Pseudomonas fluorescens does not appear to regulate the enzymes of de novo pyrimidine biosynthesis at the level of gene expression. Little or no apparent repression of pyr gene expression is observed upon addition of exogenous pyrimidines to the growth medium. The Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) were sized down and cloned into the broad host range plasmid, pKT230. Upon introduction into a P.fluorescenspyrB mutant strain, ATCase showed repression in response to exogenously fed pyrimidine compounds. Thus, it was possible to bring about changes in pyrimidine nucleotide pool levels and in transcriptional regulation of gene expression at the same time.

Cassette systems for creating intergeneric hybrid ATCases were constructed. An MluI restriction enzyme site was introduced at the carbamoylphosphate binding site within the pyrB genes of both Pseudomonas putida and Escherichia coli. Two hybrids, E. coli pyrB polar domain fused with P. putida pyrB equatorial domain and P. putida pyrB polar domain fused with E. coli pyrB equatorial domain, are possible. The intergeneric E. coli-P. putida hybrid pyrB gene was constructed and found to encode an active ATCase which complemented an E. coli Pyr- strain. These hybrids are useful for kinetic and expression studies of ATCase in E. coli.

O objetivo deste trabalho foi avaliar o efeito dos diferentes níveis de nucleotídeos sobre o desempenho de frangos de corte alimentados com probióticos. Para o trabalho, foi utilizado um delineamento inteiramente casualizado com cinco repetições dentro de cada tratamento. Foram utilizados 1050 frangos machos da linhagem Ross 308 totalizando trinta e cinco aves por boxe. As aves foram criadas até 42 dias de idade que receberam as rações experimentais a base de milho e farelo de soja contendo seis diferentes níveis de nucleotídeos (0; 100; 200; 300; 400 e 500 gramas por tonelada de ração). Os diferentes níveis de nucleotídeos foram utilizados na fase inicial (1 a 21 dias de idade) e crescimento (22 a 35 dias de idade). Durante a fase final (36 a 42 dias de idade) foi fornecido rações sem nucleotídeo para todos os tratamentos. Os resultados experimentais demonstraram que houve melhora linear no desempenho dos frangos de corte no período de 1 a 21 dias de idade, indicando que, quanto maior o nível de nucleotídeos na dieta de frangos de corte, maior foi o peso corporal das aves. A conversão alimentar também é melhorou linearmente no período de 1 a 21 dias de idade à medida que aumentou o nível de nucleotídeos na ração. O peso no período de 35 dias de idade, também teve um comportamento linear, semelhante ao período de 1 a 21 dias, indicando que, quanto maior o nível de nucleotídeos na dieta de frangos de corte, maior o desempenho das aves. Quanto aos níveis plasmáticos de ácido úrico, pôde-se observar efeito quadrático no período de 1 a 21 dias de idade, indicando o valor de 231,59 gramas de nucleotídeos por tonelada de ração, como o melhor, em níveis mínimos de ácido úrico, por outro lado, no período de 35 dias de idade, estima-se o nível de 208,99 g de nucleotídeos por tonelada de ração; como o melhor em níveis mínimos de ácido úrico no sangue. No período final (35 a 42 dias de idade) e período total (1 a 42 dias de idade) não foi possível o observar efeito dos contrastes testados em nenhum dos parâmetros avaliados. / The objective of this experiment was to evaluate the influence of different nucleotides levels in the rations of broilers containing probiotics on response of the birds and its influence on the performance. Birds were allocated in a randomized experimental design with five replications of each treatment. It was used 1,050 chicks of a day of age, males, distributed in 30 experimental boxes with 35 birds each. The chickens were reared from 1 to 42 days of age and the diets contained corn and soybean meal with one of six different nucleotídes levels (0; 100; 200; 300; 400 and 500 grams for ton of ration). The different nucleotides levels were used in the initial phase (1 to 21 days of age) and growth (22 to 35 days of age). During the final phase (36 to 42 days of age) it was supplied rations without nucleotides for all of the treatments. The experimental results demonstrated that there was improvement on broilers performance in the period from 1 to 21 days of age, demonstrating proportionality between nucleotides level in the diet of broilers and body weight of the birds. Feed conversion at 21 days of age was directly proportional to nucleotides level in the diet. Body weight at 35 days of age, also had a linear behavior, similar to the period from 1 to 21 days, indicating that, as higher the nucleotides level in the diet of broilers, higher the acting of the birds. Acid plasmatic uric levels, demonstrated quadratic effect at 21 days of age, indicating 231,59 grams of nucleotides for ton of ration, and at 35 days of age, it was considered the level of 208,99 g of nucleotides for ton of ration. In the final period (35 to 42 days of age) and total period (1 to 42 days of age) it was not demonstrated effect of the contrasts tested in none of the appraised parameters.

Thesis advisor: Evan R. Kantrowitz / The enzyme aspartate transcarbamoylase (ATCase) is an enzyme in the pyrimidine nucleotide biosynthetic pathway. It was once an attractive target for anti-proliferation drugs but has since become a teaching model due to kinetic properties such as cooperativity and allostery exhibited by the Escherichia coli form of the enzyme. ATCase from E. coli has been extensively studied over that last 60 years and is the textbook example of allosteric enzymes. Through this past research it is understood that ATCase is allosterically inhibited by CTP, the end product of pyrimidine biosynthesis, and allosterically activated by ATP, the end product of the parallel purine biosynthetic pathway. Part of the work discussed in this dissertation involves further understanding the catalytic properties of ATCase by examining an unregulated trimeric form from Bacillus subtilis, a bacterial ATCase that more closely resembles the mammalian form than E. coli ATCase. Through X-ray crystallography and molecular modeling, the complete catalytic cycle of B. subtilis ATCase was visualized, which provided new insights into the manifestation of properties such as cooperativity and allostery in forms of ATCase that are regulated. Most of the work described in the following chapters involves understanding allostery in E. coli ATCase. The work here progressively builds a new model of allostery through new X-ray structures of ATCase*NTP complexes. Throughout these studies it has been determined that the allosteric site is bigger than previously thought and that metal ions play a significant role in the kinetic response of the enzyme to nucleotide effectors. This work proves that what is known about ATCase regulation is inaccurate and that currently accepted, and taught, models of allostery are wrong. This new model of allostery for E. coli ATCase unifies all old and current data for ATCase regulation, and has clarified many previously unexplainable results. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

A radiação ultravioleta (UV) pode ser absorvida por diferentes moléculas celulares, incluindo o DNA no qual provoca distorções estruturais. As lesões mais comuns induzidas pela radiação UV são o ciclobutano de pirimidina (CPD) e o fotoproduto (6-4)-pirimidina-pirimidona [(6-4)PPs]. Estas lesões podem ser reparadas pela fotorreativação, caracterizada por ter uma única proteína (fotoliase) que remove lesões empregando luz visível (320-500 nm) como fonte de energia. Foram identificados dois tipos de fotoliases que diferem por sua especificidade ao substrato: CPD-fotoliase e (6-4)-fotoliase. Um outro mecanismo de reparo é o reparo por excisão de nucleotídeos (NER), um mecanismo que envolve múltiplos passos e proteínas. Enquanto os efeitos genotóxicos da UVC e UVB já estão relativamente esclarecidos e bem aceitos, ainda existem controvérsias sobre a genotoxicidade da radiação UVA, devido ao fato de ser fracamente absorvida pelo DNA. Alguns autores acreditam que os seus principais efeitos são gerados de forma indireta pela produção de espécies reativas de oxigênio enquanto outros acreditam que a UVA pode gerar danos ao DNA de forma direta, provocando a formação de dímeros de pirimidina. O objetivo deste trabalho foi verificar os efeitos genotóxicos da radiação UVA em fibroblastos humanos deficientes e proficientes em NER utilizando adenovírus recombinantes contendo uma ou outra fotoliase para verificar se as lesões CPD e (6-4)PP são geradas pela UVA e se elas teriam alguma importância nas respostas verificadas após irradiação. Foi verificado que as células deficientes no gene XPA são mais sensíveis à radiação UVA quando comparadas às células selvagens. Por meio da detecção imunológica, confirmamos a geração das lesões CPD, (6-4)PP e Dewar, fotoisômero da lesão (6-4)PP, após irradiação com UVA no genoma de células humanas. Empregando vetores adenovirais para transdução de fotoliase específica para lesões tipo CPD ou (6-4)PP, confirmamos que de fato essas lesões são formadas em células humanas deficientes em reparo de DNA após irradiação com UVA. Além disso, esses vírus permitiram verificar a relevância biológica dessas lesões na indução de morte celular em células XP-A irradiadas. De fato, os dados indicam que para doses baixas de radiação UVA essas lesões desempenham um importante papel na indução de morte. Não podemos descartar, porém, que lesões indiretas (provavelmente geradas por estresse oxidativo) também tenham papel na indução de morte pela radiação UVA, o que parece ser mais importante a doses médias e altas dessa radiação. / Ultraviolet radiation (UV) is absorbed by different cellular molecules, including DNA in which induces structural distortions. The most common lesions induced by UV radiation are the cyclobutane pyrimidine (CPD) and the photoproduct (6-4)-pyrimidine-pyrimidone [(6-4)PP]. These lesions can be repaired by the photoreactivation, characterized by a single protein (photolyase) that removes lesions using visible light (320-500 nm) as energy source. Two types of photolyases had been identified that differ by their substrate specificity: CPD-photolyase and (6-4)-photolyase. Another repair mechanism is the nucleotide excision repair (NER), a mechanism that involves multiple steps and proteins. While the genotoxic effects of UVB and UVC are already relatively well-understood and accepted, there is still controversy about the genotoxicity of UVA radiation, due to its low absorption by DNA. Some authors believe that the major effects are generated indirectly by the production of reactive oxygen species, while others believe that UVA can cause damage to DNA directly, inducing the formation of pyrimidine dimers. The aim of this study was to assess the genotoxic effects of UVA radiation in human fibroblasts deficient and proficient in NER, using recombinant adenovirus expressing the photolyases to verify if CPDs and (6-4)PPs are generated by UVA and whether they had any importance in the responses observed after irradiation. It was found that cells deficient in the XPA gene are more sensitive to UV radiation compared to wild type cells. By immunological detection, we confirm the generation of CPD, (6-4)PP and Dewar, photoisomer of the (6-4)PP lesion, in the genome of human cells after irradiation with UVA. Using adenoviral vectors for the transduction of photolyases specific for CPD or (6-4)PP lesions, we confirm that in fact these lesions are generated in human cells deficient in DNA repair after irradiation with UVA. Moreover, these viruses allowed us to verify the biological relevance of these lesions in the induction of cell death in irradiated XP-A cells. In fact, our data indicates that for low doses of UVA radiation, these lesions play important roles in the induction of death. We cannot rule out, however, that indirect lesions (probably caused by oxidative stress) could also have a role in the induction of death by UVA radiation, which seems to be more important in intermediate and high doses of this radiation.

The development of novel selective 5-HT6 and 5-HT7 receptor antagonists is an ever-growing area of interest among medicinal chemists. The potential of developing a therapeutic agent useful as an antipsychotic or antidepressant, as well as the possibility to develop a drug for Alzheimer’s disease and obesity has led to an increase in synthesis of possible lead compounds. The synthesis of unfused biheteroaryl derivatives is described within. The derivatives have been evaluated for binding affinity at 5-HT2A, 5-HT6 and 5-HT7 receptors. The most potent 5-HT6 receptor antagonists include a benzene ring, a hydrophobic group and a protonated nitrogen atom. The most potent and selective compound synthesized is 1-[3-butyl-5-(thienyl)phenyl]-4-methylpiperazine. The binding site of the 5-HT7 receptor is similar to that of the 5-HT6 receptor and the most selective and potent 5-HT7 receptor antagonist also contains a potonated nitrogen atom and a hydrophobic group. The difference in selectivity between the 5-HT6 and 5-HT7 receptor antagonists is the aromatic ring. The most potent 5-HT7 receptor antagonist synthesized contains a pyridine ring instead of benzene, as in the 5-HT6 receptor antagonist. The most potent and selective 5-HT7 receptor antagonist is 1-[4-(3-furyl)-6-methylpyridin-2-yl]-4-methylpiperazine. The need to increase selectivity for both 5-HT6 and 5-HT7 receptors has led to the synthesis of flexible-chain linked derivatives and the results are described within.

Les complexes de platine ont été abondamment étudiés depuis la description de l'effet trans par le chercheur russe Chernayev. Cependant, les complexes de platine contenant la pyrazine ou la pyrimidine non substituées sont rares. De nouveaux types de complexes cis et trans de platine(II) contenant des ligands mixtes, Pt(Ypy)(pz/pm)CI₂, (où Ypy =dérivé de la pyridine, pz=pyrazine et pm=pyrimidine) ont été synthétisés par la réaction de K[Pt(Ypy)Cl₃] avec (pz/pm) en milieu aqueux dans des proportions 1:1. La même synthèse dans des proportions 2:1 a conduit à des dimères où le ligand pyrazine/pyrimidine forme un pont mettant en jeu ses deux atomes d'azote dans des liaisons de coordination. Seuls les isomères trans,trans-CI₂(Ypy)Pt(µ-pz/pm)Pt(Ypy)Cl₂ ont pu être isolés. Tous les complexes ont été caractérisés par spectroscopies IR et de résonance magnétique multinucléaire (¹⁹⁵Pt, ¹H et ¹³C). La spectroscopie infrarouge (v(Pt-Cl)) ainsi que ks constantes de couplage J(¹⁹⁵Pt-¹H) et J(¹⁹⁵Pt-¹³C) donnent des informations sur la géométrie des complexes. En effet, un signal unique est observé dans la région des élongations v(Pt-CI) pour les complexes trans et deux pour ceux de configuration cis. D'autre part, les constantes de couplage ³J(¹⁹⁵Pt-¹H) sont un peu plus grandes pour les complexes de géométries cis que pour ceux de configuration trans. Les valeurs des déplacements chimiques en RMN du ¹⁹⁵Pt donnent des informations sur la densité électronique autour du platine, celles en RMN ¹H et ¹³C sur celles des ligands. L'influence de la position des groupements méthyle sur le noyau pyridine a été étudiée grâce à ces méthodes spectroscopiques. Les complexes comportant des dérivés de la pyridine substitués en 2 et en 6 montrent des résonances à plus faibles champs en RMN ¹⁹⁵Pt ce qui a été attribué à l'effet de solvant. Les signaux en RMN ¹⁹⁵Pt des monomères cis ont été observés à des champs plus élevés que ceux des analogues trans. Les dimères trans ont été observés à des champs semblables (complexes de la pyrimidine) ou un peu plus faibles (complexes de la pyrazine) que les monomères trans. Ces résultats ne plaident pas en faveur d'une rétrodonation importante Pt→N. Toutefois, les résultats en spectroscopie RMN ¹⁹⁵Pt résultent d'un ensemble de plusieurs effets électroniques, qui sont difficiles à séparer. Si donc une rétrodonation existe, elle serait faible comparativement à celle observée avec des ligands sulfoxyde ou phosphine. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Platine, Pyridine, Pyrazine, Pyrimidine, RMN, Infrarouge, Effet de solvant.

Les complexes disubstitués du Pt(II) avec des ligands azotés sont connus depuis assez longtemps, mais la chimie de coordination du platine est encore peu développée. Il y a plusieurs publications sur la chimie des dérivés de la pyrimidine, à cause de l'importance biologique de ces molécules. Cependant, il y a peu de travaux dans la littérature sur la chimie de coordination de la pyrimidine non substituée. Quelques nouvelles méthodes ont été développées dans ce projet pour la préparation de complexes contenant des ligands mixtes, amine et pyrimidine. Plusieurs amines aliphatiques et cycliques possédant un encombrement stérique différent ont été choisies pour l'étude. Les nouveaux complexes de types cis- et trans- Pt(amine)(pyrimidine)X₂ (X = Cl et I) et des dimères à pont pyrimidine de type trans, trans- X₂(amine)Pt(µ-pm)Pt(amine)X₂ ont été synthétisés et caractérisés par différentes techniques spectroscopiques. Les composés diiodo ont été synthétisés via l'intermédiaire du dimère à ponts iodo, I(amine)Pt(µ-I)₂Pt(amine)I et peut être utilisée pour la plupart des amines. La formation du dimère est très longue, car le produit initial Pt(amine)₂I₂ est insoluble dans l'eau, tout comme le dimère. La réaction entre le dimère à ponts iodo et la pyrimidine en milieux aqueux dans les proportions 1 : 2 donne des composés de type Pt(amine)(pm)I₂ d'isomérie cis. Si les ligands sont encombrés, il y aura une isomérisation cis-trans. La deuxième méthode (X = Cl) implique la formation de l'intermédiaire K[Pt(amine)CI₃], qui a été préparé par 2 méthodes différentes, dont une est limitée à des amines encombrées. Le composé ionique réagit avec la pyrimidine dans les proportions 1 : 2 pour produire Pt(amine)(pm)CI₂. Le premier produit formé est l'isomère cis, mais il peut y avoir isomérisation si l'amine est encombrée. La même réaction dans des proportions 2 : 1 a conduit à des dimères à pont pyrimidine Cl₂(amine)Pt(µpm)Pt(amine)Cl₂ de géométrie trans,trans. Les complexes ont été caractérisés à l'état solide par spectroscopie infrarouge et en solution dans l'acétone par résonance magnétique multinucléaire (¹H, ¹³C et ¹⁹⁵Pt). Ces deux techniques ont confirmé la géométrie des complexes. Les couplages J(¹⁹⁵Pt-¹H) et J(¹⁹⁵Pt-¹³C) sont plus grands pour les géométries cis que pour les isomères trans. Les constantes de couplage avec les protons pyrimidiniques ³J(¹⁹⁵Pt-H₂,₆) ont des valeurs moyennes de 25 (H₂) et 39 Hz (H₆) pour les isomères cis et 22 et 34 Hz pour les analogues trans. Ces derniers apparaissent plus blindés par rapport aux isomères cis en RMN du ¹H. Une relation linéaire a été observée entre le déplacement chimique du groupement NH₂ et l'affinité protonique des amines pour les complexes trans-Pt(RNH₂)(pm)I₂. L'augmentation de l'affinité protonique conduit à un déblindage des δ(NH₂) en RMN du ¹H et conduit aussi à un déblindage des signaux C1 en RMN du ¹³C. Les signaux en RMN du ¹⁹⁵Pt sont très semblables pour les deux isomères Pt(amine)(pm)I₂. Les signaux des dimères dichloro d'isomérie trans-trans ont été observés à des champs proches de ceux des monomères trans analogues. La spectroscopie IR a confirmé la géométrie des composés dichloro. Deux bandes v(Pt-Cl) ont été observées pour les complexes cis et une seule bande pour les composés trans. Dans la série iodo, le dimère à pont pyrimidine a été obtenu seulement avec la n-butylamine. Le dimère possède aussi une isomérie trans-trans. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Complexes de platine, Pyrimidine, Amine, RMN, Infrarouge.

The synthesis of 2,4-disubstituted pyrimidine derivatives is described. The synthetic route involved the addition reaction of lithiated intermediates, mostly heterocycles, to position 4 of 2-chloropyrimidine to give a dihydropyrimidine intermediate which was oxidized back to a pyrimidine. This was followed by nucleophilic aromatic substitution with various amines of the chlorine in the position 2. A number of compounds were prepared which showed binding towards various serotonin receptors in preliminary biological evaluation.

This thesis consists of seven research publications concerning (i) pKa studies of nucleobases in model nucleotides to understand why RNA duplexes are more stable than DNA duplexes (Paper I), (ii) the role of Me(T)-π interactions in the relative stability of DNA-RNA heteroduplexes (Paper II), (iii) pKa measurements in nucleotides with different 2′-substituents (paper III), (iv) a conformation study of constrained sugars and a pKa study of 1-thyminyl to reveal effect of sugar constraints on the pKa of the nucleobase (paper IV), (v) NMR and MD studies of 1′, 2′-oxetane constrained thymidine incorporated Dickerson Drew dodecamer (paper V), (vi) the sequence dependent pKa perturbation of 9-guaninyl moeity in single stranded (ss) DNA and RNA (paper VI), (vii) the non identical chemical nature of internucleotidic phosphates in (ss) RNA using 31P NMR (paper VI), and an alkaline hydrolysis study of phosphodiesters in ssRNAs (paper VII). The architecture of DNA and RNA molecules is determined by (a) hydrogen bonding (b) base stacking (c) a variety of additional non-covalent interactions. In paper (I) we showed that A-U and G-C base pairings in RNA are more stable than A-T and G-C base pairings in DNA by 4.3 and 1 kJ mol-1 respectively. Me(T)-π interaction plays a dominant role in the relative stability of DNA-RNA duplexes (paper II). In paper III and IV, we have shown that 1′ , 2′- oxetane and azetidine rings have strong inductive effect on pyrimidine bases, and that the H2′-sugar proton can be the marker to understand the pseudoaromaticity of pyrimidine bases, as well as increasing constraints in sugar reducing the basicity of nucleobases. A 1′, 2′-oxetane locked thymidine (T) moiety deforms the local structure of Dickerson-Drew dodecamer, d(CGCGAATTCGCG)2- investigated by High resolution NMR and MD study, as is discussed in the paper V. In papers VI and VII, we showed sequence context dependent pKa (N1) of 9-guaninyl perturbation in (ss) DNAs and RNAs and the non identical chemical nature of inter-nucleotidic phosphate groups in single stranded RNAs.