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Objetivo: Contribuir para o desenvolvimento do candidato a antitumoral (LaSOM 65) através da avaliação farmacocinética pré-clínica em roedores de diferentes doses pelas vias i.v., p.o e i.p. e avaliação da toxicidade aguda do composto. Metodologia: LaSOM 65 foi administrado a ratos Wistar nas doses de 1 mg/kg i.v. bolus (n = 8), 10 e 30 mg/kg p.o. e 30 e 90 mg/kg i.p. (n = 6/grupo). As concentrações plasmáticas foram quantificadas por CL-UV em método desenvolvido e validado. A ligação às proteínas foi determinada por ultrafiltração e a distribuição tecidual foi avaliada por homogeneizado de tecido após administração i.v. de 1 mg/kg (n = 3 animais/ponto de coleta). Para o ensaio de toxicidade aguda, dose única de 1, 2,5 e 5 mg/kg i.v. e 50, 100 e 150 mg/kg p.o. de LaSOM 65 foi administrada aos animais. Ganho de peso, massa relativa dos tecidos, determinação de parâmetros bioquímicos e hematológicos e uma observação clínica detalhada foram realizados para a avaliação de possíveis efeitos tóxicos. Resultados e Discussão: LaSOM 65 apresentou farmacocinética linear na faixa de dose de 1 a 30 mg/kg (i.v., p.o. e i.p.). Após dose i.v. apresentou um CL = 0,85 ± 0,18 L/h/kg, t1/2 = 1,8 ± 0,7 h e Vd = 1,76 ± 0,24 L/kg. Para a dose de 90 mg/kg i.p. um aumento no Vd (3,2 ± 0,8L/Kg) e t1/2 (2,9 ± 0,9 h) foi observado. O composto apresentou boa biodisponibilidade para as administrações extravasculares (58 e 50% p.o. e 73 e 61% i.p.). A ligação às proteínas foi de 84,7 ± 1,6%. O composto distribuiu-se nos tecidos investigados tendo no pulmão a maior taxa de penetração (ASCtecido/plasma = 2,7) e o cérebro a menor (ASCtecido/plasma = 0,4). Piloereção, diarréia, letargia e dispneia foram os sinais clínicos observados imediatamente após administrações i.v., os quais regrediram após 3 h. Para a via oral nenhuma alteração foi observada. Nenhum outro parâmetro avaliado demonstrou efeitos tociológicos para o LaSOM 65. Conclusões: LaSOM 65 demonstrou uma rápida distribuição tecidual e uma rápida eliminação após administração i.v. Sua boa biodisponibilidade e uma possível ausência de toxicidade apresentada por esta via permitem seu uso oral. A baixa penetração cerebral sugere que desenvolvimento no âmbito farmacotécnico será necessário visando seu uso para o tratamento de gliomas. / Purpose: To contribute with the development of a new anticancer candidate (LaSOM 65) by investigating its pre-clinical pharmacokinetics in rodents after administration of different doses by three routes (i.v., p.o. and i.p.) and acute toxicological evaluation of the compound. Methodology: LaSOM 65 was administrated to Wistar rats in the 1 mg/kg i.v. bolus (n = 8), 10 and 30 mg/kg p.o. and 30 and 90 mg/kg i.p. dose (n = 6/group). Plasma concentrations were determined by a development and validated LC-UV method. Protein binding was determined by ultrafiltration and tissue penetration was investigated in tissue homogenates after i.v. administration of 1 mg/kg (n = 3 animals/time point). For the acute toxicological assay, single administration of LaSOM 65 at the doses of 1, 2.5 and 5 mg/kg i.v. and 50, 100 and 150 mg/kg p.o. were given to the rats. Weight gain, organ relative mass, biochemical and hematological parameters and a detailed clinical observation were evaluated to determine LaSOM 65 toxicity. Results and Discussion: LaSOM 65 showed linear pharmacokinetic between 1 and 30 mg/kg (i.v., p.o and i.p.) with CL = 0.85 ± 0.18 L/h/kg, t1/2 = 1,8 ± 0,7 h and Vd = 1.76 ± 0.24 L/kg after i.v. dosing. After 90 mg/kg i.p. dosing an increase in Vd (3.2 ± 0.8 L/kg) and t1/2 (2.9 ± 0.9 h) were observed. The compound showed good bioavailability after p.o. (58 and 50%) and i.p. (73 and 61%) dosing. The protein biding was 84.7 ± 1.6%. LaSOM 65 distributed into the tissues investigated with a higher penetration ratio into lung (AUCtissue/plasma = 2.7) than into brain (AUCtissue/plasma = 0.4). Piloerection, diarrhea, lethargy and dyspnea were the clinical signs observed immediately after the i.v. administrations and they regressed 3 h post-dosing. No alterations were observed after p.o. dosing. Conclusions: LaSOM 65 showed a rapid tissue distribution and elimination after i.v. administration. Its good bioavailability together with the probably absence of toxicity for the oral route allowed its use by this route. The low brain penetration suggests that a pharmaceutical development will be necessary if LaSOM 65 is intended for the treatment of glioma.

Objetivo: Contribuir para o desenvolvimento do candidato a antitumoral (LaSOM 65) através da avaliação farmacocinética pré-clínica em roedores de diferentes doses pelas vias i.v., p.o e i.p. e avaliação da toxicidade aguda do composto. Metodologia: LaSOM 65 foi administrado a ratos Wistar nas doses de 1 mg/kg i.v. bolus (n = 8), 10 e 30 mg/kg p.o. e 30 e 90 mg/kg i.p. (n = 6/grupo). As concentrações plasmáticas foram quantificadas por CL-UV em método desenvolvido e validado. A ligação às proteínas foi determinada por ultrafiltração e a distribuição tecidual foi avaliada por homogeneizado de tecido após administração i.v. de 1 mg/kg (n = 3 animais/ponto de coleta). Para o ensaio de toxicidade aguda, dose única de 1, 2,5 e 5 mg/kg i.v. e 50, 100 e 150 mg/kg p.o. de LaSOM 65 foi administrada aos animais. Ganho de peso, massa relativa dos tecidos, determinação de parâmetros bioquímicos e hematológicos e uma observação clínica detalhada foram realizados para a avaliação de possíveis efeitos tóxicos. Resultados e Discussão: LaSOM 65 apresentou farmacocinética linear na faixa de dose de 1 a 30 mg/kg (i.v., p.o. e i.p.). Após dose i.v. apresentou um CL = 0,85 ± 0,18 L/h/kg, t1/2 = 1,8 ± 0,7 h e Vd = 1,76 ± 0,24 L/kg. Para a dose de 90 mg/kg i.p. um aumento no Vd (3,2 ± 0,8L/Kg) e t1/2 (2,9 ± 0,9 h) foi observado. O composto apresentou boa biodisponibilidade para as administrações extravasculares (58 e 50% p.o. e 73 e 61% i.p.). A ligação às proteínas foi de 84,7 ± 1,6%. O composto distribuiu-se nos tecidos investigados tendo no pulmão a maior taxa de penetração (ASCtecido/plasma = 2,7) e o cérebro a menor (ASCtecido/plasma = 0,4). Piloereção, diarréia, letargia e dispneia foram os sinais clínicos observados imediatamente após administrações i.v., os quais regrediram após 3 h. Para a via oral nenhuma alteração foi observada. Nenhum outro parâmetro avaliado demonstrou efeitos tociológicos para o LaSOM 65. Conclusões: LaSOM 65 demonstrou uma rápida distribuição tecidual e uma rápida eliminação após administração i.v. Sua boa biodisponibilidade e uma possível ausência de toxicidade apresentada por esta via permitem seu uso oral. A baixa penetração cerebral sugere que desenvolvimento no âmbito farmacotécnico será necessário visando seu uso para o tratamento de gliomas. / Purpose: To contribute with the development of a new anticancer candidate (LaSOM 65) by investigating its pre-clinical pharmacokinetics in rodents after administration of different doses by three routes (i.v., p.o. and i.p.) and acute toxicological evaluation of the compound. Methodology: LaSOM 65 was administrated to Wistar rats in the 1 mg/kg i.v. bolus (n = 8), 10 and 30 mg/kg p.o. and 30 and 90 mg/kg i.p. dose (n = 6/group). Plasma concentrations were determined by a development and validated LC-UV method. Protein binding was determined by ultrafiltration and tissue penetration was investigated in tissue homogenates after i.v. administration of 1 mg/kg (n = 3 animals/time point). For the acute toxicological assay, single administration of LaSOM 65 at the doses of 1, 2.5 and 5 mg/kg i.v. and 50, 100 and 150 mg/kg p.o. were given to the rats. Weight gain, organ relative mass, biochemical and hematological parameters and a detailed clinical observation were evaluated to determine LaSOM 65 toxicity. Results and Discussion: LaSOM 65 showed linear pharmacokinetic between 1 and 30 mg/kg (i.v., p.o and i.p.) with CL = 0.85 ± 0.18 L/h/kg, t1/2 = 1,8 ± 0,7 h and Vd = 1.76 ± 0.24 L/kg after i.v. dosing. After 90 mg/kg i.p. dosing an increase in Vd (3.2 ± 0.8 L/kg) and t1/2 (2.9 ± 0.9 h) were observed. The compound showed good bioavailability after p.o. (58 and 50%) and i.p. (73 and 61%) dosing. The protein biding was 84.7 ± 1.6%. LaSOM 65 distributed into the tissues investigated with a higher penetration ratio into lung (AUCtissue/plasma = 2.7) than into brain (AUCtissue/plasma = 0.4). Piloerection, diarrhea, lethargy and dyspnea were the clinical signs observed immediately after the i.v. administrations and they regressed 3 h post-dosing. No alterations were observed after p.o. dosing. Conclusions: LaSOM 65 showed a rapid tissue distribution and elimination after i.v. administration. Its good bioavailability together with the probably absence of toxicity for the oral route allowed its use by this route. The low brain penetration suggests that a pharmaceutical development will be necessary if LaSOM 65 is intended for the treatment of glioma.

Objetivos: Avaliar a farmacocinética e a distribuição tecidual do benzaldeído semicarbazona livre (BS) e incluso em ß-Ciclodextrinas (BS/ß-CD) após administração de diversas doses por diferentes vias de administração. Metodologia: As concentrações plasmáticas de BS foram quantificadas através de método analítico por CLAE-UV desenvolvido e validado, após administração das doses de 10 mg/kg i.v. bolus e 50 e 100 mg/kg p.o. da droga livre e 10 mg/kg i.v. bolus e 50 mg/kg p.o. da droga complexada a ratos Wistar (n = 8 animais/grupo). Os perfis plasmáticos foram avaliados individualmente pelas abordagens não-compartimental e compartimental para determinação dos parâmetros farmacocinéticos. A avaliação compartimental foi realizada utilizando software Scientist 2.0.1 (Micromath®). A ligação do BS a proteínas plasmáticas foi determinada por ultrafiltração na faixa de 1,0 a 60,0 µg/mL. O perfil de penetração tecidual da droga livre e complexada foi investigado em diferentes órgãos utilizando o método de homogeneizado de tecido até 5 h após administração de dose 10 mg/kg i.v. A penetração cerebral também foi avaliada para a droga livre e inclusa em ß-CD após a dose de 50 mg/kg até 4 h após administração (n = 3 animais/tempo coleta). Resultados e Discussão: A fração livre do BS em plasma de ratos foi de 34 ± 5%. O modelo de um compartimento descreveu adequadamente todos os perfis plasmáticos estudados. Após doses intravenosa (10 mg/kg) e oral (50 mg/kg), parâmetros farmacocinéticos como Vd (1,6 ± 0,5 e 2,2 ± 0,8 L/kg, respectivamente) e Cltot (1,4 ± 0,5 and 1,8 ± 0,5 L/h×kg, respectivamente) foram maiores para o BS complexado em relação à droga livre, embora os t1/2 (0,8 ± 0,1 h-1) mantiveram-se similares (p < 0,05). A biodisponibilidade oral do BS/ß-CD (~37%) foi aproximadamente o dobro daquela observada para a droga livre (~20%). O fator de penetração cerebral após doses intravenosa (2,8) e oral (2,5), assim como tempo de residência médio, foram maiores para a droga complexada, independente da via de administração avaliada. Conclusões: A farmacocinética do BS livre e complexado mostra uma rápida distribuição tecidual e uma rápida eliminação. A maior penetração cerebral do complexo em relação à droga livre mostra que a ß-CD é uma estrutura capaz de vetorizar, reter e modificar a liberação do BS nesse órgão, explicando os achados farmacodinâmicos prévios. / Purpose: This study aimed to investigate the pharmacokinetics and tissue distribution of benzaldehyde semicarbazone (BS) free and complexed with ß- cyclodextrin (BS/ß-CD) after administration to rodents at different doses by diverse routes. Methodology: BS plasma concentrations were determinated in Wistar rats after administration of 10 mg/kg i.v bolus and 50 and 100 mg/kg p.o. for the free drug and 10 mg/kg i.v. bolus and 50 mg/kg for the BS/ß-CD (n = 8/group), using a HPLCUV method specifically developed and validated. Individual plasma profiles obtained were evaluated by non-compartmental and compartmental approaches, using the software Scientist 2.0.1 (MicroMath®), analysis to determine the pharmacokinetic parameters. BS protein binding was determined by ultrafiltration at a concentration range of 1.0 a 60.0 µg/mL. BS tissue penetration after free or ß-CD-complexed drug administration was investigated in different tissues homogenates up to 5 h after i.v. bolus dosing of 10 mg/kg dose. Brain penetration of the free and complexed drug was also evaluated up to 4 h after administration of 50 mg/kg p.o. dose (3 animals/time point). Results and Discussion: BS free fraction in plasma was 34 ± 5%. The one-compartmental model described adequately the plasma profiles of all groups investigated. After i.v. (10 mg/kg) and p.o. (50 mg/kg) doses, pharmacokinetic parameters such as Vd (1.6 ± 0.5 e 2.2 ± 0.8 L/kg, respectively) and CLtot (1.4 ± 0.5 and 1.8 ± 0.5 L/h×kg, respectively) were higher for the BS/ß-CD than for the free drug, although the t1/2 (0.8 ± 0.1 h-1) remained the same (p < 0.05). The oral bioavailability of the BS/ß-CD (~ 37%) was approximately 2-fold of that observed for the free BS (~ 20%). The brain penetration factor after i.v. (2.8) and p.o. (2.5) doses, as well as the mean residence time, were higher after BS/ß-CD dosing than after free drug dosing, regardless of the route administrated. Conclusions: BS pharmacokinetics (free and complexed) showed a fast tissue distribution and elimination. The higher brain penetration of the drug after the administration of the complex reveals that the ß-CD may be a potential system to carrier, retain and change the delivery of BS in this organ, explaining the previous pharmacodynamic results.

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Ilex paraguariensis (erva mate) é uma planta amplamente usada na América Latina sob a forma de infusão aquosa. Dentre as propriedades atribuídas a esta planta encontra-se a atividade antioxidante que sugere um papel importante desta droga vegetal na prevenção e tratamento de doenças associadas ao estresse oxidativo como a aterosclerose, fotocarcinogênese e fotoenvelhecimento, entre outras. No entanto alguns compostos presentes nesta planta se encontram sob a forma esterificada, o que poderia dificultar a sua adequada absorção. Uma maneira de aumentar a biodisponibilidade de antioxidantes em extratos vegetais é promover a sua hidrólise visando à liberação dos compostos ativos. O objetivo deste trabalho foi estudar comparativamente o extrato hidroetanólico de Ilex paraguariensis antes e após hidrólise enzimática quanto à composição fitoquímica, atividade antioxidante in vitro e in vivo, biodisponibilidade de compostos antioxidantes e distribuição tecidual destes compostos em animais de experimentação. Para tanto o extrato foi obtido por percolação etanol:água (50% v/v) e sua hidrólise realizada por reação enzimática. A caracterização fitoquímica foi realizada por cromatografia líquida de alta eficiência (CLAE) e espectrofotometria e a atividade antioxidante dos extratos pelos métodos de DPPH e ORAC. Para os ensaios in vivo os extratos (hidrolisado e não hidrolisado) foram administrados oralmente (por gavage) a ratos Wistar machos em sistema de dose única ou doses repetidas (30 dias). Coletou-se o sangue, pele, fígado e cérebro, analisando-se a concentração dos compostos de interesse e a atividade antioxidante destes tecidos pelo método de ORAC. O extrato apresentou boa atividade antioxidante e conteúdo fenólico, sendo que estes valores não foram significativamente alterados pela hidrólise. No entanto, a hidrólise possibilitou uma maior absorção dos compostos de interesse, aumentando a atividade antioxidante plasmática. / Ilex paraguariensis (yerba mate) is a plant broadly used in Latin America as an aqueous infusion. Among its biological properties is the antioxidant activity, which suggests a important role in prevention and treatment of oxidative stress associated diseases, such as atheroclerosis, photocarcinogenesis and photoaging among other. However some of the compounds responsible for that activity are, in crude plant extract, under esterified form, which could make absorption more difficult. An approach to increase the bioavailability of antioxidants from plant extracts is to submit the extract to hydrolysis in order to release the active compounds. The goal of this work was comparatively evaluate the hydroethanolic extract of Ilex paraguariensis before and after enzimatic hydrolysis concerning phytochemical composition, in vivo and in vitro antioxidant activity, bioavailability and tissue distribution of antioxidant compounds in rats. The extract was obtained by percolation with ethanol:water (50% v/v) and the hydrolysis performed by enzymatic reaction. The phytochemical characterization was performed by high performance liquid chromatography (HPLC) and spectrophotometry and the antioxidant activity by DPPH and ORAC methods. Hydrolyzed and non hydrolyzed extracts were given orally (by gavage) to male Wistar rats in single and multiple dose (30 days) regimen. Blood, skin, liver and brain were removed, and the concentration of antioxidant compounds and antioxidant activity by ORAC method were evaluated. The crude hydroethanolic extract showed antioxidant activity and phenolic content, but these values were not significantly changed by hydrolysis. However the hydrolysis increased the absorption of the compounds and the plasma antioxidant activity.

Orientadores: Hiroshi Aoyama, Celso Eduardo Benedetti / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T20:43:42Z (GMT). No. of bitstreams: 1 Medeiros_LucianadeCamposLeite_D.pdf: 2382438 bytes, checksum: d3106e1ce3acaecc348d589d473d3411 (MD5) Previous issue date: 2008 / Resumo: Em nosso laboratório foram purificadas quatro isoformas de fosfatases ácidas, a partir de sementes de soja quiescentes, tendo sido também estudadas suas propriedades cinéticas e físico-químicas. Estudos físicos e estruturais destas enzimas requerem uma grande quantidade da proteína pura, o que exigiria várias purificações convencionais, que, em geral, são bastante trabalhosas. Devido a este fato e a existência de poucas de tais proteínas clonadas e expressas, nos propusemos a clonar e expressar uma proteína tirosina fosfatase (PTP) de soja. O estudo da cinética da PTP recombinante revelou que esta enzima possui características típicas de uma fosfatase e maior especificidade para tirosina-fosfato em relação a outros substratos analisados. Foi feito o estudo de desnaturação térmica da enzima, através de dicroísmo circular, que mostrou que a enzima recombinante apresenta baixa estabilidade térmica. Identificamos por western blot a presença da GmPTP nos diferentes tecidos de soja, germinados tanto no claro como no escuro. Estes resultados, confrontados com os obtidos no PCR quantitativo, mostram uma expressão aumentada do tecido raiz, quando comparada aos outros tecidos avaliados, que está em concordância com o encontrado na literatura, referente à importância fisiológica da enzima neste tecido. Níveis adequados de fósforo são necessários para o crescimento e desenvolvimento de todos os organismos para o bom funcionamento de funções como estrutura molecular, geração de energia e regulação metabólica. A demanda por fósforo e sua conseqüente absorção do solo pelas raízes, aumenta dramaticamente durante o período de rápido crescimento e divisão, por exemplo, na germinação de sementes. Sendo assim, como supridores importantes de fósforo para o metabolismo da planta, podemos citar as fitases e fosfatases, que, além de contribuírem para prover nutrientes, as fosfatases também desempenham papel importante no controle de diferentes funções da planta, particularmente na regulação das cascatas de transdução de sinal. Utilizamos microarranjos de quinases como ferramenta para avaliação do quinoma da raiz nas plântulas de soja germinadas na presença e ausência de luz. Assim, concluímos que fatores ambientais, como a presença de luz e disponibilidade de nutrientes fazem com que diferentes vias estejam ativas e que diferentes fitormônios estejam agindo predominantemente. Relacionando as condições de germinação das plântulas de soja com esses fatores e às quinases mais ativadas no chip, propomos um modelo de sinalização onde o hormônio ácido abscísico responde ao estresse através da ativação de vias como as de MAPKs e de cálcio, causando modulação da atividade de fosfatases e quinases, resultando em respostas como proliferação e rearranjo do citoesqueleto das raízes. Neste trabalho, nós descrevemos a clonagem, expressão de uma de soja, sua caracterização cinética, localização tecidual e investigação da possível relação com a sinalização disparada pela presença ou ausência de luz. A investigação e caracterização de PTPs em plantas podem fornecer informações relevantes no campo de pesquisa do metabolismo vegetal. Sendo assim, nós disponibilizamos dados bioquímicos relevantes os quais sugerem que as plântulas de soja contêm PTP típicas que são principalmente expressas em raízes e aparentemente desempenham papel importante durante a germinação da semente. / Abstract: Our group previously described the purification and characterization of four acid phosphatase isoforms obtained from mature soybean seeds, and determined the kinetic and physico-chemical properties of these enzymes. Structural and physical studies demand high amounts at purity levels of these enzymes, requiring efforts in laborious conventional purifications. Due to this fact and to the existence of few plant proteins that already has been cloned and expressed, we proposed to clone and express a protein tyrosine phosphatase (PTP) from soybean. The kinetic studies of the recombinant PTP revealed an enzyme with phosphatase typical features and higher specificity toward Tyr-phosphate when compared to other analysed substrates. The analysis of thermal inactivation of the enzyme was carried out by circular dichroism, which showed that GmPTP had low thermal stability. The immunolocalization of GmPTP, by western blot, identified the enzyme in different soybean tissues, which were germinated in the presence and absence of light. These results are in agreement with those obtained from the quantitative PCR results, that showed a higher expression of GmPTP in roots when compared with other evaluated tissues. The physiological importance of this tissue to the plant metabolism corroborates our results, because adequate levels of phosphorus are required for the good operation of metabolic functions, as growth and development. The phosphorus demand and the consequent soil absorption through the roots dramatically increase in periods of high growth rate, for instance, during germination. Therefore, phytases and phosphatases are important suppliers for plant metabolism and besides the contribution to provide nutrients, phosphatases could play an important role in regulation of signal transduction cascades. Kinase microarrays were employed as a strong tool to analyse the root kinome in soybean plantlets germinated in light and dark conditions. We concluded that abiotic factors as light presence and nutrients availability were responsible for the activation of different pathways and different phytormones prevalence. Relating the germination conditions of soybean plantlets to these factors and to the analysis of the most activated kinases on the chip, we propose a signaling model to explain that, when the hormone abscisic acid is the main response of stress stimuli, it triggers the activation of MAPK and calcium signaling, and the resulting modulation of kinases and phosphatases activities led to proliferation and cytoskeleton rearrangement in roots. In this work, we described the cloning and expression of a soybean PTP, its kinetic characterization, tissue distribution and investigation of a putative relationship with the signaling pathway triggered by light and dark. The investigation and characterization of PTPs from plants can add relevant information in the plant metabolism research field. Therefore, we provided a wealth biochemical data which suggest that soybean plantlets contain typical PTP, which is mainly expressed in roots and apparently plays a critical role during germination. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

La CLD est un composé toxique qui (i) pollue plus de 15% des surfaces agricoles de la Guadeloupe et de la Martinique, (ii) résiste à la dégradation biotique et abiotique, (iii) s’accumule le long de la chaine alimentaire et dont la disparition des sols par lessivage est estimée à plusieurs centaines d’années. Les animaux de rente originaires de ces zones polluées sont contaminés et l’état de l’art a révélé un manque d’information évident sur le comportement de la CLD dans l’organisme de ces animaux et moins de dix publications sur leur décontamination. Deux protocoles expérimentaux ont été conduits sur des caprins mâles en croissance afin de caractériser le comportement de la CLD dans l’organisme des ruminants non lactants et de développer des méthodes optimisant leurs processus de décontamination. Les résultats ont montré que (i) les chevreaux éliminaient plus de 70% de leur CLD tissulaire après 3 à 4 semaines de décontamination et (ii) ni leur état d’engraissement initial ni l’ajout de charbon actif ou d’huile de paraffine dans leur alimentation ne modifiaient ces taux d’excrétion. Ces études ont également montré que la CLD se comportait différemment des autres polluants lipophiles car sa concentration tissulaire (exprimée sur la base de la matière grasse) était plus importante dans le foie et les muscles des animaux contaminés que dans leurs tissus gras. Ces résultats soulèvent des questions sur les mécanismes à l'origine de la distribution tissulaire de la CLD, son métabolisme et sur l’interaction de ce dernier avec le cycle entéro-hépatique. La compréhension de ces processus permettra de mieux ajuster les stratégies de décontamination pour les rendre plus efficaces / Chlordecone (CLD) is a toxic molecule which (i) contaminates more than 15% of agricultural land in Guadeloupe and Martinique islands, (ii) resists to biotic and abiotic degradation, (iii) accumulates along the food chain and whose the disappearance by soil leaching is estimated at several hundred years. The contamination of livestock in polluted areas is a health, social and economic issue. A literature review on CLD revealed a crying lack of information on its behavior in livestock’s organism and currently, there are less than ten studies which deal with livestock’s decontamination. Two experimental protocols were developed to characterize the behavior of CLD in ruminants’ organism and to evaluate methods that can optimize the decontamination processes of these animals. Results showed that all animals have eliminated more than 70% of Chlordecone in 3 to 4 weeks and neither the initial kids’ body fatness nor the addition of activated carbon or the addition of paraffin oil in the diet during the decontamination period altered these rates of excretion. Following these studies, the lipophilic behavior of CLD in animals is, also, questioned because the results showed that the concentrations of this pollutant, expressed on the fat matter basis, were higher in the liver and the muscles than in the peri-renal fat. These results raised new questions: firstly, on the mechanisms which control the CLD tissue distribution and secondly, on the role of the CLD metabolism and its interaction with the entero-hepatic cycle. The understanding of these processes should help to better adjust the decontamination strategies in order to make them more efficient

Obesity is characterized by the accumulation of adipose tissue (AT) either in the major adipose depots, the subcutaneous (SAT) or visceral (VAT), or ectopically in other tissues and organs such as muscle or liver. Indeed, AT is more than just a big energy storage filled with lipids or an isolation layer for inner organs. Since the first AT-secreted components were discovered the general thinking about AT changed fundamentally. Today, AT is regarded as the largest endocrine organ that releases bioactive compounds named adipokines which fulfill a variety of biological functions ranging from the orchestration of hunger and satiety to the modulation of immune responses or inflammation. Obesity is almost always accompanied by noncommunicable diseases like diabetes mellitus, cardiovascular diseases or several types of cancer. Globally, more than 70 % of prematurely deaths are caused by noncommunicable diseases. So far, the mechanisms how obesity contributes to or causes these diseases are not fully understood. However, most patients with obesity suffer from symptoms of AT dysfunction as result of adipocyte hypertrophy, hypoxia, fibrosis or immune cell infiltration. Finally, obesity is linked to altered adipokine secretion which together with circulating free fatty acids (FFA) contribute to chronic AT inflammation which is believed to be a link between obesity and diseases. However, obesity is very heterogeneous, and about 20 % of all people with obesity are classified as metabolically healthy. In this context, body shape and therefore the distribution of AT within the body is of immense importance. This may also be explained by adipokine secretion patterns, as patients with more prominent subcutaneous obesity are more likely to be metabolically healthy than patients with predominant visceral AT accumulations. Therefore, it is of enormous scientific and public interest to comprehend genetic mechanisms regulating AT development and distribution, but also to better understand the concept of healthy and unhealthy obesity for a targeted obesity therapy or prevention. In chapter 1, I aimed to investigate the impact of Homeobox C9 (Hoxc9) gene knockout on AT development and distribution. Initially, Hoxc9 was determined as interesting candidate gene, because it was found twice as high expressed in subcutaneous than in visceral AT. Therefore, mice with an expected deletion of Hoxc9 in AT (ATHoxc9-/-) were generated by crossing floxed Hoxc9lox/lox with fatty acid-binding protein 4 (Fabp4)-Cre recombinase expressing mice. The study was driven by the hypothesis, that adipocyte-specific loss of Hoxc9 will result in impaired AT development or an imbalanced inguinal WAT (ingWAT) to epigonadal WAT (eWAT) ratio compared to littermate controls. Furthermore, ATHoxc9-/- mice were expected to be a potential model mimicking unhealthy obesity, due to potential smaller ingWAT vs. eWAT depots, finally contributing to a better understanding how obesity is linked to noncommunicable diseases. Mice of both sexes were included in the study and either fed a standard chow diet (CD) or a calorie-dense high-fat diet (HFD), to promote diet-induced obesity (DIO). Whereas female knockout mice did not show phenotypic abnormalities, repeated body weight measurements revealed lower weight gain for male ATHoxc9-/- mice under HFD. Additionally, the HFD fed ATHoxc9-/- mice exhibited better glucose tolerance, whereas also ATHoxc9-/- mice on CD had lower fasting glucose levels compared to littermates. Regarding the adipocyte size, ATHoxc9-/- mice are characterized by smaller adipocytes of both subcutaneous and visceral origin, whereas HFD lead to slightly enlarged eWAT adipocytes. In line with the leaner phenotype and better glucose tolerance, OLINK serum protein analyzes detected lower levels of inflammatory markers (e.g., Ccl5, Il17a or Il17f) in ATHoxc9-/- mice. However, analyzing the knockout efficiency revealed only minor, but not significant tendency of Hoxc9 reductions in AT on both, mRNA and protein level. Additionally, DNA fragments within the targeted Hoxc9 exon regions were still detectable in both ATHoxc9-/- and littermates. Based on this, I wanted to clarify whether the Fabp4-Cre model is adequate to induce an AT-specific knockout in genes expressed as early as Hoxc9. Therefore, I investigated several in vitro and ex vivo models. First, I cultured and differentiated three different adipocyte cell lines (3T3-L1, immortalized inguinal and epigonadal white adipocytes and SVF cells isolated from mouse AT depots) to investigate gene expression patterns of both Hoxc9 and Fabp4 from undifferentiated and mature adipocytes, as well as during differentiation. In all models, Hoxc9 was already expressed early in the undifferentiated stages, whereas Fabp4 expression started later during adipocyte maturation. Additionally, to translate these findings in vivo, I sacrificed pups within their first 20 days of age and dissected developing ingWAT depots. Measurements of Hoxc9 and Fabp4 gene and protein levels supported the previous in vitro results. Therefore, I conclude that the Fabp4 mediated Cre recombinase is not suited for the investigation of a gene’s role in adipose tissue development. Furthermore, this finding should be transferable in the context of developmental research in other tissues or organs. Accordingly, I strongly encourage researchers who wants to perform Cre-mediated gene knockouts to check expression patterns of both, the Cre-mediating gene and the gene of interest in appropriate cell models beforehand to save time and costs, but most importantly to focus on animal welfare. In addition, the missing control group of Fabp4-Cre mice may represent a second major limitation of the study. Several previous studies have reported phenotypes resulting from Cre expression alone. Therefore, and because the presence of the Fabp4-Cre recombinase was the only validated genetic difference between our ATHoxc9-/- (Hoxc9lox/lox, Fabp4-Cre+) and the littermate controls (Hoxc9lox/lox, Fabp4-Cre-), I concluded that the phenotypic differences observed between both groups resulted from the Fabp4-Cre genotype itself. Even while Hoxc9 was found to be expressed in earlier stages of adipogenesis than Fabp4, it was surprising and unexpected that Cre recombinase expression did not ablate Hoxc9 in mature adipocytes. The detection of potential expression differences was impeded by the overall low expression of Hoxc9 in AT, even if it was found differentially expressed between ingWAT and eWAT. To investigate the Hoxc9 impact on adipogenesis other Cre models mediated by promotors active during earlier stages of adipocyte differentiation should be used. Alternative adipocyte-specific Cre expressing mouse lines using the adiponectin (Adipoq) promotor or the less known resistin (Retn) promotor can be excluded for Hoxc9 as they are most active only in fully mature adipocytes. In contrast, platelet derived growth factor receptor α (Pdgfra) promotor controlled Cre expression is already active during the adipocyte progenitor stage and could be better suited to target Hoxc9. Yet, this Cre model accompanies with other pitfalls. In line with its expression in progenitor cells, the Hoxc9 gene will not just be deleted in cells with adipogenic fate, but also in progenitors developing into e.g., chondrocytes or osteoblasts. According to Hoxc9’s role in skeletal patterning during the embryogenesis, using this model may result in malformations or premature death. Finally, the paired related homeobox 1 (Prrx1) Cre line is a likely suitable model . This promotor was found to be active early in progenitor cells later found in ingWAT depots and may be suitable to investigate the role of Hoxc9 in AT development and distribution.:Table of contents 1 1. Introduction 2 Obesity – a Global Pandemic 2 The Human Adipose Tissue 2 Obesity and Dysfunction of Adipose Tissue 4 Society, Environment and Genetics in the Pathogenesis of Obesity and AT Distribution 6 Rationale for Chapter 1 8 The Developmental Control Genes of the Homeobox Family 8 Aim of the Study in Chapter 1 11 Rationale for Chapter 2 11 Adenylyl Cyclases 12 How Adenylyl Cyclases Impact Diabetes 14 Aim of the Study in Chapter 2 16 The Cre-loxP System and Generation Knockout Mouse Models 17 2. Chapter 1 19 3. Chapter 2 38 4. Summary 60 5. References 65 6. Appendix 75 A. Supplementary Material - Chapter 1 75 B. Supplementary Material - Chapter 2 82 Erklärung über die eigenständige Abfassung der Arbeit 95 Curriculum Vitae 96 List of Publications and Talks 97 Danksagung 99 / In chapter 2, I investigated the consequences of a whole-body ablation of adenylyl cyclase 5 (Adcy5) in mice. Genome-wide association studies (GWAS) identified human ADCY5 as a candidate for diabetes associated traits and a higher risk to develop type 2 diabetes (T2D). In several mouse models the loss of Adcy5 was described as beneficial. For instance, Adcy5-/- mice have shown improved cardiac function and an increased longevity, mimicking a phenotype resulting from caloric restriction. Furthermore, they were protected against DIO and did not develop glucose intolerance or insulin resistance. Therefore, my study focused on the AT phenotype and global AT gene expression profiles resulting from the whole-body loss of Adcy5. Again, I comprehensively investigated animals of both sexes, both under CD or HFD. Unexpectedly, our mice readily developed DIO. Moreover, female mice under HFD developed more severe obesity compared to control animals. Female C57BL/6 mice are in general not as susceptible to DIO as males from the same strain and usually develop later-onset and less severe obesity. Therefore, it was surprising that the loss of Adcy5 induced DIO-development in females, an effect known from ovariectomized mice. Since Adcy5 is highly expressed in the ovaries and the expression of the key enzyme of estrogen production, aromatase, is regulated by Adcy-generated cyclic adenosine monophosphate (cAMP), further studies are necessary to validate the hypothesis, that Adcy5 may affect DIO-susceptibility via the cAMP-aromatase-estrogen axis. Besides, our model did not reproduce the beneficial effects on energy expenditure, oxygen consumption or insulin sensitivity described by others. Our phenotype is more similar and matches to those described for Adcy3 deficient mice, where knockouts gained more weight under HFD than littermate controls. Again, a shortcoming of our study was not using littermate controls. Knockouts were generated by integrating a vector cassette into Adcy5 exon 1 of C57BL/6NTac mice. Mice of the same strain sharing more than 99% background identity were used as controls. However, this could explain the phenotype differences seen in our compared to earlier reported Adcy5 knockout models. Nevertheless, our model enabled us to investigate the impact of Adcy5 on AT morphology and gene expression independently of body weight or a metabolically healthier phenotype. Except for the female mice on HFD, all Adcy5 deficient mice exhibited an increased number of smaller adipocytes which is strongly correlated to a reduced diabetes risk by retaining insulin sensitivity. Conspicuously, all Adcy5-/- mice presented a tremendously reduced running wheel activity. Beside several genes of glucose and lipid metabolism, global gene expression analysis within AT highlighted sodium-potassium ATPase catalytic subunit alpha-3 (Atp1a3) as significantly higher expressed in Adcy5-/- mice. Mutations within Atp1a3 are known to be related to e.g., dyskinesia and could be a relevant finding to understand how the loss of Adcy5 is connected to movement disorders. Based on GWAS and other earlier studies in mice that highlighted beneficial effects resulting from its loss, ADCY5 was considered as promising drug target to treat diabetes. In summary, my work suggests that these positive effects are strongly dependent on the genetic background and also gender-dependent. Especially, significantly reduced voluntary exercise activity combined with readily developing DIO cast doubt on the beneficial potential of ADCY5 inhibitors.:Table of contents 1 1. Introduction 2 Obesity – a Global Pandemic 2 The Human Adipose Tissue 2 Obesity and Dysfunction of Adipose Tissue 4 Society, Environment and Genetics in the Pathogenesis of Obesity and AT Distribution 6 Rationale for Chapter 1 8 The Developmental Control Genes of the Homeobox Family 8 Aim of the Study in Chapter 1 11 Rationale for Chapter 2 11 Adenylyl Cyclases 12 How Adenylyl Cyclases Impact Diabetes 14 Aim of the Study in Chapter 2 16 The Cre-loxP System and Generation Knockout Mouse Models 17 2. Chapter 1 19 3. Chapter 2 38 4. Summary 60 5. References 65 6. Appendix 75 A. Supplementary Material - Chapter 1 75 B. Supplementary Material - Chapter 2 82 Erklärung über die eigenständige Abfassung der Arbeit 95 Curriculum Vitae 96 List of Publications and Talks 97 Danksagung 99