Orientador: Tomomasa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T07:05:53Z (GMT). No. of bitstreams: 1 Kota_DanielJiro_M.pdf: 555074 bytes, checksum: 910a5d4735f12d01edac8766de74e9c5 (MD5) Previous issue date: 2008 / Resumo: A bactéria Enterobacter cloacae tem se destacado como importante agente de infecção hospitalar, muitas vezes levando ao óbito de pacientes imunodeprimidos. Estudando as propriedades de virulência desta bactéria em nosso laboratório, foi detectado que 18% das amostras estudadas demonstraram atividade citotóxica sobre células Vero. A citotoxina, de peso molecular de 100 KDa, foi purificada da amostra com maior atividade citotóxica (118.9) por cromatografia de troca iônica e exclusão molecular, não mostrou atividade letal em camundongos e não causou acúmulo de fluido em intestino de camundongos recém-nascidos. Além disso, não apresentou atividade leucotóxica. Quanto à sua propriedade hemolítica, constatou-se que a toxina purificada apresentou atividade hemolítica sobre eritrócitos de coelho, carneiro e boi, sendo não hemolítica sobre eritrócitos humanos, com presença ou ausência de ~mercaptoetanol. O mecanismo de ação desta toxina parece estar relacionado com o comprometimento da mitocôndria, o que ativaria a via apoptótica e a lise da membrana das células Vero. Quand~ aquecida por 100°C por 30 minutos, esta perdeu toda sua atividade citotóxica, demonstrando ser termolábil. Apesar da similaridade em sua citotoxicidade sobre as células Vero com os efeitos causados por verotoxinas, os genes STX (Shiga-Toxin) 1 e 2 e VT (Verotoxin)1 e 2, característicos de verotoxinas, não foram amplificados pela PCR / Abstract: The bacterium Enterobacter cloacae has been recognized as an important infectious agent in hospitaIs, sometimes leading patients to obit. Studying its virulence properties in our laboratory, 18% of the strains were characterized as cytotoxic against Vero cells. The cytotoxin was purified from the most cytotoxic strain (118.9) by means ionic exchange and molecular exclusion chromatography techniques respectively, increasing its relative activity in 62.5%. The purified toxin did not display either lethal activity nor did it cause fluid accumulation in neonatal mice intestine. Furthermore, no leukotoxic activity was detected and regarding its hemolytic activity, the purified toxin showed hemolytic activity against rabbit, bovine and sheep erythrocytes, but not against human erythrocytes, with or without beta-mercaptoethanol. The toxicity mechanism seems to be associated with a failure in the mitochondria's function, which would activate the apoptotic pathway and membrane lyses. When heated for 100°C for 30 minutes, the toxin lost completely its cytotoxicity. Despite the fact that the toxin displayed similar effects in Vero cells compared to verotoxins, PCR did not amplified the STXl and -2 and VTl and 2 genes, found in verotoxin expressing strains / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Tomomasa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T01:56:06Z (GMT). No. of bitstreams: 1 Mantovani_RebecaPassarelli_M.pdf: 3691454 bytes, checksum: 62da767a8ae513cf2860f407c915aae6 (MD5) Previous issue date: 2018-08-18T22:55:56Z / Resumo: Fibrose cística (FC) é uma doença genética, autossômica recessiva, caracterizada por anormalidade no transporte eletrolítico. Achromobacter xylosoxidans é um bacilo Gram-negativo, não-fermentador e nos últimos anos tem sido encontrado em pacientes com FC, levando à doença pulmonar crônica. Neste estudo foram analisadas 17 amostras de A.xylosoxidans, isoladas de pacientes do Ambulatório de Fibrose Cística do Hospital das Clínicas (HC) da UNICAMP-SP. Os isolados foram analisados quanto as suas propriedades de virulência: atividade hemolítica, presença de hemaglutinação, atividade enzimática (caseinase, esterase, lecitina e hidrolise de elastina), adesão em célula, formação de biofilme e produção de atividade citotóxica. As amostras produziram hemolisinas, hemaglutininas e atividades enzimáticas. Catorze amostras de A.xylosoxidans (78%) formaram biofilme em microplacas, o que sugere o seu potencial em formar biofilme em cateteres e sondas. Entretanto essas bactérias não aderiram em células de pulmão NCI-H292 nas condições utilizadas. Os sobrenadantes de cultura de A.xylosoxindas apresentaram atividade citotóxica termoestável sobre célula NCI-H292, induzindo a formação de vacúolos, perda de contato celular, condensação da cromatina, núcleos picnoticos e morte celular. Foi verificado que a atividade citotóxica foi mantida mesmo após o tratamento dos sobrenadantes com polimixina B, sugerindo não ser uma endotoxina Ensaios de ELISA demonstraram significativa produção de IL-6 e IL-8 pelas células NCI-H292 após 24 horas de incubação com a fração >50kDa do sobrenadante de cultura da bacteria. Os resultados sugerem que o fator citotóxico produzido por A. xylosoxidans apresenta um importante papel na virulência, o qual provavelmente está associado à inflamação pulmonar crônica em pacientes com FC, levando a danos teciduais e declínio da função pulmonar / Abstract: Cystic fibrosis is a genetic disease, autosomal recessive, characterized by abnormal electrolyte transport. Achromobacter xylosoxidans is a Gram-negative non-fermenter and in recent years has been found in CF patients, leading to chronic lung disease. These studies were analyzed 17 samples of A.xylosoxidans isolated from patients in the Cystic Fibrosis Clinic, HC UNICAMP-SP. The isolates were analyzed for their virulence properties, such as haemolytic activity, the presence of hemagglutination, enzyme activity (caseinase, esterase, lecithin and hydrolysis of elastin), cell adhesion, biofilm formation and production of cytotoxic activity. The presence of hemolysins, hemagglutinins and enzymatic activities were not detected. Fourteen samples (78%) of A.xylosoxidans formed biofilms in microplates, suggesting the ability of bacteria to form biofilms on catheters and probes, but there was no adhesion in NCI-H292 cell lung. The culture supernatants of A.xylosoxindas isolated produced a heatstable cytotoxic factor in NCI-H292 cells, inducing the formation of vacuoles, loss of cell contact, chromatin condensation, pyknotic nuclei and cell death. ELISA assays to verify the release of IL-6 and IL-8, showed great production of these proinflammatory cytokines by NCI-H292 cells after 24 hours of incubation with the fraction> 50 kDa of culture supernatant of bacteria. It was also verified that the cytotoxic activity was maintained even after treatment with polymyxin B, suggesting may be not an endotoxin. Therefore, this cytotoxic factor produced by A. xylosoxidans may represent an important virulence factor, which is probably associated with chronic pulmonary inflammation in CF patients, leading to tissue damage and decline in lung function, decreasing the survival of these patients / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Aggregatibacter actinomycetemcomitans exerce um papel fundamental no desenvolvimento da doença periodontal crônica e agressiva. Produz fatores de virulência que o tornam capaz de colonizar e invadir os tecidos periodontais e de escapar das defesas do hospedeiro. Nesse estudo, foram determinadas as características fenotípicas e genotípicas de A. actinomycetemcomitans e a presença dos principais genes responsáveis por sua virulência. Amostras de biofilme subgengival de 65 indivíduos com doença periodontal e de 48 indivíduos sadios foram coletadas, sendo obtidos 73 isolados bacterianos. Testes de biotipagem, adesão, invasão e citotoxicidade às células orais (KB), produção de neuraminidase e biofilme, presença de cápsula e fimbrias foram avaliados, assim como, a presença dos genes flp-1 (relacionado à adesão); apaH (relacionado à invasão); promotor LTX e ltxA (produção de leucotoxina); e cdtA, cdtB, cdtC, cdtABC (produção de toxina distensora). O biotipo II foi o mais predominante nos isolados de A. actinomycetemcomitans. Sessenta e seis isolados foram capazes de aderir às células KB e dentre eles, 33 foram capazes de invadir as mesmas. Quarenta e seis isolados de A. actinomycetemcomitans foram citotóxicos às células KB, produzindo alterações na morfologia celular. Neuraminidase foi produzida por 60 dos isolados analisados, com títulos de aglutinação de 2 a 8. Cinquenta e quatro isolados foram capazes de formar grande quantidade de biofilme. Todos os isolados apresentaram cápsula, porém nenhum deles apresentou fímbrias. O gene flp-1 foi detectado em 40 isolados e o gene apaH em 52 isolados de A. actinomycetemcomitans. O promotor LTX e o gene ltxA foram encontrados em todos os isolados, sendo 64 altamente leucotóxicos e 9 fracamente leucotóxicos. O gene cdtA foi observado em 50 isolados testados; o gene cdtB, em 48 isolados; o gene cdtC, em 61 isolados e o gene cdtABC, em 40 isolados. Esses resultados sugerem que a maioria dos isolados clínicos de A. actinomycetemcomitans, provenientes de pacientes com periodontite, abrigam os principais genes responsáveis pela virulência, principalmente os relacionados à produção de toxinas. Possivelmente, a maioria dos isolados foram capazes de aderir às células orais e formar biofilmes em superfícies sólidas pelo envolvimento de outros tipos de adesinas, além das fímbrias. Além disso, dois dos três isolados de indivíduo sadio também apresentaram fatores de virulência que poderiam causar periodontite. / Aggregatibacter actinomycetemcomitans plays a key role in the development of chronic and aggressive periodontitis. This microorganism produces virulence factors which make it able to colonize and invade the periodontal tissues and escape from host defenses. In this study, the phenotypic and genotypic characteristics of A. actinomycetemcomitans and the presence of the main genes responsible for virulence were determined. Subgingival biofilm samples from 65 patients with periodontal disease and 48 healthy individuals were collected, and 73 bacterial isolates were obtained. Biotyping, adhesion, invasion and cytotoxicity to oral cells (KB), neuraminidase and biofilm production, presence of capsule and fimbriae were evaluated, as well as the presence of flp-1 (related to adhesion); apaH (related to invasion); LTX promoter and ltxA (leukotoxin production); cdtA, cdtB, cdtC, cdtABC (cytoletal distending toxin production) genes. Biotype II was the most prevalent in A. actinomycetemcomitans. Sixty-six isolates were able to adhere to KB cells and among them 33 were able to invade them. Forty-six A. actinomycetemcomitans isolates were cytotoxic to KB cells, producing changes in cell morphology. Neuraminidase was produced by 60 isolates, with agglutination titles of 2 to 8. Fifty-four A. actinomycetemcomitans were able to form large amount of biofilm. All isolates showed capsule, but not fimbriae. The flp-1 and apaH genes were detected, respectively, in 40 and 52 isolates. The LTX promoter and ltxA gene were found in all isolates, in 64 were highly leukotoxic and 9 minimally leukotoxic. The cdtA gene was observed in 50; cdtB in 48; cdtC in 61 and cdtABC in 40 A. actinomycetemcomitans. These results suggest that most of A. actinomycetemcomitans clinical isolates from periodontal patients, harbor the main genes responsible for virulence, especially those related to toxins production. Possibly, most of the isolates were able to attach to oral cells and form biofilms on solid surfaces, by the involvement of other types of adhesins, besides the fimbriae. Furthermore, two out three isolates from healthy individual also had virulence factors that could cause periodontitis.

by Wong Wah Yau. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1988. / Bibliography: leaves 219-228.

In the future, the 25 as internal loop region of Pro-RIP can be modified for the optimized recognition of proteases of other HIV strains. This approach opens a new opportunity for the anti-HIV application of maize RIP and other related type III RIPs. A modified maize RIP may also be applied to target other viruses and pathogens, for examples, hepatitis C and malaria, which are dependent on pathogen-encoded proteases for replication. / In this study, we provide an account on the generation of HIV-1 protease-sensitive maize RIP variants by first incorporating the HIV-1 protease recognition sequences to the internal inactivation region of the Pro-RIP. Among the five variants, three variants were cleaved and activated by HIV-1 protease in vitro and in vivo, resulting in an active two-chain form with N-glycosidase activity comparable to the fully active maize RIP. In addition, the variants inhibited viral replication in human T lymphocytes (C8166) infected by two T-tropic HIV-1 strains, HIV-1IIIB and HIV-1 RF/V82F/I84V, and their cytotoxicity towards uninfected cells was similar to the non-activated precursor (TAT-Pro). In comparison to TAT-Pro, variants TAT-Pro-HIV-MA/CA and Pro-TAT-Pro-HIV-p2/NC had 2- to 70-fold increase in the inhibition of p24 antigen production in the HIV-infected cells with low cytotoxicity towards uninfected C8166 cells. / Maize RIP is classified as a type III RIP. It is synthesized in the endosperm of maize as an inactive precursor (Pro-RIP), which contains a 25-amino acid internal inactivation region. During germination, a two-chain activated form (MOD) is generated by endogenous proteolysis of the internal inactivation region, whereas the two chains (16.5 and 8.5 kDa) are tightly associated without disulfide linkage. Our group has solved the crystal structures of both the Pro-RIP and MOD and found that this internal inactivation region is on the surface of the N-terminal domain in Pro-RIP . The removal of this internal inactivation region increases the inhibition of protein synthesis of rabbit reticulocyte lysate by over 600 folds. The presence of the internal inactivation region has led us to derive a novel strategy to enhance the specificity of maize RIP towards HIV-infected cells while minimizing its cytotoxic effect on normal cells. / Ribosome-inactivating proteins (RIPs) are RNA N-glycosidases which cleave the N-glycosidic bond of adenine-4324 at the alpha-sarcin/ricin (SR) loop of 28S rRNA. The depurination of the SR loop results in the inhibition of protein synthesis by impairing the binding of EF-1 or EF-2 to ribosomes. RIPs are therefore highly cytotoxic and have been used as abortificiant, anti-cancer and anti-HIV agents, either alone or as a component of immunotoxins. Many type I and II RIPs, such as MAP30, GAP30, DAP30, pokeweed antiviral protein (PAP) and ricin, have been reported to possess anti-HIV activity by inhibiting viral replication in vitro and in vivo though the anti-HIV mechanism is still unclear. / Law, Ka Yee. / Adviser: Pang-Chui Shaw. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 124-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Aggregatibacter actinomycetemcomitans exerce um papel fundamental no desenvolvimento da doença periodontal crônica e agressiva. Produz fatores de virulência que o tornam capaz de colonizar e invadir os tecidos periodontais e de escapar das defesas do hospedeiro. Nesse estudo, foram determinadas as características fenotípicas e genotípicas de A. actinomycetemcomitans e a presença dos principais genes responsáveis por sua virulência. Amostras de biofilme subgengival de 65 indivíduos com doença periodontal e de 48 indivíduos sadios foram coletadas, sendo obtidos 73 isolados bacterianos. Testes de biotipagem, adesão, invasão e citotoxicidade às células orais (KB), produção de neuraminidase e biofilme, presença de cápsula e fimbrias foram avaliados, assim como, a presença dos genes flp-1 (relacionado à adesão); apaH (relacionado à invasão); promotor LTX e ltxA (produção de leucotoxina); e cdtA, cdtB, cdtC, cdtABC (produção de toxina distensora). O biotipo II foi o mais predominante nos isolados de A. actinomycetemcomitans. Sessenta e seis isolados foram capazes de aderir às células KB e dentre eles, 33 foram capazes de invadir as mesmas. Quarenta e seis isolados de A. actinomycetemcomitans foram citotóxicos às células KB, produzindo alterações na morfologia celular. Neuraminidase foi produzida por 60 dos isolados analisados, com títulos de aglutinação de 2 a 8. Cinquenta e quatro isolados foram capazes de formar grande quantidade de biofilme. Todos os isolados apresentaram cápsula, porém nenhum deles apresentou fímbrias. O gene flp-1 foi detectado em 40 isolados e o gene apaH em 52 isolados de A. actinomycetemcomitans. O promotor LTX e o gene ltxA foram encontrados em todos os isolados, sendo 64 altamente leucotóxicos e 9 fracamente leucotóxicos. O gene cdtA foi observado em 50 isolados testados; o gene cdtB, em 48 isolados; o gene cdtC, em 61 isolados e o gene cdtABC, em 40 isolados. Esses resultados sugerem que a maioria dos isolados clínicos de A. actinomycetemcomitans, provenientes de pacientes com periodontite, abrigam os principais genes responsáveis pela virulência, principalmente os relacionados à produção de toxinas. Possivelmente, a maioria dos isolados foram capazes de aderir às células orais e formar biofilmes em superfícies sólidas pelo envolvimento de outros tipos de adesinas, além das fímbrias. Além disso, dois dos três isolados de indivíduo sadio também apresentaram fatores de virulência que poderiam causar periodontite. / Aggregatibacter actinomycetemcomitans plays a key role in the development of chronic and aggressive periodontitis. This microorganism produces virulence factors which make it able to colonize and invade the periodontal tissues and escape from host defenses. In this study, the phenotypic and genotypic characteristics of A. actinomycetemcomitans and the presence of the main genes responsible for virulence were determined. Subgingival biofilm samples from 65 patients with periodontal disease and 48 healthy individuals were collected, and 73 bacterial isolates were obtained. Biotyping, adhesion, invasion and cytotoxicity to oral cells (KB), neuraminidase and biofilm production, presence of capsule and fimbriae were evaluated, as well as the presence of flp-1 (related to adhesion); apaH (related to invasion); LTX promoter and ltxA (leukotoxin production); cdtA, cdtB, cdtC, cdtABC (cytoletal distending toxin production) genes. Biotype II was the most prevalent in A. actinomycetemcomitans. Sixty-six isolates were able to adhere to KB cells and among them 33 were able to invade them. Forty-six A. actinomycetemcomitans isolates were cytotoxic to KB cells, producing changes in cell morphology. Neuraminidase was produced by 60 isolates, with agglutination titles of 2 to 8. Fifty-four A. actinomycetemcomitans were able to form large amount of biofilm. All isolates showed capsule, but not fimbriae. The flp-1 and apaH genes were detected, respectively, in 40 and 52 isolates. The LTX promoter and ltxA gene were found in all isolates, in 64 were highly leukotoxic and 9 minimally leukotoxic. The cdtA gene was observed in 50; cdtB in 48; cdtC in 61 and cdtABC in 40 A. actinomycetemcomitans. These results suggest that most of A. actinomycetemcomitans clinical isolates from periodontal patients, harbor the main genes responsible for virulence, especially those related to toxins production. Possibly, most of the isolates were able to attach to oral cells and form biofilms on solid surfaces, by the involvement of other types of adhesins, besides the fimbriae. Furthermore, two out three isolates from healthy individual also had virulence factors that could cause periodontitis.

Orientador: Tomomassa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas,. Instituto de Biologia / Made available in DSpace on 2018-08-14T11:00:37Z (GMT). No. of bitstreams: 1 Thome_JacquelineDarcSilva_M.pdf: 1223668 bytes, checksum: 17699f260f82fb8a09136004fcc1e28a (MD5) Previous issue date: 2006 / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Ribosome-inactivating proteins (RIPs) are cytotoxins that remove a specific adenine from the sarcin-ricin loop (SRL) of large ribosomal RNA and in turn inhibit protein synthesis. Apart from N-glycosidase activity, some RIPs are found to possess antiviral activity and the suppression on human immunodeficiency virus (HIV) has been extensively studied. / Maize RIP stands out from other members for having an internal inactivation region and requires proteolytic removal to regain full activity. We have exploited the innate regulatory mechanism of maize RIP and increased its specificity towards HIV by adding the HIV protease recognition site to the inhibitory segment. Our results demonstrated the wild-type maize RIP is inhibitory on simian immunodeficiency virus (SIV) replication in rhesus macaque and showed the HIV sensitive variant undergoes specific proteolytic activation upon viral infection and exerts enhanced in vitro antiviral effects. Therapeutic applications of RIPs are often restricted by short in vivo half-life and strong allergic responses and we attempted to improve the therapeutic potential of maize RIP by coupling with polyethylene glycol (PEG). Two mutants were generated for PEGylation and the resultant MOD-PEG₂₀ₖ variant was shown to be less sensitive to antibody recognition and has a prolonged plasma half-life, suggesting it may have enhanced therapeutic values. Besides, the applicability of protease-activation system in RIPs without inactivation loop was tested using ricin A chain (RTA) as the test case and HIV recognition sites were introduced either within or at C-terminus of the protein. The C-terminal RTA variants were specifically processed and had the anti-HIV activity increased in HIV-infected cells. / The present work illustrates the potential development of maize RIP as an anti-HIV agent and shows PEGylation can serve to enhance the protein for in vivo applications. Besides, the engineering of RTA with HIV recognition site suggests the potential of the protease-activation strategy to other RIPs for activity control. / 核糖體失活蛋白（RIPs）是一種細胞毒素蛋白，能特異地水解核糖體sarcin-ricin環（SRL），通過脫嘌呤抑制核糖體的蛋白合成功能。除此功能以外，很多RIP還具有抗病毒的活性，如抗人免疫缺陷病毒（HIV）的活性。 / 玉米RIP與其他的RIP不同，它含有一段內部失活結構域，需通過蛋白水解作用移除該結構域才能成為活性體。我們利用玉米RIP的這一特性，通過對內部結構域的改造，獲得了兩個可被HIV蛋白酶特異識別的突變體。體外實驗証明HIV可以特異地識別突變體上的蛋白酶切割位點，從而將其啟動產生抗病毒活性。另外，我們以蓖麻毒素A鏈（RTA）為例，分別於蛋白質的內部和碳端插入HIV識別序列，驗證了蛋白酶啟動系統在沒有內部失活結構域的RIP中，也能通過HIV蛋白酶的切割而活化並取得抗病毒活性。我們還揭示玉米RIP活性體可以有效抑制猿免疫缺陷病毒（SIV）在感染恒河猴體內的複制。此外，我們嘗試通過與聚乙二醇（PEG）融合來優化玉米RIP的免疫原性和半衰期，成功製備了兩種融合突變體，MOD-PEG₂₀ₖ顯示較不容易被抗體識別且延長了血液半衰期。 / 綜上所述，我們的研究表明玉米RIP作為抗HIV抑制劑具有良好的研發前景，而RTA的改造展示蛋白酶啟動系統可應用於其他RIP，同時我們還證明了PEG修飾可以很好的應用於蛋白類藥物的研發。 / Au, Ka Yee. / Thesis M.Phil. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 84-95). / Abstracts also in Chinese. / Title from PDF title page (viewed on 17, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Résumé<p><p>Les tétraponérines 1 à 8 sont des alcaloïdes biosynthétisés à des fins défensives par la fourmi Tetraponera sp. originaire de Papouasie Nouvelle-Guinée. Ces alcaloïdes sont dotés de nombreuses propriétés biologiques :insecticides, cytotoxiques, neurotoxiques et antimicrobiennes.<p><p> <p><p>Récemment, le laboratoire de chimie organique s’est intéressé à l’étude de l’influence du squelette tricyclique de ces molécules sur leur activité biologique. A cet effet, la synthèse d’analogues « 666 » des tétraponérines (5-alkyldécahydro-2H,6H-dipyrido[1,2-a:1’,2’-c] pyrimidine) avait été entamée.22 Au cours de cette étude préliminaire, les intermédiaires [76] (squelette « 666 ») et [77] (squelette « iso 666 ») avaient été synthétisés avec des rendements moyens à faibles.<p><p>Dans le cadre de notre thèse, nous avons mis au point des schémas de synthèse conduisant avec de meilleurs rendements aux dérivés [76] et [77].<p><p> <p>Les paramètres de la réaction conduisant à [76] par condensation de la ∆1-pipéridéine, générée à partir du trimère de la ∆1-pipéridéine (a-tripipéridéine), avec le malonate de diéthyle ont été étudiés. Dans les meilleures conditions (pH 11.3, tampon aqueux, 17h, 20°C), le rendement en [76] obtenu est de 34% (précédemment 4%). Dans des solvants organiques, la formation du dérivé [76] n’a jamais été observée.<p><p>Bien qu’amélioré ce rendement reste faible et une autre voie de synthèse a été envisagée. Cette nouvelle approche consiste en la condensation de la ∆1-pipéridéine avec la <p>2-(2,2diéthoxyéthyl)pipéridine qui peut être préparée à partir du 2-(2-pipéridyl)éthanol commercial. Le dérivé [112] est obtenu avec un rendement de 80% pour l’étape de condensation. Les dérivés alkylés [78] ont ensuite été synthétisés en traitant l’a-aminonitrile [112] par les bromures de propyl et dodécylmagnésium.<p> <p>Nous avons ensuite étudié le mécanisme de formation du composé [77] et nous avons pu mettre en évidence que la tétrahydroanabasine [121] était un intermédiaire clé dans la formation du dérivé [77] à partir d’a-tripipéridéine. Ceci nous a permis d’améliorer le rendement de la synthèse du composé [77] à partir d’a-tripipéridéine [120] en se mettant dans des conditions favorables à la formation de la tétrahydroanabasine. Le dérivé [77] est ainsi obtenu avec un rendement de 76%. Les dérivés alkylés [79] correspondants ont été ensuite synthétisés.<p><p> <p><p>Lors de la synthèse des analogues « 666 » des tétraponérines, le composé [93] avait été isolé au cours d’essais de condensation du malonate de diéthyle sur la ∆1-pipéridéine générée par décarboxylation oxydative de la lysine par la N-bromosuccinimide. La structure de ce composé avait été proposée sur base d’une partie de ses propriétés spectroscopiques.22<p><p> <p><p>Dans le cadre de notre thèse nous avons montré que cette hypothèse de structure était incorrecte suite à une analyse complète et détaillée des spectres RMN 2D et d’une dégradation chimique. Une nouvelle hypothèse de structure ([159], squelette « 556 ») a été avancée. <p><p> <p>De plus nous avons pu mettre en évidence que la 3-bromo-1-pipéridéine [152] est un intermédiaire dans la formation de [159]. Sur cette base, nous avons mis au point un nouveau schéma de synthèse totale de [159] au départ du 5-amino-1-pentanol (rendement global 42%) qui confirme la nouvelle structure proposée.<p><p> <p><p>En vue de synthétiser les analogues alkylés « 556 » des tétraponérines, nous avons synthétisé la 2-acétonyl-3-bromo-1-carbométhoxypipéridine [201] et la 3-bromo-1-carbométhoxy-2-(2-oxo-1-dodécyl)pipéridine [208]. Après déprotection, réarrangement via un ion aziridinium et condensation avec la ∆1-pipéridéine, nous avons obtenu les dérivés acétonyle « 556 » [204] et 2-oxododécyle « 556 » [209]. Faute de temps, nous n’avons pas pu mettre au point la réduction de leurs fonctions cétones en méthylènes. <p><p> <p><p>Enfin, pour compléter les résultats des tests biologiques de notre étude structure-activité, nous avons synthétisé la cis- et trans-2-méthyl-6-n-pentylpipéridine (dérivés à courte chaîne des cis- et trans-solénopsines B) et la tétraponérine-5 (T-5).<p><p>Les différents composés synthétisés au cours de ce travail ont été évalués pour leur activité cytotoxique sur une souche de cellules cancéreuses humaines du colon (HT.29).<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

No / The identification of an agent that is selectively activated by a cytochrome P450 (CYP) has the potential for tissue specific dose intensification as a means of significantly improving its therapeutic value. Towards this goal, we disclose evidence for the pathway of activation of a duocarmycin analogue, ICT2700, which targets CYP1A1 for biological activity.