O Hemocentro São Paulo é responsável por manter um banco de dados com informações sobre cada doação ou tentativa de doação de sangue. No entanto, os dados desse banco de dados não possuem a qualidade requerida pelas ferramentas/técnicas de análise. Por essa razão, fica difícil utilizar tais dados para estabelecer relações sistemáticas entre as variáveis armazenadas. A principal contribuição desta tese é a descrição da proveniência para atributos selecionados usando critérios de classificação definidos por especialistas. Este trabalho mostra que é possível fazer investigações detalhadas usando a descrição dos dados sem a necessidade de alterar a estrutura do banco de dados. Durante o período de 1996 a 2006, 1.469.505 doadores foram responsáveis por mais de 2.8 milhões de doações. Após a descrição da proveniência, foram obtidos 252.301 doadores do sexo masculino e 133.056 doadores do sexo feminino e que atenderam aos critérios de inclusão usados nesta tese. Dos 385.357 doadores incluídos na análise, 21.954 (5,7%) tiveram suas doações adiadas devido a seus baixos níveis de hematócrito, 3.850 (1,5%) eram do sexo masculino e 18.104 (13,6%) do sexo feminino. Os resultados obtidos demonstram que, embora os intervalos de espera entre as doações de sangue sejam grandes entre os doadores do sexo feminino e masculino, as mulheres são recusadas mais cedo, por risco de desenvolver anemia, do que os homens. Aproximadamente 12,84% das mulheres e 1,21% dos homens desenvolveriam hematócrito baixo antes da sétima doação. Os dados sugerem que indivíduos com baixo nível de hematócrito devem esperar mais tempo antes de executarem a próxima doação. Portanto, é importante compreender se existe uma ligação entre a doação de sangue e a diminuição no nível de hematócrito, a fim de evitar resultados indesejáveis para os doadores de sangue. O modelo de proveniência apresentado nesta tese não foi definido de acordo com os modelos de proveniência genéricos já implementados. Esta tese apresenta um modelo de proveniência que foi capaz de acrescentar informações semânticas para adquirir conhecimento de um experimento in silico. Um dos principais objetivos foi desenvolver uma abordagem baseada em declarações, tentando responder a importantes questionamentos biológicos. O modelo descrito combina ricas informações em cada processo usando declarações, e se baseia no conhecimento de especialistas. Esta tese também utilizou estatística descritiva e Análise de Sobrevivência. Finalmente, com a validação do modelo em um domínio conhecido, é pretendido expandir esse método para outros sistemas de informação voltados para hemoterapia. / The São Paulo Blood Center is responsible to maintain a database with information on each donation. However, this database does not have the quality required by techniques of analysis. For this reason, it is difficult to use it directly to establish systematic relationships between the variables. The main contribution of this paper is a provenance description of attributes selected using classification criteria defined by specialists. We show that it is possible to make detailed investigations using the data description without the need to change the structure of the database. During 1996 2006, 1,469,505 donors were responsible for more than 2.8 million of donation. After the provenance description, we obtained 252,301 male and 133,056 female that met our inclusion criteria. Of the 385,357 donors included in the analysis, 21,954(5.7%) were deferred due to low hematocrit, 3,850(1.5%) were males and 18,104(13.6%) were females. Our results show that, although the intervals between donations for female and male donors are wider, women presented anemia earlier than men. Approximately 12,84% of the females and 1,21% of the males would develop low hematocrit before the 7th donation. Our data suggest that individuals with low hematocrit level should wait longer before the next donation. Therefore, it is important to understand if there is a connection between blood donation and decrease in hematocrit level in order to prevent undesirable outcomes to blood donors. The provenance model presented here was not defined according to the generic provenance models already implemented. This thesis presents a provenance model that is able to add semantic information to acquire knowledge of an in silico experiment. One of the main purposes is to develop an approach based on declarations in order to answer biological questions. The provenance model described in this paper combines rich information for each process using the declarations, each having expert knowledge as a basis. To evaluate this provenance model we use descriptive statistics and Survival Analysis. Finally, with the validation of the model in a known domain, we intent to apply and validate this provenance model to other hemotherapy information systems.

Mutageniciteten för olika ämnen bestäms vanligtvis med hjälp av konventionella in vivo och in vitro metoder. Att övergå till in silico metoder vore både etiskt och miljö- mässigt gynnsamt. Flera olika parametrar kan användas för att förutse mutagenicitet. Bland dessa ingår energin för lägsta oockuperade molekylorbitalen (LUMO), aktiveringsenergin, lokala min/max i elektrostatiska potentialen (Vs,min/Vs,max), lokala min i ”electron attachment energy” (Es,min ), med flera. Aktiveringsenergin är en parameter som kan förutspå mutagenicitet med hög noggrannhet, förutsatt att reaktionsmekanismen antingen är känd eller kan antas. Däremot är beräkningskostnaden för aktiveringsenergin hög. Mutagen-X (MX) och dess analoger (föreningar med samma grundstruktur men olika funktionella grupper, totalt 29 olika föreningar), är föreningar vars mutagenicitet har bestämts med hjälp av traditionella metoder men har nyligen också kunnat bestämmas genom kvantkemiskmodellering. Kvantkemiskmodellering har med framgång implementerats i studien av Kari Tuppurainen [1]; där LUMO har bestämts för MX och dess analogier. Utöver detta visades även en statistiskt signifikant korrelation mellan LUMO för MX och dess analoger och den biologiska aktiviteten som bestämdes med hjälp av Ames test. Målet med detta arbete har varit att undersöka vare sig mutageniciteten för MX och dess analogier kan även bestämmas genom att beräkna Es,min, Vs,max och aktiveringsenergin. Reaktionen som undersöktes var en Michael additionsreaktion  mellan amingruppen på kvävebasen guanin och betapositionen på MX och dess analogier. Av den anledningen utvärderades parametrarna (Es,min, Vs,max och aktiveringsenergin) vid betapositionen. De beräknade värdena för Es,min var korrelerade med biologiska aktiviteten. Även aktiveringsenergierna för MX och dess analogier beräknades vid betapositionen och korrelerades sedan med biologiska aktiviteten och Es,min värdena. Om en statistiskt signifikant korrelation observerades mellan aktiveringsenergin och Es,min värdena, hade detta varit en indikation att Es,min värden kan användas för att ersätta aktiveringsenergin. Es,min värden har en jämförelsevis låg beräkningskostnad. Dock observerades ingen statistiskt signifikant korrelation mellan Es,min värdena och biologiska aktiviteten. Vidare observerades ingen statistiskt signifikant korrelation mellan aktiveringsenergin och biologiska aktiviteten och/eller Es,min värdena. Därmed fanns flera indikationer att den tänkta reaktionsmekanismen var felaktig. Under efterforskningen hittades en studie som visade att en-elektronreduktionsmekanismen var den mest troliga reaktionsmekanismen för den undersökta reaktionen. Detta kan vara en förklaring till varför en statistiskt signifikant korrelation kunde observeras mellan LUMO och biologiska aktiviteten, medan ingen korrelation observerades för Es,min, Vs,max och aktiveringsenergin mot biologiska aktiviteten. Avsaknaden av en korrelation för dessa parametrar anses bero på att den föreslagna reaktionsmekanismen var felaktig. Vidare studier hade behövts för att undersöka dessa parametrars förmåga att kunna förutse mutagenicitet. / Mutagenicity of various compounds is traditionally predicted by conventional in vivo and in vitro methods. However, transitioning to in silico methods would be beneficial both ethically and environmentally. The descriptors that can be used to predict mutagenicity are the lowest unoccupied molecular orbital (LUMO) energy, activation energy, the local minimum / maximum electrostatic potential energy (Vs,min/Vs,max), the minimum local electron attachment energy (Es,min), etc. The activation energy is a descriptor that can predict mutagenicity accurately provided that the reaction mechanism is known or can be assumed. However, determining the activation energy is computationally costly. Mutagen-X (MX) and its analogues (compounds with the same backbone but different functional groups, 29 compounds in total), are compounds of which the mutagenicity had been characterized by traditional means but recently also using an in silico method – molecular modeling. Molecular modeling had been successively employed in the study by Kari Tuppurainen [Source]; the LUMO of MX and its analogues had been computed and, importantly, the obtained values demonstrated a statistically significant correlation with the biological activity determined using Ames test. The aim of this thesis was to investigate whether the mutagenicity of MX and its analogues could also be determined by computing Es,min, Vs,max and the activation energy. The studied reaction was a Michael addition reaction between an amine group on the guanine nucleobase and the beta position of MX and its analogues. Therefore, the studied parameters (Es,min, Vs,max and the activation energy) were evaluated at the beta position. The computed Es,min values were correlated with the biological activity. Activation energies for MX and its analogues were also computed at the beta position and then correlated with the biological activity and Es,min values.  If a highly statistically significant correlation between the activation energy and Es,min values at the beta position would have been observed, that would indicate that Es,min values  could be used as a substitute for the activation energy. Es,min values have comparatively low computational cost. However, no statistically significant correlation between Es,min values and the biological activity was observed. Furthermore, no statistically significant correlation was observed between the activation energy and biological activity and/or Es,min values, respectively. Thus, there were several indications that the proposed reaction mechanism was incorrect. After consulting literature, we learned that the one electron reduction mechanism would be a more probable reaction mechanism. This could be an explanation as to why a highly statistically significant correlation could be observed for LUMO vs. the biological activity, whereas no correlation was observed for Es,min, Vs,max, the activation energy versus the biological activity. The absence of a correlation for these parameters is thought to be due to the proposed reaction mechanism being inaccurate. Additional studies would have to be performed to further investigate the predictive abilities for mutagenicity of the studied parameters.

L’infection au papillomavirus humain (HPV) est connue pour être le principal facteur causal d’une série de maladies aussi bien bénignes (condylomatose ano-génitale, papillomatose lyringée, et autres) que malignes (cancer du col de l’utérus, certains cancers ORL, et autres). Deux vaccins prophylactiques (Gardasil® et Cervarix®) sont sur la marché depuis à peu près quatre ans pour prévenir cette infection. Le présent travail de thèse comportait trois objectifs principaux : i) étudier in-silico l’immunogénicité du vaccin Gardasil® ; ii) étudier in-silico l’histoire naturelle d’une infection à HPV et iii) évaluer in-silico le potentiel de l’hypothèse thérapeutique suivante : l’administration intramusculaire du vaccin Gardasil® chez des patients atteints d’une papillomatose laryngée induirait un effet bénéfique car l’arrivée des immunoglobulines au tissu affecté empêcherait l’HPV de compléter son cycle de vie et, par conséquent, la maladie de se propager. Les principales conclusions sont : i) pour qu’une papillomatose laryngée ne s’étende pas il faudrait, d’après nos simulations, que le taux d’IgGs sériques soit maintenu au-dessus de 200 mMU/mL ; ii) pour rester, sur une période de 10 ans, le plus longtemps possible au-dessus de ce seuil (d´effet thérapeutique), en administrant la quantité minimale de vaccin, il faudrait, d’après nos simulations, suivre le protocole suivant : l’immunisation de base (à 0, 2 et 6 mois), suivie de trois rappels successifs tous les six mois jusqu’au 24ème mois, suivis d’un rappel 18 mois plus tard ; iii) par ailleurs, il semble inutile (voire contreproductif), d’après nos simulations, de modifier le schéma traditionnel de base (0-2-6 mois) / Two prophylactic vaccines have demonstrated to prevent infections with the human papillomavirus (HPV). Thus, they have been in the market for the last four years, or so. The three main objectives of the present project were: i) to study in-silico the immunogenicity of one of these vaccines (Gardasil®); ii) to study in-silico the natural history of an HPV infection, and iii) to assess in-silico the potential of the following therapeutic hypothesis : the intramuscular administration of Gardasil® to patients already suffering from a recurrent respiratory papillomatosis would result in a better prognosis thanks to the fact that the HPV-specific immunoglobulins that would bathe the affected tissue would impede the virus to complete its life cycle and, therefore, the disease to progress. The main conclusions are: i) according to our simulations, the minimum serum IgG titer required for hampering the progression of a recurrent respiratory papillomatosis would be 200 mMU/mL ; ii) in order to keep, within a time window of ten years, the anti-HPV IgG titer over the just-mentioned therapeutic-effect threshold, the biggest possible fraction of time and through the administration of the smallest possible number of booster doses, it would be necessary, according to our simulations, to adopt the following vaccination schedule: the basic three doses (at months 0, 2 and 6), followed by three successive booster doses, every six months, until reaching the 24th month, followed by a late final booster dose, 18 months later. iii) incidentally, it would seem to be inappropriate, according to our simulations, to modify the original initial vaccination schedule (at months 0, 2 and 6)

Selon une vision populaire, l’évolution serait un processus de « progrès » qui s’accompagnerait d’un accroissement de la complexité moléculaire des êtres vivants. Cependant, les programmes de séquençage des génomes ont révélé l’existence d’espèces dont les lignées ont, au contraire, subi une réduction massive de leur génome. Ainsi, chez les cyanobactéries Prochlorococcus et Pelagibacter ubique, certaines lignées ont subi une réduction de 30% de leur génome. Une telle évolution « à rebours », dite évolution réductive, avait déjà été observée pour des bactéries endosymbiotiques, pour lesquelles la sélection naturelle n’est pas assez efficace pour éliminer les mutations délétères comme les pertes de gènes. Cela vient notamment du fait que ces bactéries endosymbiotiques subissent, à chaque reproduction de leur hôte, une réduction drastique de leur taille de population. Cette explication semble peu plausible pour des cyanobactéries marines comme Prochlorococcus et Pelagibacter, qui ont un mode de vie libre et qui font partie des bactéries les plus abondantes des océans. D’autres hypothèses ont ainsi été proposées pour expliquer l’évolution réductive comme l’adaptation à un environnement stable et pauvre en nutriments, des forts taux de mutation, mais aucun de ces hypothèses ne semble capable d’expliquer toutes les caractéristiques génomiques observées. Dans cette thèse, nous nous intéressons au cas de l’évolution réductive chez Prochlorococcus, pour laquelle de nombreuses séquences et données sont disponibles. Deux approches sont utilisées pour cette étude : une analyse phylogénétique des génomes de Prochlorococcus, et une approche théorique de simulation où nous testons différents scénarios évolutifs pouvant conduire à une évolution réductive. La combinaison de ces deux approches permet finalement de proposer un scénario plausible pour expliquer l'évolution réductive chez Prochlorococcus. / Given a popular view, evolution is an incremental process based on an increase of molecular complexity of organisms. However, some organisms have undergo massive genome reduction like the endosymbionts. In this case the reduction can be explained by the Muller’s ratchet due to the endosymbiont lifestyle with small population and lack of recombination. However, in some marine bacteria, like Prochlorococcus et Pelagibacter, lineage have undergo up to 30% of genome reduction. Their lifestyle is almost the opposite to the one of the endosymbionts and reductive genome evolution can not be easily explicable by the Muller’s ratchet. Some other hypothesis has been proposed but none can explain all the observed genomic characteristics. In the thesis, I am interested in the reductive evolution of Prochlorococcus. I used two approaches: a theoretical one using simulation where different scenarios are tested and an analysis of Prochlorococcus genomes in a phylogenetic framework to determine the causes and characteristics of genome reduction. The combination of these two approaches allows to propose an hypothetical evolutive history for the reductive genome evolution of Prochlorococcus.

Introdução: É cada vez mais preocupante a escassez de água e a qualidade dos recursos hídricos. Nas últimas décadas diversos estudos têm apontado a presença de fármacos em água destinada ao consumo humano, caracterizando a existência de rotas de exposição que podem representar riscos para a saúde humana e meio ambiente. Devido a escassez de dados sobre o comportamento ambiental e toxicidade na exposição crônica a baixas doses a fármacos, sua ocorrência em água é uma preocupação para comunidade científica, reguladores e população. Diversos estudos recentes têm sugerido critérios para a definição de fármacos prioritários, ou seja, abordagens com componentes ou fatores que atribuem grau de relevância aos contaminantes. Devido aos altos custos e necessidade de testes adicionais, uma das alternativas propostas para avaliação do comportamento ambiental e toxicidade têm sido os modelos in silico. Objetivos: Estudar o comportamento ambiental e o potencial de toxicidade de ingredientes farmacêuticos ativos(IFAs) para fins de identificação de contaminantes prioritários por meio da aplicação de ferramentas computacionais e modelos in silico. Métodos: Foram selecionados fármacos considerados relevantes para o Brasil conforme critérios de inclusão específicos. Para os fármacos selecionados, foram realizadas buscas de dados experimentais em bases de dados de agências internacionais e predições de propriedades físico-químicas, transporte e mobilidade no ambiente, persistência, bioacumulação e toxicidade, através de modelos in silico e ferramentas desenvolvidas pela USEPA e OECD: EPISuite (v 4.1, EPA, 2011), PBT Profiler (EPA, v.2.0, 2012) TEST© - Toxicity Estimation Software Tool (v.4.1, EPA, 2012) e QSAR Toolbox (v.3.2, OECD de 2013). Foi utilizado o método de priorização do software ToxPi GUI (Carolina Center for Computational Toxicology/Universidade da Carolina do Norte), para obtenção de um Índice de Prioridade Toxicológica (Toxicological Priority Index-ToxPi) e definição dos agentes prioritários com base nos resultados das predições e dados da literatura Resultados: Foram obtidos dados e predições de propriedades físico-químicas, transporte e mobilidade no ambiente, persistência, bioacumulação e toxicidade para os 39 IFAs selecionados. Com base nestes dados, foi obtido um perfil de priorização baseado no ToxPi. Conclusão: Fármacos de diferentes classes podem apresentar características físico-químicas e de comportamento ambiental que lhes conferem alto potencial de exposição ambiental, e apesar do uso seguro nas condições posológicas, há diversos ingredientes ativos com potencial de toxicidade e que podem representar alta preocupação em exposições crônicas As ferramentas computacionais podem ser uma importante ferramenta para avaliação do comportamento ambiental e da toxicidade e identificação preliminar de agentes prioritários. / Introduction: It is a subject of more concern the shortage of water and quality of water sources. For the past decades a variety of studies have pointed the presence of pharmaceuticals in water intended for human consumption, characterizing routes of human and environmental exposure over potential health risks. Due to lack of environmental fate and toxicity data on chronic exposure to low doses of pharmaceuticals, their occurrence in water worries the scientific community, regulators and population. Many recent studies have suggested criteria for the definition of prioritary pharmaceuticals, i.e. approaches of components and factors that attach the relevance of contaminants. Also due to high costs and the necessity for additional tests, one of the alternatives suggested for the assessment of environmental fate and toxicity are in silico models. Objective: To study the environmental fate and the potential of toxicity in active pharmaceutical ingredients (API) in order to identify the prioritary contaminants through the application of computational tools and in silico models. Methods: Pharmaceuticals considered relevant to Brazil were selected according to specific inclusion criteria. For selected pharmaceuticals, experimental data searches were made in databases of international agencies and predictions of physicochemical, transport and mobility properties in the environment, persistence, bioaccumulation and toxicity, through in silico models and tools developed by the USEPA and OECD: EPISuiteTM (v 4.1, EPA, 2011), PBT Profiler (EPA, v.2.0, 2012) TEST© Toxicity Estimation Software Tool (v.4.1, EPA, 2012) and QSAR Toolbox (v.3.2, OECD, 2013). The priorization method of software ToxPi GUI (Carolina Center for Computational Toxicology/University of North Carolina) was used in order to obtain a Toxicological Priority index - ToxPi and a definition of prioritary agents based on results of the predictions and data from literature. Results: Data and predictions of physicochemical, transport and mobility properties in the environment, persistence, bioaccumulation and toxicity were obtained to the 39 selected APIs. It was obtained a profile of priorization based on ToxPi with these data. Conclusion: Different classes of pharmaceuticals may have physicochemical and environmental fate properties that give them high potential of environmental exposure, and in spite of the safe use in posological conditions, there are several active ingredients with toxicity potential that may represent high concern in chronic exposures. The computational tools may be an important tool for environmental fate and toxicity assessments in order to identify preliminary prioritary agents.

Introdução: É cada vez mais preocupante a escassez de água e a qualidade dos recursos hídricos. Nas últimas décadas diversos estudos têm apontado a presença de fármacos em água destinada ao consumo humano, caracterizando a existência de rotas de exposição que podem representar riscos para a saúde humana e meio ambiente. Devido a escassez de dados sobre o comportamento ambiental e toxicidade na exposição crônica a baixas doses a fármacos, sua ocorrência em água é uma preocupação para comunidade científica, reguladores e população. Diversos estudos recentes têm sugerido critérios para a definição de fármacos prioritários, ou seja, abordagens com componentes ou fatores que atribuem grau de relevância aos contaminantes. Devido aos altos custos e necessidade de testes adicionais, uma das alternativas propostas para avaliação do comportamento ambiental e toxicidade têm sido os modelos in silico. Objetivos: Estudar o comportamento ambiental e o potencial de toxicidade de ingredientes farmacêuticos ativos(IFAs) para fins de identificação de contaminantes prioritários por meio da aplicação de ferramentas computacionais e modelos in silico. Métodos: Foram selecionados fármacos considerados relevantes para o Brasil conforme critérios de inclusão específicos. Para os fármacos selecionados, foram realizadas buscas de dados experimentais em bases de dados de agências internacionais e predições de propriedades físico-químicas, transporte e mobilidade no ambiente, persistência, bioacumulação e toxicidade, através de modelos in silico e ferramentas desenvolvidas pela USEPA e OECD: EPISuite (v 4.1, EPA, 2011), PBT Profiler (EPA, v.2.0, 2012) TEST© - Toxicity Estimation Software Tool (v.4.1, EPA, 2012) e QSAR Toolbox (v.3.2, OECD de 2013). Foi utilizado o método de priorização do software ToxPi GUI (Carolina Center for Computational Toxicology/Universidade da Carolina do Norte), para obtenção de um Índice de Prioridade Toxicológica (Toxicological Priority Index-ToxPi) e definição dos agentes prioritários com base nos resultados das predições e dados da literatura Resultados: Foram obtidos dados e predições de propriedades físico-químicas, transporte e mobilidade no ambiente, persistência, bioacumulação e toxicidade para os 39 IFAs selecionados. Com base nestes dados, foi obtido um perfil de priorização baseado no ToxPi. Conclusão: Fármacos de diferentes classes podem apresentar características físico-químicas e de comportamento ambiental que lhes conferem alto potencial de exposição ambiental, e apesar do uso seguro nas condições posológicas, há diversos ingredientes ativos com potencial de toxicidade e que podem representar alta preocupação em exposições crônicas As ferramentas computacionais podem ser uma importante ferramenta para avaliação do comportamento ambiental e da toxicidade e identificação preliminar de agentes prioritários. / Introduction: It is a subject of more concern the shortage of water and quality of water sources. For the past decades a variety of studies have pointed the presence of pharmaceuticals in water intended for human consumption, characterizing routes of human and environmental exposure over potential health risks. Due to lack of environmental fate and toxicity data on chronic exposure to low doses of pharmaceuticals, their occurrence in water worries the scientific community, regulators and population. Many recent studies have suggested criteria for the definition of prioritary pharmaceuticals, i.e. approaches of components and factors that attach the relevance of contaminants. Also due to high costs and the necessity for additional tests, one of the alternatives suggested for the assessment of environmental fate and toxicity are in silico models. Objective: To study the environmental fate and the potential of toxicity in active pharmaceutical ingredients (API) in order to identify the prioritary contaminants through the application of computational tools and in silico models. Methods: Pharmaceuticals considered relevant to Brazil were selected according to specific inclusion criteria. For selected pharmaceuticals, experimental data searches were made in databases of international agencies and predictions of physicochemical, transport and mobility properties in the environment, persistence, bioaccumulation and toxicity, through in silico models and tools developed by the USEPA and OECD: EPISuiteTM (v 4.1, EPA, 2011), PBT Profiler (EPA, v.2.0, 2012) TEST© Toxicity Estimation Software Tool (v.4.1, EPA, 2012) and QSAR Toolbox (v.3.2, OECD, 2013). The priorization method of software ToxPi GUI (Carolina Center for Computational Toxicology/University of North Carolina) was used in order to obtain a Toxicological Priority index - ToxPi and a definition of prioritary agents based on results of the predictions and data from literature. Results: Data and predictions of physicochemical, transport and mobility properties in the environment, persistence, bioaccumulation and toxicity were obtained to the 39 selected APIs. It was obtained a profile of priorization based on ToxPi with these data. Conclusion: Different classes of pharmaceuticals may have physicochemical and environmental fate properties that give them high potential of environmental exposure, and in spite of the safe use in posological conditions, there are several active ingredients with toxicity potential that may represent high concern in chronic exposures. The computational tools may be an important tool for environmental fate and toxicity assessments in order to identify preliminary prioritary agents.

Staphylococcus aureus resistente à meticilina (MRSA, Methicillin-Resistant Staphylococcus aureus) é um dos principais responsáveis por infecções nosocomiais, sendo identificado também em infecções associadas à comunidade. Embora potentes fármacos anti-estafilocócicos estejam disponíveis, as infecções causadas por este patógeno continuam a apresentar significativa morbidade e mortalidade devido ao aparecimento de cepas com resistência a múltiplos fármacos, incluindo vancomicina e teicoplanina. Compostos 5-nitro-heterocíclicos com estrutura análoga à nifuroxazida, antimicrobiano utilizado em infecções gastrintestinais, têm apresentado satisfatória atividade in vitro frente a estas cepas multirresistentes, sendo importante e necessária a avaliação de sua biodisponibilidade oral como próximo estágio no desenvolvimento de um novo fármaco, visando à seleção eficiente e ao aprimoramento da estrutura molecular. Neste contexto, o presente estudo tem por objetivo empregar ensaios in vitro, utilizando células Caco-2, e métodos in silico, utilizando descritores moleculares VolSurf, a fim de analisar a permeabilidade de análogos à nifuroxazida com atividade antimicrobiana apresentando, principalmente, atividade potencial frente a cepas multirresistentes de S. aureus. Empregou-se o método de MTT (brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difenil tetrazólio) para a avaliação da citotoxicidade. Nos estudos de permeabilidade in vitro foram utilizadas membranas de células Caco-2 cultivadas em placas Transwel® por 21 dias. A quantificação das frações permeadas foi realizada por cromatografia a líquido de alta eficiência com detecção UV (CLAE-UV), com métodos validados de acordo com a Resolução 899/03. Os estudos in silico foram realizados por meio de análise exploratória, pelo método de consenso de análise de componentes principais (CPCA, Consensus Principal Component Analysis), e análise de regressão, por quadrados mínimos parciais (PLS, Partial Least Squares). As células Caco-2 apresentaram viabilidade adequada para a realização dos estudos de permeabilidade frente a todos os derivados da nifuroxazida, exceto o derivado MeTIO (5-nitro-2-tiofilideno 4-metilbenzidrazida). Os valores de permeabilidade aparente (Papp) obtidos para os análogos à nifuroxazida indicam que estes possuem alta permeabilidade. Os modelos obtidos por CPCA e PLS foram capazes de separar as moléculas em grupos de compostos de baixa, média e alta permeabilidade, sendo os análogos à nifuroxazida classificados como compostos de alta permeabilidade. Para os compostos em estudo, as propriedades determinantes da permeabilidade através de células Caco-2, de acordo com os modelos, seriam de natureza topológica e estérica, sendo possível a previsão externa qualitativa e quantitativa da permeabilidade através de células Caco-2. / Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens responsible for nosocomial infections, also identified in community-associated infections. Although potent anti-staphylococcal drugs are available, infections caused by this bacteria continues to show significant morbidity and mortality due to the emergence of strains with resistance to multiple drugs, including vancomycin and teicoplanin. The 5-nitro-heterocyclic derivatives of nifuroxazide, which is an antimicrobial used to treat gastrointestinal infections, have shown satisfactory in vitro activity against multidrug-resistant strains of S. aureus. As a next step in the development of a new drug, it is important and necessary the evaluation of the oral bioavailability to achieve an efficient selection and refinement of the molecular structure. In this context, this study aims to develop in vitro assays through Caco-2 cells, and in silico approaches, using VolSurf molecular descriptors, in order to analyze the permeability of 5-nitro-heterocyclic compounds analogues to nifuroxazide with antimicrobial activity, especially showing promising activity against multidrug-resistant Staphylococcus aureus. The MTT (bromide 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyl tetrazolium) method was employed to perform the cytotoxicity evaluation. Caco-2 cell monolayers cultivated for 21 days in Transwel® plates were used for the in vitro permeability assays. The quantification of the permeated fractions was done by High Performance Liquid Chromatography with UV detection (HPLC-UV), with validated methods according to the Resolution 899/03. In silico studies were performed through exploratory analysis by consensus principal component analysis (CPCA) and regression analysis by partial least squares (PLS). Caco-2 cells showed suitable cell viability for the permeability studies against all nifuroxazide analogues except the MeTIO (5-nitro-2-thiophilydene 4-methylbenzidrazide). The nifuroxazide derivatives apparent permeability values (Papp) obtained indicate these are high permeable compounds. The models obtained by CPCA and PLS were able to separate the molecules into groups of low, medium and high permeabilities, and the nifuroxazide analogues were classified as high permeable substances. The identified properties for the permeability through Caco-2 cells to the studied compounds were topologic and sterical, and it is possible to perform qualitative and quantitative external predictions with these models for the permeability through Caco-2 cells.

La diversité des maladies des patients de réanimation fait que l’environnement technologique de soins intensifs est composé de nombreux systèmes de monitorage de constantes physiologiques, permettant à l’équipe soignante de déterminer un traitement adapté au patient. De plus en plus de systèmes ont une fonction d’assistance à la prescription ou aux soins afin de réduire la charge de travail et mentale des infirmières exigées par certains traitements ou protocoles. Ces systèmes informatisés facilitent l’intégration du protocole et de l’information disponible dans les signaux et peuvent aboutir à des systèmes de complexité élevée. La question de l’évaluation de la qualité de la réalisation et de la balance bénéfice/risque pour les patients liée à l’usage de nouveaux systèmes d’assistance se pose alors. Un problème consiste à mener cette évaluation a priori dès la conception du protocole ou de l’algorithme. Ce travail de thèse donne un exemple de méthode pour un système d’assistance au contrôle de la glycémie des patients de réanimation : l’évaluation est menée depuis la conception de l’algorithme de contrôle jusqu’à une étude clinique de grande ampleur. L’origine du dispositif étudié vient de ce que les patients présentent souvent une hyperglycémie liée au stress à leur arrivée en réanimation (l’augmentation de l’insulino-résistance, l’administration de certaines de drogues ou la déficience en insuline inhibent la réponse physiologique à l’augmentation de la glycémie). Un problème vient alors de ce que, d’une part, l’hyperglycémie prolongée étant associée à une morbidité voire une mortalité accrue, contrôler la glycémie est bénéfique, et d’autre part réduire la glycémie fait courir le risque d’épisodes hypoglycémiques pendant le séjour en réanimation. Dans ce cas, optimiser la balance bénéfice/risque est encore un problème ouvert. Bien qu’il existe de nombreux travaux sur la modélisation de la pharmacodynamique du système glucose-insuline, peu de travaux exploitent ces modèles pour fournir un système de contrôle fonctionnel, testé et industrialisable. La thèse présente un système de contrôle glycémique ainsi qu’une méthode d’évaluation généralisable à d’autres systèmes, qui teste numériquement les performances techniques et cliniques de ce type de systèmes sur des patients virtuels. Les résultats d’une étude clinique réelle sont aussi présentés. / The variety of ICU patients diseases implies that technological environment of critical care is composed of many vital signs monitoring systems, allowing the medical team to determine appropriate treatment to the patient. More and more systems have a decision-making support function to reduce the mental and physical workload of nurses required for certain treatments or protocols. These computerized systems facilitate the integration of the protocol and the information available in the signals and can lead to systems of high complexity. The issue of assessing the performances and the benefit/risk balance for the patient related to the use of new support systems arises. The problem is to conduct this evaluation a priori, during the design process of the protocol or algorithm. This work provides an example of a method to support the control of blood glucose in the ICU system evaluation is conducted for the design of the control algorithm to a large-scale clinical study.The origin of the studied device comes from the fact that patients often experience hyperglycemia due to stress upon their arrival in the ICU (increased insulin resistance, administration of interacting drugs or insulin deficiency inhibit the physiological response to the blood sugar increase). A problem then is that, on the one hand, as sustained hyperglycemia is associated with an increased morbidity or mortality, controlling glycaemia reduces risks, but, on the other hand, reducing blood sugar exposes to the risk of hypoglycemia during the ICU stay. In this case, optimizing benefit/risk ratio is still an open problem.Although there are many studies on modeling the pharmacodynamics of glucose-insulin system, few works use these models to provide a functional, tested and industrialized control system. The thesis presents a glycemic control system and a generalized method of evaluation with other systems, which tests digitally technical and clinical performances of such systems on virtual patients. The results of a real clinical trial are also presented.

Staphylococcus aureus resistente à meticilina (MRSA, Methicillin-Resistant Staphylococcus aureus) é um dos principais responsáveis por infecções nosocomiais, sendo identificado também em infecções associadas à comunidade. Embora potentes fármacos anti-estafilocócicos estejam disponíveis, as infecções causadas por este patógeno continuam a apresentar significativa morbidade e mortalidade devido ao aparecimento de cepas com resistência a múltiplos fármacos, incluindo vancomicina e teicoplanina. Compostos 5-nitro-heterocíclicos com estrutura análoga à nifuroxazida, antimicrobiano utilizado em infecções gastrintestinais, têm apresentado satisfatória atividade in vitro frente a estas cepas multirresistentes, sendo importante e necessária a avaliação de sua biodisponibilidade oral como próximo estágio no desenvolvimento de um novo fármaco, visando à seleção eficiente e ao aprimoramento da estrutura molecular. Neste contexto, o presente estudo tem por objetivo empregar ensaios in vitro, utilizando células Caco-2, e métodos in silico, utilizando descritores moleculares VolSurf, a fim de analisar a permeabilidade de análogos à nifuroxazida com atividade antimicrobiana apresentando, principalmente, atividade potencial frente a cepas multirresistentes de S. aureus. Empregou-se o método de MTT (brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difenil tetrazólio) para a avaliação da citotoxicidade. Nos estudos de permeabilidade in vitro foram utilizadas membranas de células Caco-2 cultivadas em placas Transwel® por 21 dias. A quantificação das frações permeadas foi realizada por cromatografia a líquido de alta eficiência com detecção UV (CLAE-UV), com métodos validados de acordo com a Resolução 899/03. Os estudos in silico foram realizados por meio de análise exploratória, pelo método de consenso de análise de componentes principais (CPCA, Consensus Principal Component Analysis), e análise de regressão, por quadrados mínimos parciais (PLS, Partial Least Squares). As células Caco-2 apresentaram viabilidade adequada para a realização dos estudos de permeabilidade frente a todos os derivados da nifuroxazida, exceto o derivado MeTIO (5-nitro-2-tiofilideno 4-metilbenzidrazida). Os valores de permeabilidade aparente (Papp) obtidos para os análogos à nifuroxazida indicam que estes possuem alta permeabilidade. Os modelos obtidos por CPCA e PLS foram capazes de separar as moléculas em grupos de compostos de baixa, média e alta permeabilidade, sendo os análogos à nifuroxazida classificados como compostos de alta permeabilidade. Para os compostos em estudo, as propriedades determinantes da permeabilidade através de células Caco-2, de acordo com os modelos, seriam de natureza topológica e estérica, sendo possível a previsão externa qualitativa e quantitativa da permeabilidade através de células Caco-2. / Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens responsible for nosocomial infections, also identified in community-associated infections. Although potent anti-staphylococcal drugs are available, infections caused by this bacteria continues to show significant morbidity and mortality due to the emergence of strains with resistance to multiple drugs, including vancomycin and teicoplanin. The 5-nitro-heterocyclic derivatives of nifuroxazide, which is an antimicrobial used to treat gastrointestinal infections, have shown satisfactory in vitro activity against multidrug-resistant strains of S. aureus. As a next step in the development of a new drug, it is important and necessary the evaluation of the oral bioavailability to achieve an efficient selection and refinement of the molecular structure. In this context, this study aims to develop in vitro assays through Caco-2 cells, and in silico approaches, using VolSurf molecular descriptors, in order to analyze the permeability of 5-nitro-heterocyclic compounds analogues to nifuroxazide with antimicrobial activity, especially showing promising activity against multidrug-resistant Staphylococcus aureus. The MTT (bromide 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyl tetrazolium) method was employed to perform the cytotoxicity evaluation. Caco-2 cell monolayers cultivated for 21 days in Transwel® plates were used for the in vitro permeability assays. The quantification of the permeated fractions was done by High Performance Liquid Chromatography with UV detection (HPLC-UV), with validated methods according to the Resolution 899/03. In silico studies were performed through exploratory analysis by consensus principal component analysis (CPCA) and regression analysis by partial least squares (PLS). Caco-2 cells showed suitable cell viability for the permeability studies against all nifuroxazide analogues except the MeTIO (5-nitro-2-thiophilydene 4-methylbenzidrazide). The nifuroxazide derivatives apparent permeability values (Papp) obtained indicate these are high permeable compounds. The models obtained by CPCA and PLS were able to separate the molecules into groups of low, medium and high permeabilities, and the nifuroxazide analogues were classified as high permeable substances. The identified properties for the permeability through Caco-2 cells to the studied compounds were topologic and sterical, and it is possible to perform qualitative and quantitative external predictions with these models for the permeability through Caco-2 cells.

Εξήντα έξη χρόνια μετά την εμφάνιση της πρώτης δημοσίευσης που αφορούσε στη θεραπεία με τη χρήση μουσταρδών αζώτου και ενώσεις που ανήκουν στη θεραπευτική αυτή κατηγορία, όπως το κυκλοφωσφαμίδιο και η μελφαλάνη εξακολουθούν να αποτελούν πρώτης γραμμής χημειοθεραπευτικούς παράγοντες τόσο κατά της λευχαιμίας όσο και έναντι άλλων συμπαγών όγκων. Οι διλειτουργικοί αλκυλιωτικοί παράγοντες, κατηγορία στην οποία ανήκουν και οι μουστάρδες αζώτου, επάγουν το θάνατο των καρκινικών κυττάρων και κατά συνέπεια και την κυτταροτοξική τους δράση μέσω της δημιουργίας διαμοριακών χιαστί τύπου δεσμών μεταξύ των κλώνων του DNA. Οι φαρμακολογικές τους δράσεις σχετίζονται κυρίως με την σύνθεση του DNA των διαιρούμενων κυττάρων, προκαλώντας ουσιαστικά μια γενική αναστολή της κυτταρικής διαίρεσης. Ωστόσο, εκτός από την αναμενόμενη και επιθυμητή θεραπευτική δράση, προκαλούν τοξικές παρενέργειες, οι οποίες σχετίζονται με μεταλλαγή των κυττάρων της αρχέγονης σειράς, εμφάνιση καρκινογένεσης και τερατογένεσης. Το εύρος των μειονεκτημάτων που παρουσιάζουν και ειδικά η συστηματική τοξικότητα σε συνδυασμό με την υψηλή χημική in vivo δραστικότητα τους και την μειωμένη εκλεκτικότητα τους, είχαν ως αποτέλεσμα τον περιορισμό της χρήσης τους. Παρόλα αυτά κατέχουν εξέχουσα θέση στη θεραπευτική έναντι μιας μεγάλης ποικιλίας τύπων καρκίνου, συμπεριλαμβανομένου και των αιματολογικών νεοπλασμάτων. Στην προσπάθεια επίλυσης του προβλήματος της αυξημένης τοξικότητας και της απουσίας εκλεκτικότητας των ενώσεων της κατηγορίας αυτής, διάφορες μουστάρδες αζώτου συζεύχθηκαν μέσω εστερικού δεσμού με ποικίλους στεροειδικούς σκελετούς, στρατηγική που αποδείχθηκε ουσιαστικά ιδιαιτέρως αποτελεσματική. Αποτελέσματα μελετών τόσο της ερευνητικής μας ομάδας όσο και άλλων, υποστηρίζουν πως ο στεροειδικός σκελετός δεν μπορεί πλέον να θεωρηθεί μόνο ως απλός φορέας για την αποτελεσματική μεταφορά της αλκυλιωτικής ομάδας στις θέσεις στόχους του DNA, εφόσον μικρές δομικές τροποποιήσεις του επιφέρουν σημαντικές διαφοροποιήσεις στην αντιλευχαιμική δράση των τελικών στεροειδών εστέρων. Επιπρόσθετα, οι μελέτες αυτές έδειξαν ότι η παρουσία της –NHCO- ομάδας είτε ως ενδοκυκλικής λακταμικής είτε ως εξωκυκλικής αμιδικής στον Δ-δακτύλιο του στεροειδικού τμήματος είναι καθοριστικός παράγοντας για την εμφάνιση κυτταροτοξικής ή αντινεοπλασματικής δράσης στεροειδών εστέρων της χλωραμβουκίλης και αναλόγων αυτής. Μέχρι σήμερα έχει προκύψει ένας εκτεταμένος αριθμός στεροειδών εστέρων, από την σύζευξη απλών και τροποποιημένων στεροειδών με αρκετές διαφορετικές μουστάρδες αζώτου. Η πλειονότητα αυτών των παραγώγων έχουν υποβληθεί σε in vitro και in vivo βιολογικές δοκιμές έναντι των λευχαιμίων Ρ388 και L1210 και αρκετά από αυτά έχουν αποδειχθεί αποτελεσματικοί αντινεοπλασματικοί παράγοντες. Σε μια πρόσφατη 3D-QSAR μελέτη τριάντα οκτώ στεροειδών εστέρων τριών διαφορετικών μουσταρδών αζώτου με απλούς και τροποποιημένους στεροειδικούς σκελετούς, έγινε προσπάθεια διερεύνησης των 3D σχέσεων χημικής δομής-βιολογικής δραστικότητας, με σκοπό την εύρεση των απαραίτητων στερεοηλεκτρονικών χαρακτηριστικών των ενώσεων που συμβάλλουν στην εκδήλωση της αντιλευχαιμικής δράσης των. Η ανάλυση στηρίχθηκε στην χρήση των μεθοδολογιών CoMSIA και CoMFA. Τα δεδομένα που ελήφθησαν από τις 3D-QSAR αναλύσεις αξιοποιήθηκαν στη συνέχεια από ένα υπολογιστικό πρόγραμμα de novo σχεδιασμού και ανακάλυψης νέων βιοδραστικών μορίων, την εφαρμογή LeapFrog. Με βάση τα είκοσι βελτιωμένα στεροειδικά εστερικά παράγωγα που προτάθηκαν από το LeapFrog, επιλέχθηκαν να σχεδιαστούν και να συντεθούν οι ακόλουθες αρωματικές μουστάρδες αζώτου: α) 2-[π-Ν,Ν-δις(2-χλωροαιθυλ)αμινοφαινυλο]βουτυρικό οξύ (7), β) 2-ακεταμιδο-2-[π-Ν,Ν-δις(2-χλωροαιθυλ)αμινοφαινυλο]οξικό οξύ (30), γ) 2-ακεταμιδο-2-[ο-Ν,Ν-δις(2-χλωροαιθυλ)αμινοφαινυλο]οξικό οξύ (31) και δ) το 3-ακεταμιδο-3-[π-Ν,Ν-δις(2-χλωροαιθυλ)αμινοφαινυλο] προπανοϊκό οξύ (42), καθώς και τα εστεροποιημένα παράγωγα αυτών, με επιλεγμένους απλούς και τροποποιημένους στεροειδικούς σκελετούς. Η σύζευξη των νεων μουσταρδών με επιλεγμένες στεροειδικές αλκοόλες απέδωσε δεκαεπτά νέα εστερικά ανάλογα, η σύνθεση των οποίων είχε ως απώτερο σκοπό τη βελτίωση του θεραπευτικού δείκτη των μουσταρδών αζώτου καθώς και τον εμπλουτισμό της βιβλιοθήκης των αποτελεσμάτων που αφορούν στη σχέση χημικής δομής-βιολογικής δραστικότητας των ενώσεων της κατηγορίας αυτής. Συγκεκριμένα, για τη σύνθεση των παραπάνω μουσταρδών αζώτου, εφαρμόστηκε η κλασσική μεθοδολογία σύνθεσης. Ωστόσο, αυτή αποδείχθηκε αποδοτική μονό κατά τη σύνθεση της μουστάρδας 7, παρέχοντας το τελευταίο σε συνολική απόδοση 45%. Για τις υπόλοιπες μουστάρδες ακολουθήθηκαν εναλλακτικές πορείες σύνθεσης λόγω προβλημάτων που παρουσιάστηκαν κατά την εφαρμογή της κλασσικής μεθοδολογίας. Κρίσιμο στάδιο κατά τη σύνθεση των μουσταρδών αζώτου 30 και 31, αποτέλεσε η αρχική προστασία της ελεύθερης αμινομάδας της DL-α-φαινυλγλυκίνης (16) που χρησιμοποιήθηκε ως πρώτη ύλη της συνθετικής πορείας. Η διερεύνηση και η βελτιστοποίηση των πειραματικών συνθηκών ήταν απαραίτητη και κατά το στάδιο της χλωρίωσης των Ν,Ν-δις(2-υδροξυαιθυλ)-παραγώγων 26 και 27 για τον σχηματισμό της χαρακτηριστικής Ν,Ν-δις(2-χλωροαιθυλαμινο)-ομάδας των παραγώγων 30 και 31. Κατά τη σύνθεση του 3-ακεταμιδο-3-[π-Ν,Ν-δις(2-χλωροαιθυλ)αμινοφαινυλο]προπανοϊκού οξέος (42) ήταν απαραίτητη η βελτιστοποίηση παρασκευής των ενδιαμέσων 33 και 37. Μετά από διερευνητικές προσπάθειες κατέστη εφικτή η βελτίωση της απόδοσης σύνθεσης του παραγώγου 33 από 54% σύμφωνα με τη βιβλιογραφία σε 98%, καθώς και η σύνθεση του ακετάμιδο παραγώγου 37 τόσο σε λιγότερα συνθετικά στάδια και σε καλύτερη απόδοση σε σχέση με τα δημοσιευμένα βιβλιογραφικά δεδομένα. Σε επόμενο στάδιο της μελέτης σχεδιάσθηκε η εστεροποίηση των μουστάρδων 7, 30 και 42 με επιλεγμένους στεροειδικούς σκελετούς. Για τη σύνθεση των τελικών εστερικών στεροειδικών παραγώγων εφαρμόστηκε η μέθοδος των μικτών ανυδριτών, η οποία περιλαμβάνει την αντίδραση της αρωματικής μουστάρδας αζώτου με ελαφρά περίσσεια 2,4,6-τριχλωροβενζοϋλοχλωρίδιου παρουσία τριαιθυλαμίνης, το σχηματισμό του αντίστοιχου μικτού ανυδρίτη και στη συνέχεια in situ αντίδρασή του με τη στεροειδική αλκοόλη παρουσία 4-διμεθυλαμινοπυριδίνης. Τελικώς κατέστη εφικτή η σύνθεση δεκαεπτά τελικών στεροειδών εστέρων από τη σύζευξη των μουσταρδών αζώτου, 7 και 42 με επιλεγμένες στεροειδείς αλκοόλες. Παρά τις διερευνητικές προσπάθειες που καταβλήθησαν δεν κατέστη εφικτή η εστεροποίηση της μουστάρδας 30. Το σύνολο των τελικών στεροειδών εστέρων προωθήθηκαν σε βιολογικές μελέτες για την αποτίμηση της αντινεοπλασματικής τους δράσης. Τα διαθέσιμα αποτελέσματα αυτών των μελετών σχετίζονται με τα έντεκα στεροειδικά εστερικά παράγωγα της μουστάρδας 7 (2-PHE-BU). Τα μελετηθέντα αυτά παράγωγα επέδειξαν μειωμένη τοξικότητα και μια οριακή αντινεοπλασματική δραστικότητα έναντι της λευχαιμίας Ρ388, στην οποία είχαν μελετηθεί, συγκριτικά με την ελέυθερη αρωματική μουστάρδα (2-PHE-Bu). Ωστόσο, τα ληφθέντα βιολογικά αποτελέσματα ήταν υποδεέστερα των αντίστοιχων δεδομένων που είχαν συλλεχθεί κατά τη μελέτη του PHE και των στεροειδικών εστέρων αυτού έναντι και πάλι της λευχαιμίας Ρ388. Το γεγονός αυτό πιθανόν να οφείλεται στη μειωμένη ενζυμική υδρόλυση που υφίστανται in vivo τα παράγωγα αυτά, λόγω της στερεοχημικής παρεμπόδισης που προκαλεί η αιθυλική αλυσίδα γύρω από τον εστερικό δεσμό σύνδεσης μουστάρδας-στεροειδούς, γεγονός που συνεπάγεται μειωμένη απελευθέρωση της μουστάρδας αζώτου. Στο σημείο αυτό αξίζει να αναφερθεί πως αν και, τόσο οι νέες αρωματικές μουστάρδες αζώτου, όσο και τα στεροειδικά τους παράγωγα είχαν προταθεί ως υποψήφια παράγωγα με βελτιωμένη βιοδραστικότητα κατά την πραγματοποίηση de novo σχεδιασμού από το υπολογιστικό πρόγραμμα LeapFrog, τα αποτελέσματα των βιολογικών δοκιμών δεν επιβεβαιώσαν τις συγκεκριμένες ποτάσεις. Πιθανότατα, παράμετροι που δεν ελήφθησαν υπόψη κατά τον de novo σχεδιασμό από το LeapFrog, όπως η μεταβολική τροποποίηση των ενώσεων να εμπλέκονται και να καθορίζουν την βιοδραστικότητα του τελικών στεροειδικών εστέρων. Θεωρείται ότι μια αναθεώρηση κρίσιμων παραμέτρων όπως η χημική ποικιλομορφία και ο αριθμός των ενώσεων που εισέρχονται ως δεδομένα στις μελέτες 3D-QSAR μπορεί να αποδώσει μεγαλύτερης αξιοπίστιας μοντέλα CoMFA και CoMSIA. Τα νέα αυτά μοντέλα κατά την εισαγωγή τους σε υπολογιαστικά προγράμματα de novo σχεδιασμού μπορούν να συμβάλλουν στο σχεδιασμό νεών ενώσεων με πιθανά υπέρτερη βιοδραστικότητα. / Sixty-six years after the serendipitous discovery and the first publication referred to nitrogen mustard therapy, nitrogen mustards such as cyclophosphamide and melphalan are still front-line chemotherapeutic agents for the treatment of leukemia and various solid tumors. Several chemotherapeutics act as DNA-damaging agents resulting in cell cycle arrest and cell death of the uncontrollably proliferating cancer cells. Among them, bifunctional DNA alkylating agents, such as the nitrogen mustards, form intrestrand crosslinks (ICLs), extremely cytotoxic lesions, blocking DNA replication and transcription. However, cellular responses triggered by ICLs can cause resistance in tumor cells, limiting the efficacy of such treatment. Cytotoxicity, mutagenesis, and clastogenesis are attributed to their ability to damage DNA. Although, these drugs remain some of the most commonly prescribed chemotherapies for the treatment of various solid and hematological malignancies, particularly in combination with other classical or target therapeutics in multi-agent regimens, severe side effects on normal tissues comprise drawbacks of their use. These are attributed to their low selectivity to alkylate specific DNA bases due to their high inherent reactivity, resulting in the non-specific alkylation of other cellular nucleophilic species such as amino acid residues or low molecular weight thiols. Several approaches have been explored to reduce the toxicity and increase the therapeutic efficacy of nitrogen mustards. Among them, the generation of DNA-directed alkylating agents via the chemical linkage of nitrogen mustards with molecules of increased DNA-binding affinity and the synthesis of nitrogen mustard prodrugs led to interesting results. The chemical linkage of nitrogen mustards to carrier molecules (e.g steroids) with affinity for specific binding sites (nuclear receptors) has been used aiming at the improvement of the antineoplastic treatment. Our ongoing studies in this field have demonstrated that steroidal esteric derivatives of aromatic nitrogen mustards increase the damaging effects on specific DNA sequences and achieve better selectivity and reduced toxicity compared to nitrogen mustards themselves. Steroidal skeletons, which incorporate a –NHCO- moiety are considered more appropriate modules than the common or non-modified steroids, since their esters with aromatic nitrogen mustards, such as chlorambucil and its analogs, have been proved potent antileukemic agents. To this direction, our group has published a series of studies related with steroidal esters of aromatic nitrogen mustards as antineoplastic, especially antileukemic agents. Our efforts have succeeded in the identification of potent and promising derivatives with enhanced activity and reduced toxicity compared to the corresponding nitrogen mustards against in vitro and in vivo experimental tumors. Extensive structure-activity relationship (SAR) studies have demonstrated unique structural features of both the steroidal part and the nitrogen mustard which contribute substantially to the bioactivity of the target steroidal esters Furthermore, recent 3D QSAR/CoMFA and CoMSIA studies led to the generation of related models which indicated the influence of stereoelectronic and physicochemical parameters on the antileukemic activity of target compounds. The reliability of both models was evaluated and their predictive ability on the activity of a test set of compounds proved satisfactory. Furthermore, based on the proposed CoMFA model and using the de novo ligand design routine LeapFrog of SYBYL, a series of candidate molecules with potentially optimal bioactivity was proposed, creating new challenges in further investigation of this class of compounds. Prompted by the aforementioned results and extending our structure-activity relationship studies, we decided to investigate if the incorporation of in silico predictive nitrogen mustards on various simple and modified steroids might result to steroidal esters with improved antineoplastic activity. The selection of the nitrogen mustards which were synthesized and used in this study was based on the predictive antileukemic activity of their corresponding steroidal esters and the synthetic accessibility of the intermediate and target compounds. Thus, we designed and synthesized a new series of steroidal esters containing the aromatic nitrogen mustards 2-[4-N,N-bis(2-chloroethyl)amino-phenyl]butanoic acid (2-PHE-BU, 7), 2-acetamido-2-[p-N,N-bis(2-chloroethyl)amino-phenyl]acetic acid (30), 2-acetamido-2-[o-N,N-bis(2-chloroethyl)amino-phenyl]acetic acid (31) and 3-acetamido-3-[p-N,N-bis(2-chloroethyl)amino-phenyl]propanoic acid (42). The chemical linkage of nitrogen mustards 7 and 42 with simple and modified steroids led to the synthesis of seventeen new steroidal esters aiming at the investigation of new compounds with enhanced antineoplastic activity and higher therapeutic index. The synthesis of the aforementioned nitrogen mustards was based on a classic synthetic approach. However, this procedure proved efficient only for the synthesis of compound 7, in an overall yield of 45%. Key step for the synthesis of nitrogen mustards 30 and 31 was the protection of the free amino-group of DL--phenyglycine. Optimization of the experimental conditions were also necessary for the chlorination of N,N-bis(2-hydroxyethyl) intermediates 26 and 27, which led to the corresponding nitrogen mustards 30 and 31. During the synthesis of the 3-acetamido-3-[p-N,N-bis(2-chloroethyl)amino]propanoic acid (42), optimization of the procedure for the preparation of the intermediates 33 and 37 was also necessary. Our attempts resulted in the preparation of compound 37 in less steps and higher yield (98%) compared to that referred in the literature. The synthesis of the final steroidal esteric derivatives was based on the well established method of mixed anhydrides, which involves the reaction of the aromatic nitrogen mustard with a slight excess of 2,4,6-trichlorobenzoyl chloride in the presence of triethylamine, followed by the addition of steroidal alcohol in the presence of 4-dimethylaminopyridine. The newly synthesized alkylating steroidal esters exhibited reduced toxicity and slightly improved antileukemic activity against P388 leukemia bearing mice compared to the free nitrogen mustard 2-PHE-BU. Nevertheless, they did not prove superior to already synthesized, structurally related steroidal esters of PHE indicating that the enzymatic hydrolysis and the liberation of the nitrogen mustard in vivo is possibly disfavored due to the steric hindrance of the ethyl group around the formed ester bond. Nevertheless, the biological results obtained in the present study clearly indicate that except of the important stereoelectronic requirements which are considered and incorporated as data in LeapFrog routine, other parameters are possibly implicated and determine the bioactivity of the target steroidal esters. We expect that a careful revision of the implicated parameters such as the chemical diversity and the number of the tested compounds will allows us the generation of new CoMFA and CoMSIA models which subsequently will contribute to higher predicted reliability of the in silico design by software packages such as LeapFrog.