This thesis is a comparative study of gene arrangements in Pseudomonas species, and is organized into three major sections. The first section compares gene arrangements for different pathways in Pseudomonas aeruginosa PAO1 to determine if the gene arrangements are similar to previous studies. It also serves as a reference for pyrimidine gene arrangements in P. aeruginosa. The second part compares the physical, and genetic maps of P. aeruginosa PAO1 with the genome sequence. The final section compares pyrimidine gene arrangements in three species of Pseudomonas. Pyrimidine biosynthesis and salvage genes will be aligned for P. aeruginosa PAO1, P. putida KT2440, and P. syringae DC3000. The whole study will gives insight into gene patterns in Pseudomonas, with a focus on pyrimidine genes.

The objective of this research was to study the pyrimidine salvage pathways of Rhizobium. Three approaches were used to define the pyrimidine salvage pathways operative in two species of Rhizobium, R. meliloti and R. leguminosarum . The first approach was to ascertain the pyrimidine bases and nucleosides that could satisfy the pyrimidine requirement of pyrimidine auxotrophs. Uracil, cytosine, uridine or cytidine all satisfied the absolute pyrimidine requirement. The second approach was to select for mutants resistant to 5-fluoropyrimidine analogues which block known steps in the interconversion of the pyrimidine bases and nucleosides. Mutants resistant to 5-fluorouracil lacked the enzyme uracil phosphoribosyltransferase (upp ) and could no longer use uracil to satisfy their pyrimidine requirement. Mutants resistant to 5-fluorocytosine, while remaining sensitive to 5- fluorouracil, lacked cytosine deaminase (cod) and thus could no longer use cytosine to satisfy their pyrimidine auxotrophy. The third approach used a reversed phase HPLC column to identify the products that accumulated when cytidine, uridine or cytosine was incubated with cell extracts of wild type and analogue resistant mutants of Rhizobium. When cytidine was incubated with cell extracts of Rhizobium wild type, uridine, uracil and cytosine were produced. This Indicated that Rhizobium had an active cytidine deaminase (cdd) and either uridine phosphorylase or uridine hydrolase. By dialyzing the extract and reincubating it with cytidine, uridine and uracil still appeared. This proved that it was a hydrolase ( nuh ) rather than a phosphorylase that degraded the nucleoside. Thus, Rhizobium was found to contain an active cytidine deaminase and cytosine deaminase with no uridine phosphorylase present. The nucleoside hydrolase was active with cytidine, uridine and to a far lesser extent with purines, adenosine and inosine. When high concentrations of cytidine were added to mutants devoid of hydrolase, cytosine was produced from cytidine - 5-monophosphate by the sequential action of uridine ( cytidine ) kinase and nucleoside monophosphate glycosylase. Both ft meliloti and ft leguminosarum had identical salvage pathways.

Four main series of novel heterocyclic compounds were successfully syniliesised. Two of these series were found to be post-emergence herbicides with the activities of each being based on a different mode of action. The (pyrazole-4-yl)alkanones are inhibitors of protoporphyrinogen oxidase, an enzyme in chlorophyll biosynthesis, whereas alkyl 3-arylsulfonylamino- 3-methyllhio-2-(pyrimidin-2-ylcarbamoyl)acrylates and pyrimidin-2-yl 3-(2- chlorophenyl)sulfonyl-amino-3-methylthio-2-cyanoacrylamides (collectively termed "vinylogous sulfonylureas") are inhibitors of acetohydroxy acid synthase (AHAS). an enzyme in branched-chain amino acid biosynthesis. Both these enzymes are established targets for current commercial herbicides. Studies of the utility of 2-(l-ethoxyalkylidene)-3-oxoaIkanenitriles (acrylonilriles) in heterocycle synthesis were facilitated by the recent development of a convenient route to these starting materials. Acrytonitriles were reacted with different hydrazines to give (pyrazol-4-yl)alkanones and pyrazole-4-carbonitriles in varying proportions depending on the reaction conditions and the substituents on the reactants. Although distinction between alternative 3- and 5-substituted pyrazoles is a perennial problem in pyrazole synthesis, in this case the products of these reactions were successfully characterised and identified using a range of n.m.r. spectroscopy techniques. Once the herbicidal mode of action of the (pyrazol-4-yl)alkanones had been confirmed, synthesis of a series of analogues allowed the structural elements contributing to biological activity to be identified. The reaction of acrylonitriles with bidetate nucleophiles such as thiourea gave novel pyrimidines. but these compounds were not herbicidal. The vinylogous sulfonylureas were synthesised using established procedures to obtain novel compounds structurally related to the commercial herbicide chlorsulfuron. The biological activity of the vinylogous sulfonylureas was found to be sensitive to apparently minor changes in structure, but x-ray crystallographically-generated structures of an active and an inactive member of the series revealed marked differences in conformation. Some of the vinylogous sulfonylureas were used as synthons for pyrazole and pyrazolopyrimidine derivatives. Although these compounds did not exhibit herbicidal activity, this synthesis provided the basis for some interesting chemistry. Unexpected elimination of the arylsulfonylamino group was observed when a vinylogous sulfonyurea was treated with methyl hydrazine. In order to confirm the identity of the 3-methylthiopyrazole product, model compounds were synthesised using alternative routes. The resulting pairs of 3- and 5-substituted pyrazoles were characterised using n.m.r spectroscopy.

Parmi les diverses méthodes de synthèse décrites dans la littérature, la réaction des o-aminoesters avec orthoesters, suivie de cyclisation par une amine aliphatique ou aromatique constitue une voie efficace pour la formation de pyrimidines condensées à d'autres hétérocycles. Cette séquence réactionnelle a été appliquée pour l'obtention des isoxazolo[5,4-d]pyrimidiones et des triazolo[4,5-d]pyrimidinones. Cependant, dans certains cas, lorsque l'imidate intermédiaire porte un groupement méthyle, le processus de cyclisation peut être totalement inhibé. Une séquence alternative consiste en la condensation de l'orthoester avec un o-aminoamide. De nombreuses isoxazolo- et triazolopyrimidinones non décrites ont été formées selon ces deux méthodes qui permettent d'introduire simulténaément des substituants en position 5 et 6 sur le cycle pyrimidinone. La première méthode, réalisée au départ d'o-aminonitriles conduit à des iminopyridines qui sont transformées en aminopyrimidines par réarrangement de Dimroth. Une étude en spectrométrie de masse et en pyrolyse-éclair sous vide a révélé que les dérivés de l'isoxazole sont d'excellents précurseurs de nouveaux hétérocumulènes RN=C=C=C=X. Ces cumulènes sont classés en trois catégories selon que X est en O, NH ou NR'. - Formation des iminopropadiénones, RN=C=C=C=O. - Formation des diiminopropadiènes, RN=C=C=C=NH. - Formation des diiminopropadiènes, RN=C=C=C=NR'. Nous nous sommes également intéressés à la spectrométrie de masse et à la pyrolyse de quelques triazolopyrimidines. En impact électronique, les 3-phényltriazolopyrimidines perdent de l'azote pour générer une structure alpha-cétocétèninime ou alpha-iminocétène tandis que la pyrolyse conduit au système pyrimidoindole.

This manuscript presents a thorough investigation and description of metabolic control dynamics in vivo and in silico using as a model de novo pyrimidine biosynthesis. Metabolic networks have been studied intensely for decades, helping develop a detailed understanding of the way cells carry out their biosynthetic and catabolic functions. Biochemical reactions have been defined, pathway structures have been proposed, networks of genetic control have been examined, and mechanisms of enzymatic activity and regulation have been elucidated. In parallel with these types of traditional biochemical analysis, there has been increasing interest in engineering cellular metabolism for commercial and medical applications. Several different mathematical approaches have been developed to model biochemical pathways by combining stoichiometric and/or kinetic information with probabilistic analysis, or deciphering the comparative logic of metabolic networks using genomic-derived data. However, most of the research performed to date has relied on theoretical analyses and non-dynamic physiological states. The studies described in this dissertation provide a unique effort toward combining mathematical analysis with dynamic transition experimental data. Most importantly these studies emphasize the significance of providing a quantitative framework for understanding metabolic control. The pathway of de novo biosynthesis of pyrimidines in Escherichia coli provides an ideal model for the study of metabolic control, as there is extensive documentation available on each gene and enzyme involved as well as on their corresponding mechanisms of regulation. Biochemical flux through the pathway was analyzed under dynamic conditions using middle-exponential growth and steady state cultures. The fluctuations of the biochemical pathway intermediates and end products transitions were quantified in response to physiological perturbation. Different growth rates allowed the comparison of rapid versus long-term equilibrium shifts in metabolic adaptation. Finally, monitoring enzymatic activity levels during metabolic transitions provided insight into the interaction of genetic and biochemical mechanisms of regulation. Thus, it was possible to construct a robust mathematical model that faithfully represented, with a remarkable predictability, the nature of the metabolic response to specific environmental perturbations. These studies constitute a significant contribution to the fields of quantitative biochemistry and metabolic control, which can be extended to other cellular processes as well as different organisms.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--Illinois State University, 1978. / Title from title page screen, viewed Jan. 24, 2005. Dissertation Committee: Fritz Schwalm (chair), Herman Brockman, Harry Huizinga, Mathew Nadakavukaren, Arlan Richardson. Includes bibliographical references (leaves 96-105) and abstract. Also available in print.

Thesis (M.S.)--The University of North Carolina at Greensboro, 2009. / Directed by Lakshmi P. Kotra; submitted to the Dept. of Chemistry and Biochemistry. Title from PDF t.p. (viewed May 17, 2010). Includes bibliographical references (p. 41-43).

FGFR3 est un récepteur cellulaire impliqué dans de nombreux processus biologiques chez l’homme. La dérégulation de l’activité tyrosine kinase de FGFR3 est à l’origine d’anomalies de croissance osseuse et de certains cancers (myélome multiple, vessie). Le blocage de ce récepteur par des inhibiteurs compétitifs de l’ATP constitue une approche thérapeutique intéressante. La première partie de la thèse a concerné la préparation d’analogues du PD173074, inhibiteur nanomolaire de FGFR3, issu de l’industrie. Basée sur un squelette pyrido[2,3-d]pyrimidine, une chimiothèque de triazoles a été obtenue via une réaction de cycloaddition 1,3-dipolaire catalysée au cuivre (I). Parmi les 27 analogues synthétisés, le meilleur inhibiteur restaure ex vivo la croissance osseuse de fémurs de souris naines. Dans une seconde partie, nous avons développé deux nouvelles séries d’inhibiteurs de type pyrazolooxadiazole et pyrazolo[4,3-d]pyrimidine, à partir d’un précurseur pyrazole commun. Des procédures micro-ondes rapides et efficaces ont été utilisées pour conduire aux composés cibles. L’étude des relations structure-activité, réalisée sur une soixantaine de composés, a été validée par modélisation moléculaire. Le composé le plus actif présente une CI50 submicromolaire / FGFR3 is a cellular receptor involved in many human biological processes. Deregulated tyrosine kinase activity of the FGFR3 is at the origin of abnormal bone growth and some cancers (multiple myeloma, bladder). Blocking this receptor by ATP competitive inhibitors may constitute an interestingtherapeutic approach. The first part of this thesis concerned the preparation of analogues of PD173074, a nanomolar inhibitor of FGFR3, stemming from the industry. Based on pyrido [2,3-d] pyrimidine skeleton, a library of triazoles was obtained via copper (I)-catalyzed 1,3-dipolar cycloaddition. Among the 27 analogues synthesized, the best inhibitor restores bone growth of dwarf mice femurs ex vivo. In a second part, we have developed two new series of inhibitors based on pyrazolooxadiazole and pyrazolo [4,3-d] pyrimidine skeletons, derived from a common pyrazole precursor. Rapid and efficient microwave procedures were used to yield the target compounds. The study of structure-activity relationships, performed on sixty compounds, was confirmed by molecular modeling. The most active compound has a submicromolar IC50 value.

Pyrimidine salvage pathways are essential to all cells. They provide a balance of RNA synthesis with the biosynthetic pathway in pyrimidine prototrophs and supply all the pyrimidine requirements in auxotrophs. While the pyrimidine biosynthetic pathway is found in almost all organisms and is nearly identical throughout nature, the salvage pathway often differs from species to species, with aspects of salvage seen in every organism. Thus significant taxonomic value may be ascribed to the salvage pathway. The pyrimidine salvage pathways were studied in 55 microorganisms. Nine different salvage motifs, grouped I-IX, were identified in this study based on the presence of different combinations of the following enzymes: cytidine deaminase (Cdd), cytosine deaminase (Cod), uridine phosphorylase (Udp), uracil phosphoribosyltransferase (Upp), uridine hydrolase (Udh), nucleoside hydrolase (Nuh), uridine/cytidine kinase (Udk), 5'-nucleotidase and CMP kinase (Cmk).