Le paclitaxel (Taxol®) et son analogue hémisynthétique le docetaxel (Taxotere®) appartiennent à une famille d'agents antitumoraux dont l'originalité réside dans leur structure tétracyclique inusuelle et complexe, et surtout dans leur mécanisme d'action. Ils ont en effet pour cible principale une protéine constitutive du fuseau mitotique, la tubuline.<br /> Bien que très utilisés en clinique (cancers du sein, de l'ovaire, du poumon), ils présentent un inconvénient majeur pour leur emploi : leur grande toxicité et leur mode d'administration induisent de graves effets secondaires. De plus, des phénomènes de résistance apparaissent également.<br /> Une méthode efficace d'amélioration de ces composés est de les transformer en prodrogues moins cytotoxiques et plus hydrosolubles. L'intérêt prépondérant de cette transformation apparaît dans la possibilité de délivrer ces produits de manière mieux ciblée, plus spécifique des tumeurs. Elles pourront alors être utilisés dans le cadre du concept PMT (Prodrug Mono Therapy) ou dans une stratégie immunociblée de type ADEPT (Antibody Directed Enzyme Prodrug Therapy).<br /><br /> Dans cette optique, des prodrogues glucuronylées du paclitaxel et du docetaxel ont été conçues et synthétisées. L'espaceur employé est un espaceur double, reliant un carbamate de p-hydroxybenzyle à une chaîne diamine. Ces prodrogues ont été évaluées biologiquement (stabilité, cytotoxicité, hydrolyse enzymatique) pour valider cette approche. L'utilisation du nouvel espaceur a donné des résultats satisfaisants, en particulier pour les hydrolyses enzymatiques qui ont été améliorées par rapport à celles des prodrogues décrites précédemment. La conception et la synthèse de différents espaceurs applicables à la préparation d'autres prodrogues sont également présentées.

Epoxy fatty acids (EXA) are valuable to industry as they are used in synthesizing plasticizers such as of poly vinyl chloride, resins, adhesives, coating materials such as paint, lubricant, lubricant additives, insecticides, insect repellants, crop oil concentrates and formulations of carriers for slow release pesticides and herbicides. There is interest in developing commercial oilseeds accumulating epoxy fatty acids to at least 50% of the seed oil. Soybeans are the most widely cultivated oilseed and its oil has high levels of linoleic acid which can be a substrate for epoxygenase enzymes. Cahoon et al., expressed a cytochrome P450 enzyme (CYP726A1) from Euphorbia lagascae in soybean somatic embryos and found that the epoxy fatty acid, vernolic acid, reached ~8% of the total fatty acids in transgenic somatic embryos. Rabbit Livers possess a cytochrome P450, CYP2C2, which catalyzes the same epoxidation reaction as the E. lagascae enzyme but might be less likely to be influenced by regulatory machinery in plant cells. This CYP2C2 gene was placed in a plant expression vector under a seed-specific promoter and used to transform soybean, Glycine max, somatic embryos. The ten putative transgenic clones observed after 4-5 weeks were separated and proliferated under selection. glucuronidase (GUS) assays and PCR analyses performed on selected clones were positive. However vernolic acid in total lipids and specific lipid classes was not detected as analyzed by GC. In vitro enzyme assay performed on microsomes isolated from mature somatic embryos at three weeks of maturation using [14C] 18:2 PC as substrate showed presence of [14C] methyl vernoleate. Preliminary analyses on toxicity of epoxy fatty acids and corresponding diols in bacteria, yeast and caco-2 cells showed that leukotoxin diol (LD) most toxic.

Au quotidien, nous sommes exposés à une multitude de xénobiotiques, des molécules exogènes à un organisme vivant et qui sont considérées toxiques pour ce dernier. Parmi tous ces xénobiotiques, le Bisphénol-A (BPA), un xénoestrogène, est l'un de ceux attirants le plus l'attention de la communauté scientifique. Des travaux effectués antérieurement par notre laboratoire ont mis en évidence des effets cytotoxiques sur les cellules placentaires (Benachour et Aris, 2009). À de très faibles concentrations, le BPA induit une augmentation significative de l'apoptose et de la nécrose des cytotrophoblastes. De plus, à ces mêmes concentrations, le BPA provoque une augmentation de l'expression et de la sécrétion de facteur de nécrose tumorale alpha (TNF-alpha), une cytokine clef dans les phénomènes d'inflammation. À un niveau cellulaire, la nécrose, l'apoptose ainsi que l'augmentation de la sécrétion de TNF-alpha sont des observations décrites dans les cas de prééclampsie (PE), de diabète gestationnel (DG) et de retard de croissance intra-utérin (RCIU). Ces travaux laissent croire à une plus grande accumulation du BPA chez les femmes ayant développé l'une de ces complications, ainsi qu'un rôle du BPA dans le développement physiopathologique de ces complications. Pour vérifier cette hypothèse, quatre groupes ont été formés, un groupe témoin où aucune pathologie n'est connue, un groupe de femmes ayant développé la prééclampsie, un groupe de femmes ayant développé un diabète gestationnel et finalement un groupe de femmes ayant eu une grossesse compliquée par un retard de croissance intra-utérin. Chacun de ces groupes était formé de 23 candidates sélectionnées dans une cohorte préexistante, la cohorte PÉRICARD. Pour chaque candidate, le Bisphénol A a été quantifié dans le sérum maternel, dans l'homogénat placentaire ainsi que dans le sérum foetal. Les dosages ont été effectués par chromatographie gazeuse couplée à un spectromètre de masse, l'une des méthodes les plus sensibles existantes pour la quantification du Bisphénol A. Nos résultats ont permis de mettre en évidence une accumulation différentielle du BPA dans le placenta de femmes ayant une grossesse avec prééclampsie et ayant mené à un retard de croissance intra-utérin. Le placenta de ces femmes a accumulé une plus forte quantité BPA que les femmes du groupe témoins. Ces résultats entérinent donc l'hypothèse d'une implication du BPA dans la prééclampsie et dans le retard de croissance intra-utérin.

Modifying substrate specificity of β-glucuronidase (GUS) would be helpful in various enzyme prodrug systems in delivering drug dose to the site of action in the cancer treatment. Due to the presence of endogenous enzyme in human tissues, GUS-based Antibody-Directed Enzyme Prodrug Therapy (ADEPT) requires a novel substrate to avoid undesirable systemic activation. GUS is a glycosyl hydrolase, highly specific towards the glucuronide derivatives. It catalyzes the glycosidic cleavage of β-D-glucuronides to β-D-glucuronic acid and aglycone moiety. In order to gain insight on the substrate specificity of GUS, C6 carboxyl group of glucuronic acid was modified to C6 carboxamide (amide derivative). We have examined amide derivatized substrates with a variety of different aglycone groups including p-nitrophenyl, phenyl and 4-methylumbelliferone to further probe the activity profile of GUS. In an effort to optimize GUS activity, docking studies have been performed which indicated that amino acid point mutations near C6 carboxyl group of glucuronic acid could improve binding of the derivatized substrates. As a result point mutations to Arg-562 and Lys-568 which make the active site less positively charged either by glutamine or glutamate lead to an enzyme with much lower native substrate activity but abolished activity for the amide-derivatized substrate. This research study showed that there is still a further need of finding appropriate mutations required to make glucuronamide a better substrate for the mutated version of GUS.

The purpose of our study was to describe the dynamics of E. coli in the rivers of the Seine watershed. This watershed is characterized by a poor microbiological water quality due to an important population density, industrial activities and intensive agriculture. The main objectives of our study were to obtain field microbiological data describing the system and to understand the underlying mechanisms in order to build a model able to describe and predict the fate of the fecal bacteria at the scale of the whole watershed. This model was built in order to help management of microbiological water quality. <p>To be able to describe and modelize the fate of fecal bacteria at the scale of such a large and complex system, it was first necessary to identify and quantify the sources of contamination and to be able to describe the processes affecting these bacteria once discharged to the rivers. <p>The quantification of E. coli in the natural aquatic systems through the traditional culture-based techniques has been strongly questioned during the last years because they have a long response delay (24 to 48 h) and they do not take into account the viable but not culturable bacteria (VBNC) which nevertheless could represent a health thread. For this reason we developed in this study two alternative methods: the direct (without passing through a cultivation step) measurement of the â-D-glucuronidase activity and a fluorescent in situ hybridization with an E. coli specific probe coupled with a viability test. The first has the main advangate to give a very rapid evaluation of the E. coli content of the water while the second one was able to enumerate VBNC E. coli. <p>The study of the sources of contamination of the rivers revealed the predominance, in this anthropogenicised watershed, of the point sources (effluents from wastewater tretament plants). Diffuse sources soil leaching and surface runoff) may nevertheless be locally important and have also be quantified. The influence of the land use on these diffuse sources has been quantified demonstrating that soil leaching and surface runoff of grazed areas was mainly responsible of fecal contamination in rural areas.<p>The attachment of E .coli to particles in the river waters was studied as it can influence the fate of this kind of bacteria in the aquatic systems. A linear regression was found between the percentage of particle-associated E. coli and the SM content of the water. Settling velocities of particle-associated E. coli were determined. The mortality rate of E. coli was analysed following various perspectives: the river order, the attachment of E. coli to particles, the importance of the cultivability loss in the disappearance of these bacteria and the importance of protozoan grazing. The results confirmed the importance of grazing and showed that the abondance of free living E. coli bacteria decreased two times more rapidly than particle-associated E. coli. In the Seine river, we showed that the net loss of culturable E. coli was 1.5 time higher than the net loss of viable E. coli. Finally, no relationship was found between mortality rate and river order. <p>These results have been used to build two models CF-SENEQUE and CF-SiAM-3D which describe E. coli dynamics respectively for the whole Seine river watershed and for the Seine for the estuary. Comparisons model calcultaions with field data of fecal coliforms abundances showed that these models correctly simulated the longitudinal distribution of fecal colifoms in the main rivers of the Seine watershed and in the estuary. These models were used to test the impact on the microbiological water quality of various scenarii of wastewater management .<p> / Doctorat en sciences, Spécialisation biologie animale / info:eu-repo/semantics/nonPublished

As Mucopolissacaridoses são erros inatos do metabolismo, fazem parte das doenças lisossômicas de depósito e ocorrem devido à deficiência na atividade de enzimas que catalisam a degradação de glicosaminoglicanos. O objetivo desse estudo foi aperfeiçoar o diagnóstico bioquímico das Mucopolissacaridoses dos tipos I, VI e VII, estabelecendo o uso do tampão fosfato de sódio 20 mmol/L pH 7,0 (tampão universal - TU) e outros parâmetros bioquímicos. Nesse trabalho foi aprimorada a técnica de medida de atividade da beta-glicuronidase (GUSB), enzima deficiente na MPS VII, reduzindo a quantidade de reagentes em 4 vezes e a utilização do tamanho dos picotes de sangue impregnado em papel filtro (SPF) para 1,2 mm. Estudamos a cinética da atividade da GUSB determinando o pH ótimo (4,4), Km (1,25 mM), Vmáx (594,48 nmol/h/mL), termoestabilidade (inativação significante da enzima a partir de 60 min a 60 ºC) e tempo e temperatura de armazenamento (até 30 dias à 4, 25 e 37 ºC, acima de 60 dias à -20 ºC) e estabelecemos um intervalo de referência para a atividade da GUSB em amostras de indivíduos saudáveis nessa metodologia (174,4 nmol/h/mL a 781,9 nmol/h/mL). Estabelecemos o uso do TU para determinação das atividades da alfa-iduronidase (IDUA), arilsulfatase B (ASB) e GUSB medindo a atividade enzimática em SPF eluído nesse tampão e correlacionamos com a técnica espectrofluorimétrica já padronizada para cada enzima em SPF de 1,2 mm em amostras de indivíduos saudáveis As correlações foram positivas e os coeficientes de validação da técnica estavam dentro dos limites aceitáveis. As médias de atividade determinadas para indivíduos saudáveis foram: 14,65 + 4,35 nmol/h/mL (IDUA), 22,51 + 5,09 nmol/h/mL (ASB) e 531,92 + 121,05 nmol/h/mL (GUSB). Foram analisados parâmetros bioquímicos envolvidos em estresse oxidativo no plasma de indivíduos com MPS VI e comparados com MPS I e controles saudáveis. A medida da atividade da SOD não diferiu entre os grupos, a atividade de CAT encontrava-se diminuída tanto em MPS VI quanto em MPS I e a dosagem de TBARS estava aumentada em ambas as MPS em relação aos controles. A partir desse estudo, foi possível padronizarmos e aperfeiçoarmos novas técnicas para o diagnóstico laboratorial para a MPS I, VI e VII além de introduzir o estresse oxidativo como um possível marcador no uso da terapia de reposição enzimática. / Mucopolysaccharidoses are inborn errors of metabolism, being part of lysosomal storage diseases and occuring due to deficiency in the activity of enzymes that catalyze the degradation of glycosaminoglycans. The aim of this study was to improve the biochemical diagnosis of Mucopolysaccharidoses of types I, VI and VII, establishing the use of 20 mmol/L sodium phosphate buffer pH 7.0 (universal extraction buffer - UEB) and other biochemical parameters. In this work, the activity measurement technique of beta-glucuronidase (GUSB), enzyme deficient in MPS VII, has been improved, reducing the amount of reagents in 4 times and using the size of dried blood spots (DBS) for 1.2 mm. We studied the kinetics of GUSB activity by determining the optimum pH (4.4), Km (1.25 mM), Vmax (594.48 nmol/h/mL), thermostability (significant inactivation of the enzyme from 60min at 60 ºC) and storage time and temperature (up to 30 days at 4, 25 and 37 °C, above 60 days at -20 °C) and established a reference range for GUSB activity in samples from healthy subjects in this methodology (174.4 nmol/h/mL at 781.9 nmol/h/ mL). We established the use of TU to determine the activities of alpha-iduronidase (IDUA), arylsulfatase B (ASB) and GUSB by measuring the enzymatic activity in DBS eluted in this buffer and correlated with the standardized spectrofluorometric technique for each enzyme in DBS of 1.2 mm in samples from healthy individuals Correlations were positive and the validation coefficients of the technique were within acceptable limits. The activity means determined for healthy individuals were 14.65 ± 4.35 nmol/h/mL (IDUA), 22.51 ± 5.09 nmol/h/mL (ASB) and 531.92 ± 121.05 nmol/h/mL (GUSB). Biochemical parameters involved in oxidative stress in the plasma of individuals with MPS VI and compared to MPS I and healthy controls were analyzed. Measurement of SOD activity did not differ between groups, CAT activity was decreased in both MPS VI and MPS I and the TBARS dosage was increased in both MPS compared to controls. From this study, it was possible to standardize and improve new techniques for laboratory diagnosis for MPS I, VI and VII, besides introducing oxidative stress as a possible marker in the use of enzyme replacement therapy.

As Mucopolissacaridoses são erros inatos do metabolismo, fazem parte das doenças lisossômicas de depósito e ocorrem devido à deficiência na atividade de enzimas que catalisam a degradação de glicosaminoglicanos. O objetivo desse estudo foi aperfeiçoar o diagnóstico bioquímico das Mucopolissacaridoses dos tipos I, VI e VII, estabelecendo o uso do tampão fosfato de sódio 20 mmol/L pH 7,0 (tampão universal - TU) e outros parâmetros bioquímicos. Nesse trabalho foi aprimorada a técnica de medida de atividade da beta-glicuronidase (GUSB), enzima deficiente na MPS VII, reduzindo a quantidade de reagentes em 4 vezes e a utilização do tamanho dos picotes de sangue impregnado em papel filtro (SPF) para 1,2 mm. Estudamos a cinética da atividade da GUSB determinando o pH ótimo (4,4), Km (1,25 mM), Vmáx (594,48 nmol/h/mL), termoestabilidade (inativação significante da enzima a partir de 60 min a 60 ºC) e tempo e temperatura de armazenamento (até 30 dias à 4, 25 e 37 ºC, acima de 60 dias à -20 ºC) e estabelecemos um intervalo de referência para a atividade da GUSB em amostras de indivíduos saudáveis nessa metodologia (174,4 nmol/h/mL a 781,9 nmol/h/mL). Estabelecemos o uso do TU para determinação das atividades da alfa-iduronidase (IDUA), arilsulfatase B (ASB) e GUSB medindo a atividade enzimática em SPF eluído nesse tampão e correlacionamos com a técnica espectrofluorimétrica já padronizada para cada enzima em SPF de 1,2 mm em amostras de indivíduos saudáveis As correlações foram positivas e os coeficientes de validação da técnica estavam dentro dos limites aceitáveis. As médias de atividade determinadas para indivíduos saudáveis foram: 14,65 + 4,35 nmol/h/mL (IDUA), 22,51 + 5,09 nmol/h/mL (ASB) e 531,92 + 121,05 nmol/h/mL (GUSB). Foram analisados parâmetros bioquímicos envolvidos em estresse oxidativo no plasma de indivíduos com MPS VI e comparados com MPS I e controles saudáveis. A medida da atividade da SOD não diferiu entre os grupos, a atividade de CAT encontrava-se diminuída tanto em MPS VI quanto em MPS I e a dosagem de TBARS estava aumentada em ambas as MPS em relação aos controles. A partir desse estudo, foi possível padronizarmos e aperfeiçoarmos novas técnicas para o diagnóstico laboratorial para a MPS I, VI e VII além de introduzir o estresse oxidativo como um possível marcador no uso da terapia de reposição enzimática. / Mucopolysaccharidoses are inborn errors of metabolism, being part of lysosomal storage diseases and occuring due to deficiency in the activity of enzymes that catalyze the degradation of glycosaminoglycans. The aim of this study was to improve the biochemical diagnosis of Mucopolysaccharidoses of types I, VI and VII, establishing the use of 20 mmol/L sodium phosphate buffer pH 7.0 (universal extraction buffer - UEB) and other biochemical parameters. In this work, the activity measurement technique of beta-glucuronidase (GUSB), enzyme deficient in MPS VII, has been improved, reducing the amount of reagents in 4 times and using the size of dried blood spots (DBS) for 1.2 mm. We studied the kinetics of GUSB activity by determining the optimum pH (4.4), Km (1.25 mM), Vmax (594.48 nmol/h/mL), thermostability (significant inactivation of the enzyme from 60min at 60 ºC) and storage time and temperature (up to 30 days at 4, 25 and 37 °C, above 60 days at -20 °C) and established a reference range for GUSB activity in samples from healthy subjects in this methodology (174.4 nmol/h/mL at 781.9 nmol/h/ mL). We established the use of TU to determine the activities of alpha-iduronidase (IDUA), arylsulfatase B (ASB) and GUSB by measuring the enzymatic activity in DBS eluted in this buffer and correlated with the standardized spectrofluorometric technique for each enzyme in DBS of 1.2 mm in samples from healthy individuals Correlations were positive and the validation coefficients of the technique were within acceptable limits. The activity means determined for healthy individuals were 14.65 ± 4.35 nmol/h/mL (IDUA), 22.51 ± 5.09 nmol/h/mL (ASB) and 531.92 ± 121.05 nmol/h/mL (GUSB). Biochemical parameters involved in oxidative stress in the plasma of individuals with MPS VI and compared to MPS I and healthy controls were analyzed. Measurement of SOD activity did not differ between groups, CAT activity was decreased in both MPS VI and MPS I and the TBARS dosage was increased in both MPS compared to controls. From this study, it was possible to standardize and improve new techniques for laboratory diagnosis for MPS I, VI and VII, besides introducing oxidative stress as a possible marker in the use of enzyme replacement therapy.

As Mucopolissacaridoses são erros inatos do metabolismo, fazem parte das doenças lisossômicas de depósito e ocorrem devido à deficiência na atividade de enzimas que catalisam a degradação de glicosaminoglicanos. O objetivo desse estudo foi aperfeiçoar o diagnóstico bioquímico das Mucopolissacaridoses dos tipos I, VI e VII, estabelecendo o uso do tampão fosfato de sódio 20 mmol/L pH 7,0 (tampão universal - TU) e outros parâmetros bioquímicos. Nesse trabalho foi aprimorada a técnica de medida de atividade da beta-glicuronidase (GUSB), enzima deficiente na MPS VII, reduzindo a quantidade de reagentes em 4 vezes e a utilização do tamanho dos picotes de sangue impregnado em papel filtro (SPF) para 1,2 mm. Estudamos a cinética da atividade da GUSB determinando o pH ótimo (4,4), Km (1,25 mM), Vmáx (594,48 nmol/h/mL), termoestabilidade (inativação significante da enzima a partir de 60 min a 60 ºC) e tempo e temperatura de armazenamento (até 30 dias à 4, 25 e 37 ºC, acima de 60 dias à -20 ºC) e estabelecemos um intervalo de referência para a atividade da GUSB em amostras de indivíduos saudáveis nessa metodologia (174,4 nmol/h/mL a 781,9 nmol/h/mL). Estabelecemos o uso do TU para determinação das atividades da alfa-iduronidase (IDUA), arilsulfatase B (ASB) e GUSB medindo a atividade enzimática em SPF eluído nesse tampão e correlacionamos com a técnica espectrofluorimétrica já padronizada para cada enzima em SPF de 1,2 mm em amostras de indivíduos saudáveis As correlações foram positivas e os coeficientes de validação da técnica estavam dentro dos limites aceitáveis. As médias de atividade determinadas para indivíduos saudáveis foram: 14,65 + 4,35 nmol/h/mL (IDUA), 22,51 + 5,09 nmol/h/mL (ASB) e 531,92 + 121,05 nmol/h/mL (GUSB). Foram analisados parâmetros bioquímicos envolvidos em estresse oxidativo no plasma de indivíduos com MPS VI e comparados com MPS I e controles saudáveis. A medida da atividade da SOD não diferiu entre os grupos, a atividade de CAT encontrava-se diminuída tanto em MPS VI quanto em MPS I e a dosagem de TBARS estava aumentada em ambas as MPS em relação aos controles. A partir desse estudo, foi possível padronizarmos e aperfeiçoarmos novas técnicas para o diagnóstico laboratorial para a MPS I, VI e VII além de introduzir o estresse oxidativo como um possível marcador no uso da terapia de reposição enzimática. / Mucopolysaccharidoses are inborn errors of metabolism, being part of lysosomal storage diseases and occuring due to deficiency in the activity of enzymes that catalyze the degradation of glycosaminoglycans. The aim of this study was to improve the biochemical diagnosis of Mucopolysaccharidoses of types I, VI and VII, establishing the use of 20 mmol/L sodium phosphate buffer pH 7.0 (universal extraction buffer - UEB) and other biochemical parameters. In this work, the activity measurement technique of beta-glucuronidase (GUSB), enzyme deficient in MPS VII, has been improved, reducing the amount of reagents in 4 times and using the size of dried blood spots (DBS) for 1.2 mm. We studied the kinetics of GUSB activity by determining the optimum pH (4.4), Km (1.25 mM), Vmax (594.48 nmol/h/mL), thermostability (significant inactivation of the enzyme from 60min at 60 ºC) and storage time and temperature (up to 30 days at 4, 25 and 37 °C, above 60 days at -20 °C) and established a reference range for GUSB activity in samples from healthy subjects in this methodology (174.4 nmol/h/mL at 781.9 nmol/h/ mL). We established the use of TU to determine the activities of alpha-iduronidase (IDUA), arylsulfatase B (ASB) and GUSB by measuring the enzymatic activity in DBS eluted in this buffer and correlated with the standardized spectrofluorometric technique for each enzyme in DBS of 1.2 mm in samples from healthy individuals Correlations were positive and the validation coefficients of the technique were within acceptable limits. The activity means determined for healthy individuals were 14.65 ± 4.35 nmol/h/mL (IDUA), 22.51 ± 5.09 nmol/h/mL (ASB) and 531.92 ± 121.05 nmol/h/mL (GUSB). Biochemical parameters involved in oxidative stress in the plasma of individuals with MPS VI and compared to MPS I and healthy controls were analyzed. Measurement of SOD activity did not differ between groups, CAT activity was decreased in both MPS VI and MPS I and the TBARS dosage was increased in both MPS compared to controls. From this study, it was possible to standardize and improve new techniques for laboratory diagnosis for MPS I, VI and VII, besides introducing oxidative stress as a possible marker in the use of enzyme replacement therapy.