε κορεσμένη συγκέντρωση σπιραμυκίνης, η τιμή της φαινομενικής Vmax για το M1 RNA αυξάνεται κατά 2,4 φορές και η τιμή της φαινομενικής K μειώνεται κατά 5 φορές. / -

No description available.

No description available.

During protein synthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of mRNA-programmed ribosomes in a GTP-dependent manner. To enable future studies on the functional and structural requirement of EF-Tu’s function, a Cysteine-free variant of EF-Tu was constructed suitable for subsequent labelling of the protein and use in kinetic studies. Here, the kinetic properties of three Cysteine-less EF-Tu variants are reported, demonstrating that only the variant with the Alanine substitution in position 81 retains wild-type activity with respect to the interaction with guanine nucleotides, aa-tRNA and the ribosome. To explore a possible tRNA independent pathway for the GTPase activation signal, three residues in domain II of EF-Tu (Arginine 279, Glycine 280, Arginine 283) were mutated; the activity of EF-Tu variants were analyzed. Results suggest that these residues are indeed required for efficient ribosome-dependent stimulation of the GTPase activity of EF-Tu. / x, 85 leaves : ill. (some col.) ; 29 cm

Bacteriophages (hereafter referred as phages) can translate their mRNAs efficiently by maximizing the use of codons decoded by the most abundant tRNAs of their bacterial hosts. Translation efficiency directly influences phage fitness and evolution. Reengineered phages find application in controlling their host population in both health and industry. The objective of this thesis work is to examine the factors shaping codon choices of single stranded DNA (ssDNA) and double stranded DNA (dsDNA) Escherichia coli phages. In chapter two, we employed two indices, rRSCU (correlation in relative synonymous codon usage between phages and their hosts) and CAI (codon adaptation index) to measure codon adaptation in phages. None of the analyzed ssDNA phages encode tRNAs while some dsDNA phages encode their own tRNAs. Both rRSCU and CAI are negatively correlated with number of tRNA genes encoded by these dsDNA phages. We observed significantly greater rRSCU for dsDNA phages (without tRNAs) than ssDNA phages. In addition, we propose that ssDNA phages have evolved a novel codon adaptation strategy to overcome the disruptive effect of their high C→T mutation rates in codon adaptation with host. In chapter three, we formulated an index phi to measure selection by host translation machinery and to present explicit linear and nonlinear models to characterize the effect of C→T mutation and host-tRNA-mediated selection on phage codon usage. The effect of selection (phi) on codon usage is detectable in most dsDNA and ssDNA phage species. C→T mutations also interfere with nonsynonymous substitutions at second codon positions, especially in ssDNA phages. Strand asymmetry along with the accompanying local variation in mutation bias can significantly affect codon adaptation in both dsDNA and ssDNA phages.

This thesis describes the use of functional genomics screens in yeast to study anticancer drug action and intracellular transport. The yeast Saccharomyces cerevisiae provides a particularly useful model system for global drug screens, due to the availability of knockout mutants for all yeast genes. A complete collection of yeast deletion mutants was screened for sensitivity to monensin, a drug that affects intracellular transport. A total of 63 deletion mutants were recovered, and most of them were in genes involved in transport beyond the Golgi. Surprisingly, none of the V-ATPase subunits were identified. Further analysis showed that a V-ATPase mutant interacts synthetically with many of the monensin-sensitive mutants. This suggests that monensin may act by interfering with the maintenance of an acidic pH in the late secretory pathway. The second part of the thesis concerns identification of the underlying causes for susceptibility and resistance to the anticancer drug 5-fluorouracil (5-FU). In a functional genomics screen for 5-FU sensitivity, 138 mutants were identified. Mutants affecting tRNA modifications were particularly sensitive to 5-FU. The cytotoxic effect of 5-FU is strongly enhanced in these mutants at higher temperature, which suggests that tRNAs are destabilized in the presence of 5-FU. Consistent with this, higher temperatures also potentiate the effect of 5-FU on wild type yeast cells. In a plasmid screen, five genes were found to confer resistance to 5-FU when overexpressed. Two of these genes, CPA1 and CPA2 encode the two subunits of the arginine-specific carbamoyl-phosphate synthase. The three other genes, HMS1, YAE1 and YJL055W are partially dependent on CPA1 and CPA2 for their effects on 5-FU resistance. The specific incorporation of [14C]5-FU into tRNA is diminished in all overexpressor strains, which suggest that they may affect the pyrimidine biosynthetic pathway.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 119-137).

The information flow through the regulatory networks in biological systems has been a rapidly growing field of research. Translation, being a very important regulatory check point, presents itself as a legitimate process for investigation. Only few regulatory factors and pathways are currently delineated that regulate translation through intermediary components in a remote manner, with global implications. In this context, this thesis studies the proteomics and transcriptomics data of Escherichia coli (E. coli) mutants, defective in translation, with the aim to unravel such regulatory factors or pathways and thus probe their regulatory aspects. Two main mics techniques are the backbone of this study; proteomics and transcriptomics. These provide a holistic view of cell states which allow us to investigate the regulation happening at the translation as well as at the transcription level. Two different proteomics techniques are used to resolve the proteomes; two-dimensional gel electrophoresis (2DE) and LC-coupled mass spectrometry. These have been introduced in the first chapter. Transcriptomics and proteomics being an evolving field, most of the techniques need optimization before applied for actual experiments and data acquisition. As part of our experimental strategy, we performed both transcriptomics and proteomics experiments in parallel. During application of 2DE based proteomics, we observed significant deficiencies in the 2DE technique itself, which we addressed as our first priority. We ran numerous optimization protocols to arrive at an optimized protocol to remove acidic region streaking (ARS) in 2DE, which is a well-known artifact. We describe the development of the modified protocol and discuss the detailed comparative analyses with recently published 2DE gels confirming the efficacy of the method in Chapter 2. The optimized 2DE technique developed by us was exploited in combination with MALDI mass spectrometry for the comparative proteomic analysis between the wild type E. coli and a mutated (ΔmetZWV::kan) strain. The proteomics results and its functional validation revealed a direct link between the flux of 10-formyltetrahydrofolate and the regulation of purine metabolism. The experimental observations were computationally modelled using flux balance analysis to understand the mechanistic detail involved in the remote regulation driven by purine metabolism and other peripheral pathways. The experimental details and the computational modelling are covered in chapter three. To gather wider perspective on the regulatory links in the E. coli organism, related to translation, we extended the omics studies using microarray technique on newer mutant strains. Our experiments aimed at obtaining differential transcript levels in the whole cell and the polysomal fraction of the E. coli cells. Three different E. coli mutants were used in this study; infC135, PthTs and folD122, which were defective in translation initiation, recycling and one carbon metabolism, respectively. The analysis revealed important routes of metabolic regulation. Few of them are worth mentioning; for example, purine and 10-fTHF metabolism that controls macromolecular synthesis, energy generation and inter-conversion of metabolites through pyruvate and also flagellar biosynthesis which is remote to translation. Transcriptomics data available from GEO database was analyzed as a background and based on the analysis we propose which of the differentially expressed genes are of generic in nature or unique to our mutants. These interesting observations about regulatory pathways are discussed in chapter four. To validate our transcriptomics results at the proteomics level and with a higher sensitivity than 2DE proteomics, we studied the whole cell proteomics data from two E. coli mutants, infC135 and PthTs, using high resolution FT-ICR mass spectrometry. Although a small number of differentially expressed proteins compared to microarray data, we could correlate the results with our transcriptomics data, especially, the proteins in the catabolic pathways. We elaborate the aforesaid study in chapter five. At the end we summarize the above omics studies to notice the following aspects emerging out. Translation, being a fundamental and essential process for the cell, disturbing it from any angle should affect many other processes which might seem remotely or not at all related to protein synthesis. This is evident from the whole study; we have been able to see some regulations which are very close to translation, but most are not directly related to translation. Apart from this we were able to point out routes of regulation which might control the amount of macromolecules synthesized, utilization of energy and metabolites and flagellar biogenesis. Another aspect is that we were able point out the gap in information between our regulation of pathways close to and remote to protein biosynthesis. Lastly, few master regulators were pointed out which might have potential functions in addition to what is known till date. A concluding discussion about these aspects has been discussed in the sixth chapter.

Les approches bioinformatiques développées au cours de cette thèse ont permis d’une part le développement de banques de données concernant les ARNt classiques ainsi que les ARNt mitochondriaux de métazoaires. Celles-ci sont basées sur de nouveaux outils pour la détection de gènes d’ARNt «bizarres» et des alignements de séquences basés sur les propriétés structurales préservées. Les analyses des séquences collectées ont conduit non seulement à une vision globale de la diversité des ARNt dans les génomes mitochondriaux couvrant l’ensemble des groupes taxonomiques des métazoaires, mais également une meilleure connaissance de l’organisation des génomes et d’en proposer des liens évolutifs. Elles ont également permis de confirmer et d’élargir l’existence d’ARNt les plus petits connus à ce jour et de poser les bases de compréhension des repliements tridimensionnaux des ARNt mitochondriaux. Ces travaux permettent de mieux appréhender la compréhension des relations structure/fonction des ARNt mitochondriaux humains, et en particulier les dysfonctionnements dans les pathologies mitochondriales. / The bioinformatic approaches presented in this thesis include the development of databases for classical tRNAs and the mitochondrial tRNAs of metazoans. They are based on new tools for the detection of "bizarre" tRNA genes and sequences, and for the calculation of alignments based on their structural features. The analysis of collected sequences have led to an global overview on the diversity of tRNAs in mitochondrial genomes covering all taxonomic groups of metazoans, but also to a better understanding of genome organization and their evolution. The present study revealed the existence of the smallest known tRNA so far and provides the basis for understanding the three-dimensional folding of mitochondrial tRNA. This work helps to better understand the structure/function relationships of human mitochondrial tRNAs and, in particular, the dysfunctions in mitochondrial pathologies.