Cette thèse vise à développer une approche basée sur le concept de Graphe Condensé de Réaction (GCR) capable de (i) sélectionner un espace optimal de descripteurs séparant au mieux différentes classes de réactions, et (ii) de préparer de nouveaux descripteurs pour la modélisation « structure–réactivité ». Cette méthodologie a été appliquée à la recherche par similarité dans une base de données contenant 8 classes de réaction différentes; et à la cartographie de son espace chimique en utilisant des cartes de Kohonen et de cartes topographiques génératives. La seconde partie de la thèse porte sur le développement de modèles prédictifs pour le pKa et pour des conditions optimales pour différents types de réaction de Michael impliquant à la fois les descripteurs d’effet électronique et des descripteurs calculés sur les GCR. / This thesis aims to develop an approach based on the Condensed Graph of Reaction (CGR) method able to (i) select an optimal descriptor space the best separating different reaction classes, and (ii) to prepare special descriptors to be used in obtaining predictive structure-reactivity models. This methodology has been applied to similarity search studies in a database containing 8 different reaction classes, and to visualization of its chemical space using Kohonen maps and Generative Topographic Mapping. Another part of the thesis concerns development of predictive models for pKa and for optimal conditions for different types of Michael reaction involving both CGR-based and Electronic Effect Descriptors.

The present work describes new aspects of organic and supramolecular chemistry. The scientific contribution consists of two parts, which focus on the development of receptors for the sulfate anion and quantitative assessment of stacking interactions between an anthracene dye and nucleobases in an aqueous solution. In Chapter 1, basic concepts concerning supramolecular chemistry and recognition of cations and anions are discussed, as well as modern methods for the determination of binding constants. Particular attention is paid to fluorescence sensing of ions and underlying mechanisms of binding-induced fluorescence responses. Chapter 2 is dedicated to the design and synthesis of new fluorescent sulfate receptors functioning in aqueous solution. After a short review of the most effective sulfate receptors/probes created so far, a new design of PET probes for sulfate sensing is presented. The syntheses and anion binding properties of new compounds are described. The experimental data obtained for the receptors are discussed in detail to reveal the origin of high selectivity towards sulfate. Chapter 3 explores the importance of nucleobase–arene stacking interactions in recognition of nucleotides by synthetic receptors. Various experimental and theoretical approaches are presented to assess dispersion interactions between aromatic rings and nucleobases in the receptor–nucleotide complexes.

cAMP and Ca2+ are key regulators of exocytosis in many cells, including insulin-secreting pancreatic β-cells. Glucose-stimulated insulin secretion from β-cells is pulsatile and driven by oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i), but little is known about the kinetics of cAMP signaling and the mechanisms of cAMP action. Evanescent wave microscopy and fluorescent translocation biosensors were used to monitor plasma membrane-related signaling events in single MIN6-cells and primary mouse β-cells. Glucose stimulation of insulin secretion resulted in pronounced oscillations of the membrane phospholipid PIP3 caused by autocrine activation of insulin receptors. Glucose also triggered oscillations of the sub-plasma membrane cAMP concentration ([cAMP]pm). These oscillations were preceded and enhanced by elevations of [Ca2+]i, but conditions raising cytoplasmic ATP triggered [cAMP]pm elevations without accompanying changes in [Ca2+]i. The [cAMP]pm oscillations were also synchronized with PIP3 oscillations and both signals were suppressed after inhibition of adenylyl cyclases. Protein kinase A (PKA) was important for promoting concomitant initial elevations of [cAMP]pm and [Ca2+]i, and PKA inhibitors diminished the PIP3 response when applied before glucose stimulation, but did not affect already manifested PIP3 oscillations. The glucose-induced PIP3 oscillations were markedly suppressed in cells treated with siRNA against the cAMP-dependent guanine nucleotide exchange factor Epac2. Pharmacological activation of Epac restored PIP3 responses after adenylyl cyclase or PKA inhibition. Glucose and other cAMP-elevating stimuli induced redistribution of fluorescence-tagged Epac2 from the cytoplasm to the plasma membrane. This translocation was modulated by [Ca2+]i and depended on intact cyclic nucleotide-binding and Ras-association domains. In conclusion, glucose generates cAMP oscillations in β-cells via a concerted action of Ca2+ and metabolically generated ATP. The oscillations are important for the magnitude and kinetics of insulin secretion. While both protein kinase A and Epac is required for initiation of insulin secretion the cAMP-dependence of established pulsatility is mediated by Epac2.

This thesis concerns studies of aqueous soft matter systems, especially surfactant micelle systems. The aim has been to study the molecular arrangement and electronic structure of the constituents of, as well as transport properties in such a system. The molecular arrangement and electronic structure has been studied by means of X-ray absorption spectroscopy (XAS) and resonant inelastic X-ray spectroscopy (RIXS). The transport properties have been investigated by low-frequency dielectric spectroscopy (LFDS) and small angle X-ray scattering (SAXS) as well as a theoretical modelling. The latter was based on Fick’s laws of the release from binary surfactant system and was validated by experiments. The RIXS and XAS measurements show the electronic structure in bulk water and the influence of the chemical surrounding of the water molecule in bulk water and of the water molecules confined in a micelle lattice. The spectra are highly dependent on the molecular arrangement in such systems. For glycine and sodium polyacrylate RIXS and XAS spectra show features which are unique for carboxyl and carboxylate groups and such measurements can thus be used for fingerprinting. The LFDS and SAXS measurements show a strong correlation between structure in a surfactant/poly-ion system and apparent mobility of surfactants. This conclusion is in line with earlier observations. By the theoretical modelling a predictive model for the surfactant release from a binary surfactant micelle system has been obtained and the importance of different factors for surfactant release has been further clarified.

Saccharomyces cerevisiae are unicellular organisms that are highly adaptable to acute changes in nutrient availability. The two main signaling pathways that allow S. cerevisiae to sense and respond to changes in glucose availability in the environment are the conserved cAMP/PKA and AMPK/Snf1 kinase-dependent pathways. The conserved TORC1 pathway is primarily responsible for allowing cells to respond to the availability of nitrogen. Studies have shown that S. cerevisiae, but not mammalian and plant cells, regulate nuclear tRNA trafficking in response to nutrient stress. Here, we show that the yeast species of the Saccharomyces genus, but not Schizosaccharomyces pombe and Kluyveromyces lactis specifically regulate nuclear tRNA export in response to nutrient stress, providing further evidence that regulation of nuclear tRNA export in response to nutrient availability is not evolutionarily conserved. We also established that amino acid and nitrogen starvation affects nuclear export of a subset of tRNAs in S. cerevisiae. Inhibition of TORC1 signaling by rapamycin treatment, which simulates nitrogen starvation, also affects nuclear export of the same subset of tRNAs, suggesting that the TORC1 signaling pathway plays a role in regulating nuclear export of the tRNAs in response to nitrogen level. Regulation of nuclear export of these tRNAs by nitrogen deprivation is most likely due to an effect on the function of the nuclear tRNA export receptors, as overexpression of the tRNA export receptor, Los1p, restores export of the tRNAs during nitrogen starvation. These findings suggest that the TORC1 signaling pathway may, in part, regulate nuclear export of the tRNAs by affecting the function of the tRNA export receptors. In contrast to amino acid and nitrogen starvation, glucose depletion affects nuclear export of all tRNA species in S. cerevisiae. Evidence obtained suggests that nuclear retention of tRNA in cells deprived of glucose is due to a block in nuclear re-import of the nuclear tRNA export receptors. Retention of the receptors in the cytoplasm is not caused by activation of Snf1p, but by the inactivation of PKA during glucose deprivation. Furthermore, regulation of nuclear re-import of the receptors is not due to phosphorylation of the tRNA export receptors by PKA. However, PKA phosphorylates known components of the tRNA export machinery. A model that is consistent with the data is that PKA and an unknown mechanism regulate the activity of these components or an unidentified protein(s) to control nuclear re-import of the receptors in response to glucose availability.

La protéine kinase AMPc-dépendante (PKA) joue un rôle crucial dans la régulation neurohormonale de la fonction cardiaque. L'activation aiguë de la PKA est bénéfique car elle conduit à une augmentation de la contraction cardiaque en phosphorylant les acteurs clés du couplage excitation-contraction. En revanche, son activation chronique est délétère et ces effets semblent faire intervenir la régulation de protéines nucléaires pouvant conduire au remodelage hypertrophique et à l'insuffisance cardiaque. La localisation subcellulaire de la PKA, assurée par des protéines d'ancrage (AKAPs), est importante pour la rapidité et la spécificité d'action des hormones mettant en jeu la voie de l'AMPc. Les niveaux d'AMPc sont régulés par l'activité des adénylate cyclases et des phosphodiestérases (PDEs), et l'état de phosphorylation des protéines cibles de la PKA dépend de l'activité des Ser/Thr phosphatases (PPs). Dans le cœur, les PDEs les plus importantes dégradant l'AMPc sont les PDE3 et les PDE4. Les principales PPs cardiaques sont PP1, PP2A et PP2B. Dans une première partie de mon travail, j'ai mis au point, dans les cardiomyocytes de rats adultes, une mesure de l'activité de la PKA en temps réel dans les compartiments cytoplasmiques et nucléaires. J'ai utilisé pour cela des sondes de type AKAR (A-kinase activity reporters) basées sur le transfert d'énergie de fluorescence (FRET) et localisées spécifiquement dans le noyau ou dans le cytoplasme par des séquences d'adressage ou d'exclusion nucléaires. J'ai ainsi pu montrer qu'une stimulation maintenue des récepteurs β-adrénergiques active la PKA de façon plus importante dans le cytoplasme que dans le noyau, et que cette activation se développe lentement au niveau nucléaire que dans le cytoplasme. De ce fait, une stimulation brève des récepteurs β-adrénergiques active maximalement la PKA dans le cytoplasme, mais de façon marginale dans le noyau. Dans une seconde partie de l'étude, je me suis intéressée au rôle des PDE3 et PDE4 ainsi qu'à celui de PP1, PP2A et PP2B dans la régulation de l'activité PKA cytoplasmique et nucléaire, en réponse à une stimulation β-adrénergique. J'ai montré que la PDE4, mais pas la PDE3, régule l'activité de la PKA cytoplasmique et nucléaire. L'utilisation de souris invalidées pour les gènes Pde4b et Pde4d a révélé que l'isoforme PDE4B est prédominante pour la modulation de l'activité PKA cytoplasmique, alors que les deux isoformes PDE4B et PDE4D contribuent à la régulation de l'activité PKA nucléaire. Finalement, j'ai montré que la PP1 et la PP2A, mais pas la PP2B, participent à la terminaison des réponses β-adrénergiques dans le cytoplasme, alors qu'au niveau nucléaire, la PP1 semble jouer un rôle majeur. En conclusion, ce travail a mis en évidence le rôle des phosphodiestérases et des phosphatases dans l'intégration différentielle des réponses PKA à une stimulation β-adrénergique dans le cytoplasme et le noyau de cardiomyocytes adultes.

Cyclic-AMP dependent protein kinase A (PKA) regulates traffic of multiple proteins at different stages along the constitutive secretory pathway. PKA effects are regulated by protein phosphatases, which reverse the actions of PKA by dephosphorylation of PKA-substrates. Localization of specific PKA effects is mediated by the binding of A-kinase anchoring proteins (AKAPs). Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, and represents a large proportion of total protein constitutively secreted from macrophages. The signalling and trafficking pathways regulating secretion of apoE are unknown. Catalase is a peroxisomal enzyme which contributes to defence against hydrogen peroxide (H2O2). The primary hypothesis of this thesis is PKA and related protein phosphatase pathways are involved in the regulation of apoE secretion. The secondary hypothesis is that these pathways also regulate cellular clearance of H2O2. In Chapter Three, I have investigated the role of PKA in apoE secretion from primary human macrophages. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI14-22) all decreased basal secretion of apoE by between 50-80% whereas apoE mRNA or cellular protein are unaffected. Disruption of PKA-AKAP anchoring also significantly inhibited apoE secretion from human macrophages. Secretion of apoE was not immediately stimulated by PKA activity, suggesting that although PKA activity may be permissive for apoE secretion, it is in itself insufficient to stimulate apoE secretion above basal levels. Data from confocal microscopy and live cell imaging revealed PKA inhibition paralysed apoE vesicular movement from and to the plasma membrane. In Chapter Four, I investigated the effects of protein phosphatase 2B (PP2B) inhibition on apoE secretion by cyclosporin A (CsA). This was found to dose- and time-dependently inhibit secretion of apoE from primary human macrophages and increased cellular accumulation of apoE without affecting apoE mRNA levels. The role of PP2B in regulating apoE secretion was confirmed by using additional peptide and chemical inhibitors of PP2B. This effect was independent of the known inhibition of ABCA1 by CsA. Live cell imaging and confocal microscopy all demonstrated that inhibition of PP2B did not affect the apparent cellular distribution of apoE. Biochemical and microscopy studies indicated distinct mechanisms for PKA and PP2B regulation of apoE secretion. Chapter Five identified PKA-anchoring AKAPs in human macrophages, and investigated AKAP220 expression and its role in PKA-dependent processes relevant to atherosclerosis. AKAP220 protein was absent in human monocytes but was detectable after their differentiation into macrophages, with stable expression during late stages of maturation. It was also present in Chinese Hamster Ovary cells (CHO) cells. AKAP220 silencing had no effects on lipoprotein cholesteryl ester accumulation, total cellular apoE levels, apoE secretion or cholesterol efflux from human macrophages. Confocal microscopy in CHO cells revealed peroxisomal localisation of AKAP220. Catalase activity was confirmed to be PKA-regulated process, and AKAP220 was found to be a negative regulator of catalase activity, such that cell lysate catalase activity increased during AKAP220 silencing. AKAP220 silencing also decreased basal secretion of H2O2, detected using a sensitive and specific Amplex?? Red assay kit from intact CHO monolayers. In conclusion, this thesis has provided evidence that apoE secretion occurs via PKA- and PP2B-dependent pathways in human macrophages, and has identified the A-kinase anchoring protein AKAP220 as a regulator of cellular H2O2 clearance. These results will provide a basis for future investigations into the roles of PKA-related pathways in apoE secretion and catalase activity.

Cyclic-AMP dependent protein kinase A (PKA) regulates traffic of multiple proteins at different stages along the constitutive secretory pathway. PKA effects are regulated by protein phosphatases, which reverse the actions of PKA by dephosphorylation of PKA-substrates. Localization of specific PKA effects is mediated by the binding of A-kinase anchoring proteins (AKAPs). Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, and represents a large proportion of total protein constitutively secreted from macrophages. The signalling and trafficking pathways regulating secretion of apoE are unknown. Catalase is a peroxisomal enzyme which contributes to defence against hydrogen peroxide (H2O2). The primary hypothesis of this thesis is PKA and related protein phosphatase pathways are involved in the regulation of apoE secretion. The secondary hypothesis is that these pathways also regulate cellular clearance of H2O2. In Chapter Three, I have investigated the role of PKA in apoE secretion from primary human macrophages. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI14-22) all decreased basal secretion of apoE by between 50-80% whereas apoE mRNA or cellular protein are unaffected. Disruption of PKA-AKAP anchoring also significantly inhibited apoE secretion from human macrophages. Secretion of apoE was not immediately stimulated by PKA activity, suggesting that although PKA activity may be permissive for apoE secretion, it is in itself insufficient to stimulate apoE secretion above basal levels. Data from confocal microscopy and live cell imaging revealed PKA inhibition paralysed apoE vesicular movement from and to the plasma membrane. In Chapter Four, I investigated the effects of protein phosphatase 2B (PP2B) inhibition on apoE secretion by cyclosporin A (CsA). This was found to dose- and time-dependently inhibit secretion of apoE from primary human macrophages and increased cellular accumulation of apoE without affecting apoE mRNA levels. The role of PP2B in regulating apoE secretion was confirmed by using additional peptide and chemical inhibitors of PP2B. This effect was independent of the known inhibition of ABCA1 by CsA. Live cell imaging and confocal microscopy all demonstrated that inhibition of PP2B did not affect the apparent cellular distribution of apoE. Biochemical and microscopy studies indicated distinct mechanisms for PKA and PP2B regulation of apoE secretion. Chapter Five identified PKA-anchoring AKAPs in human macrophages, and investigated AKAP220 expression and its role in PKA-dependent processes relevant to atherosclerosis. AKAP220 protein was absent in human monocytes but was detectable after their differentiation into macrophages, with stable expression during late stages of maturation. It was also present in Chinese Hamster Ovary cells (CHO) cells. AKAP220 silencing had no effects on lipoprotein cholesteryl ester accumulation, total cellular apoE levels, apoE secretion or cholesterol efflux from human macrophages. Confocal microscopy in CHO cells revealed peroxisomal localisation of AKAP220. Catalase activity was confirmed to be PKA-regulated process, and AKAP220 was found to be a negative regulator of catalase activity, such that cell lysate catalase activity increased during AKAP220 silencing. AKAP220 silencing also decreased basal secretion of H2O2, detected using a sensitive and specific Amplex?? Red assay kit from intact CHO monolayers. In conclusion, this thesis has provided evidence that apoE secretion occurs via PKA- and PP2B-dependent pathways in human macrophages, and has identified the A-kinase anchoring protein AKAP220 as a regulator of cellular H2O2 clearance. These results will provide a basis for future investigations into the roles of PKA-related pathways in apoE secretion and catalase activity.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-12-12T18:02:43Z No. of bitstreams: 1 GuttoRaffysonSilvaDeFreitas_TESE.pdf: 4320273 bytes, checksum: eaf5ef8d3670982fdb7ddbad48ace3c4 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-12-13T18:43:50Z (GMT) No. of bitstreams: 1 GuttoRaffysonSilvaDeFreitas_TESE.pdf: 4320273 bytes, checksum: eaf5ef8d3670982fdb7ddbad48ace3c4 (MD5) / Made available in DSpace on 2017-12-13T18:43:50Z (GMT). No. of bitstreams: 1 GuttoRaffysonSilvaDeFreitas_TESE.pdf: 4320273 bytes, checksum: eaf5ef8d3670982fdb7ddbad48ace3c4 (MD5) Previous issue date: 2017-08-18 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / As quinoxalinas comp?em uma classe de compostos heteroc?clicos de nitrog?nio com um amplo espectro de aplica??es, em diversas ?reas. Neste contexto, destacam-se as atividades biol?gicas para diversos fins. Atualmente, os m?todos de qu?mica computacional tem se mostrado uma ferramenta de grande relev?ncia para realizar investiga??es de propriedades estruturais, eletr?nicas e termodin?micas de diversos compostos heteroc?clicos, incluindo derivados quinoxal?nicos. No presente trabalho, estas propriedades foram investigadas em tr?s abordagens distintas, tendo como base Teoria do Funcional de Densidade (DFT). Na primeira parte, foi realizada uma investiga??o acerca dos aspectos estruturais, eletr?nicos e energ?ticos do composto 6,7-dinitroquinoxalina-2,3-diona (DNQX), um importante antagonista do neuroreceptor glutamato iGluR2, o qual possui papel importante em processos relacionados ao aprendizado e mem?ria. Mais especificamente, foram avaliados os poss?veis taut?meros do DNQX, constatando que a forma dicarbonilada ? a de menor energia. Foi realizado um estudo dos valores de pKa associados ?s forma??es das esp?cies mono e diani?nicas, em fase gasosa e aquosa, a partir de diferentes metodologias. No cap?tulo seguinte, foram estudadas rea??es de substitui??o nucleof?lica arom?tica envolvendo 6,7-dicloroquinoxalina-2,3-diona (DCQX) e os amino?lcoois: etanolamina e dietanolamina, em DMF. Foram calculadas as varia??es de energia livre associadas ? forma??o dos poss?veis produtos de mono e dissubstitui??o, assim como os estados de transi??o para forma??o dos mesmos. Os resultados foram discutidos em termos de aspectos estruturais, como base em trabalhos previamente relatados na literatura, de onde foi poss?vel sugerir que estas rea??es ocorrem via mecanismos concertados para dupla substitui??o do cloro tanto por etanolamina, ambas por ataque nucleof?lico do nitrog?nio, quanto por dietanolamina, por ataque nucleof?lico do nitrog?nio e do oxig?nio. Ademais, as an?lises dos valores de ?G de rea??o e de ativa??o sugerem que a forma??o dos produtos ciclizados ? mais favorecida, para ambos os casos. Por fim, foram avaliadas as propriedades estruturais e termodin?micas de diversos complexos de cobre (II) com o quimiossensor N-(2-aminofenil)-3-[(1S,2S)-1,2,3-trihidroxi-propil]quinoxalina-2-carboxamida (AAQX), no intuito de entender a intera??o desta quinoxalina com o referido metal, via forma??o de um complexo metal-ligante 2:1. Devido ao tamanho dos complexos, os c?lculos foram realizados com a combina??o de m?todos DFT e semi-emp?rico, a fim de reduzir o custo computacional. Os resultados sugerem que complexos com estrutura tetra?drica s?o mais favor?veis e a substitui??o do ligante cloreto por ?gua n?o forma complexos est?veis. Com o desenvolvimento do trabalho, foram obtidos resultados satisfat?rios associados ao uso da DFT para o estudo de diferentes propriedades destes derivados quinoxal?nicos. / Quinoxaline are part of a family of heterocyclic compounds with a wide spectra of applications in many subjects, for instance biological activities. Nowadays, quantum chemistry methods has been proving of particular importance, in order to shed light on the electronic structure and the thermodynamics of many of such heterocyclic molecules, among them quinoxaline. In this work, we shall investigate such properties using three Density Functional Theory (DFT) based approaches. First, we have studied the geometries, electronic structure and energies of 6,7-dinitroquinoxaline-2,3-dione (DNQX), an important antagonist for the glutamate receptor iGluR2, deeply related with processes of learning and memory. In particular, from those possible DNQX investigated, the minima is found as being the diamide form. We have also carried out a study concerning the pKa of formation for both mono and dianionics, in gas phase and solution, by means of a few different methodologies. In the following chapter, we have investigated the nucleophilic substitution reaction between 6,7-Dinitroquinoxaline-2,3-dione (DNQX) and amino alcohols, namely: ethanolamine and diethanolamine, in DMF. We have calculated the free energy change associated to formation of all relevant products. The results are presented in terms of geometries, based on previous works, from which was possible to find enough evidences suggesting that such processes happen by means of concerted reactions for double substitution of chlorine atoms either by ethanolamine and diethanolamine. Moreover, the values of ?G of formation and activation tends to demonstrate that cyclic products are preferable. Finally, we have evaluated the structure and thermodynamics of several complex of Cu (II) with N-(2-aminophenyl)-3-[(1S,2S)-1,2,3-trihydroxy-propyl]quinoxaline-2-carboxamide (AAQX), in order to proper understand the metal-quinoxalinedione interaction via formation of a complex metal-ligand 2:1. Due the size of the complex, all calculations were performed using a hybrid DFT/semi-empirical approach, to reduce the computational effort. The results show tetrahedral geometries being more favorable and substitution of a ligand by water giving rise to unstable complexes. All along this work DFT has been heavily used in order to investigate the properties of quinoxalinedione-like derivatives.

Quantum Chemical Topology (QCT) descriptors, calculated from ab initio wave functions, have been utilised to model pKa and mutagenicity for data sets of pharmaceutically relevant compounds. The pKa of a compound is a pivotal property in both life science and chemistry since the propensity of a compound to donate or accept a proton is fundamental to understanding chemical and biological processes. The prediction of mutagenicity, specifically as determined by the Ames test, is important to aid medicinal chemists select compounds avoiding this potential pitfall in drug design. Carbocyclic and heterocyclic aromatic amines were chosen because this compounds class is synthetically very useful but also prone to positive outcomes in the battery of genotoxicity assays.The importance of pKa and genotoxic characteristics cannot be overestimated in drug design, where the multivariate optimisations of properties that influence the Absorption-Distribution-Metabolism-Excretion-Toxicity (ADMET) profiles now features very early on in the drug discovery process.Models were constructed using carboxylic acids in conjunction with the Quantum Topological Molecular Similarity (QTMS) method. The models produced Root Mean Square Error of Prediction (RMSEP) values of less than 0.5 pKa units and compared favourably to other pKa prediction methods. The ortho-substituted benzoic acids had the largest RMSEP which was significantly improved by splitting the compounds into high-correlation subsets. For these subsets, single-term equations containing one ab initio bond length were able to accurately predict pKa. The pKa prediction equations were extended to phenols and anilines.Quantitative Structure Activity Relationship (QSAR) models of acceptable quality were built based on literature data to predict the mutagenic potency (LogMP) of carbo- and heterocyclic aromatic amines using QTMS. However, these models failed to predict Ames test values for compounds screened at GSK. Contradictory internal and external data for several compounds motivated us to determine the fidelity of the Ames test for this compound class. The systematic investigation involved recrystallisation to purify compounds, analytical methods to measure the purity and finally comparative Ames testing. Unexpectedly, the Ames test results were very reproducible when 14 representative repurified molecules were tested as the freebase and the hydrochloride salt in two different solvents (water and DMSO). This work formed the basis for the analysis of Ames data at GSK and a systematic Ames testing programme for aromatic amines. So far, an unprecedentedly large list of 400 compounds has been made available to guide medicinal chemists. We constructed a model for the subset of 100 meta-/para-substituted anilines that could predict 70% of the Ames classifications. The experimental values of several of the model outliers appeared questionable after closer inspection and three of these have been retested so far. The retests lead to the reclassification of two of them and thereby to improved model accuracy of 78%. This demonstrates the power of the iterative process of model building, critical analysis of experimental data, retesting outliers and rebuilding the model.