The small envelope of hepatitis B virus (HBV) can self-assembles into virus-like particles (VLPs) and they are highly immunogenic. The use of hepatitis B surface antigen (HBsAg) as a vector to deliver foreign CTL epitopes has met with little success due to the constraints of HBsAg stability and secretion imposed by the insertion of foreign sequence into critical regions. In this study, the efficacy of the small HBsAg envelope protein to deliver foreign CTL epitopes using a protective CTL epitope of human respiratory syncytial virus (RSV) was investigated. The strategy of deleting a DNA sequence encoding HBsAg-specific CTL epitopes at different sites and replacing with DNA sequence encoding RSV CTL epitope resulted in recombinant HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and RSV protective responses in vivo when these recombinant HBsAg DNAs were used to immunised mice. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease relevant protective CTL responses. They also suggest the applicability of the approach to derive recombinant HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The use of HBsAg VLPs has been used globally as administered vaccine for hepatitis B virus infection makes it an attractive vector candidate to deliver immunogens for other diseases. Since the HBsAg DNAs we tested formed recombinant HBsAg VLPs, our results have implications for the development of vaccination strategies using either recombinant HBsAg DNA or VLP vaccines.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Cytotoxic T lymphocytes (CTL) are major effector cells in the adaptive immune response against intracellular pathogens and cancers, killing targets with high precision. Precision is achieved through the specificity of the clonally expressed T cell receptor (TCR). TCRs recognise a specific peptide chain loaded into a major-histocompatability complex, triggering signalling, inducing the CTL to attach and kill target cells. Key stages in this attack are the initial conjugation followed by polarisation and docking of the centrosome to the junction of the two cells, the immune synapse (IS). This focuses secretion of the cytolytic components, perforin and granzyme, from modified lysosomes to kill the target cell. My PhD has utilised amino acid substitutions in the target peptide to alter its signal strength and shown this alters the subsequent killing efficiency of a target population. I developed new imaging and analysis techniques to investigate the effect of TCR signal strength at each step of the killing process. I show the first step, conjugation, is reduced for a percentage of cells with dwell times decreasing as TCR signal strength decreased. The next key step of centrosome polarisation and docking at the IS was also impaired for an increasing proportion of cells as TCR signalling reduced. Impaired centrosome docking reduced efficient granule recruitment to the IS, necessary for target killing. Centrosome docking was linked with the TCR-induced intracellular calcium flux, the duration of which increases with the strength of TCR signalling. This demonstrates how the process of CTL killing can be fine-tuned by the quality of antigen.

Orientador: José Mauricio Sforcin / Resumo: Própolis e seus compostos fenólicos são conhecidos por apresentarem propriedades antioxidantes e anticâncer. Recentemente, os mecanismos de ação da propolis têm sido objeto de investigação. Este estudo teve como objetivo elucidar os efeitos da própolis e três compostos fenólicos (ácido cafeico - Caf, ácido dihidrocinâmico - Cin; ácido p-cumárico - Cou) na mesma proporção que são encontrados em nossa amostra de própolis, isoladamente ou em combinação, sobre células de carcinoma epidermóide de laringe humano (HEp-2). A viabilidade celular, tipo de morte e parada do ciclo celular, geração de espécies reativas de oxigênio (ROS) e a possível capacidade da própolis em induzir o efluxo de doxorrubicina (DOX) via inibidor de glicoproteína-P (P-gp) foram avaliadas. A própolis exerceu um efeito citotóxico em células HEp-2 e apresentaram um valor de IC50 igual a 80 µg/mL, enquanto que os compostos isolados (isoladamente ou em combinação) não mostraram efeito sobre a viabilidade celular após 72 h. Assim, concentrações mais elevadas destes compostos foram testadas e Caf (IC50: 1.332 µM) induziu necrose em células HEp-2, enquanto que a própolis induziu apoptose em células HEp-2, ambos, provavelmente devido à geração de ROS. A amostra de própolis induziu parada do ciclo celular na fase G2/M e Caf na fase S. Própolis ou seus componentes, com exceção de Caf, pode agir como um potencial inibidor de P-gp por modulação da atividade da P-gp, inibindo o efluxo de DOX. Sendo assim, os dados sugerir... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Propolis and its phenolic compounds are known for their antioxidant and anticancer properties. Propolis mechanisms of action have been the subject of research recently. This study aimed to elucidate the effects of propolis and three phenolic compounds (caffeic acid – Caf; dihydrocinnamic acid – Cin; p-coumaric acid – Cou) in the same proportion they are found in our propolis sample, alone or in combination, towards human larynx epidermoid carcinoma (HEp-2) cell. Cell viability, apoptosis/necrosis and cell cycle arrest, generation of reactive oxygen species (ROS) and the ability of propolis to induce doxorubicin (DOX) efflux using a P-glycoprotein (P-gp) inhibitor (verapamil) were assayed. Propolis exerted a cytotoxic effect in HEp-2 cells and exhibited an IC50 value of 80 µg/mL, whereas the isolated compounds (alone or in combination) had no effect on cell viability after 72 h. Hence, higher concentrations of these compounds were tested and Caf (IC50: 1.332 µM) induced necrosis in HEp-2 cells, while propolis induced apoptosis, both, probably due to ROS generation. Propolis induced cell cycle arrest at G2/M phase and, Caf at S phase. Propolis or its components, except Caf, can act as a P-gp inhibitor by modulating P-gp activity and inhibiting the efflux of DOX. Altogether, data suggested that propolis exerted cytotoxic effects against HEp-2 cells and some mechanisms are discussed. Its potential as an antitumor drug should be investigated in further assays. / Mestre

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As lactonas sesquiterpÃnicas apresentam estruturas quÃmicas diversificadas, bem como uma grande variedade de atividades biolÃgicas, dentre as quais se destaca a atividade citotÃxica e antitumoral. O objetivo do presente trabalho foi avaliar o potencial citotÃxico de trÃs novos derivados da α-santonina (1): 3-oxo-7αH,6H-eudesma-1,4,11-trien-6,12-olideo (2), 11,13-dehidrolumissantonina (3) e 10α-acetoxi-3-oxo-1,7αH,6H-guai-4,11-dien-6,12-olideo (4) e estudar seus efeitos sobre a proliferaÃÃo celular, ciclo celular e eventos apoptÃticos. Todos os novos derivados inibiram a proliferaÃÃo das cÃlulas tumorais, pelo ensaio do MTT, exceto o protÃtipo (α-santonina), apÃs 72 h de incubaÃÃo. As linhagens HL60 (leucemia) e HCT-8 (cÃlon) mostraram maior sensibilidade ao tratamento com os novos derivados, cujos valores de CI50 para HL60 foram 1,14 (0,23-2,77); 2,30 (1,87-2,84) e 1,60 (1,09-2,35) ÂM e HCT-8 iguais a 2,92(0,98-4,86); 1,96 (1,64-2,29)e 0,36 (0,16-0,79) ÂM, para os compostos 2, 3 e 4, respectivamente. Dois dos trÃs derivados foram menos citotÃxicos para as cÃlulas mononucleares do sangue perifÃrico (PBMC) com CI50 igual a 10,75 (4,6-23,3) ÂM (3) e CI50 igual a 16,77 (7,3-36,8) ÂM (4). PorÃm, o composto 2 apresentou menor seletividade (CI50 igual a 3,24 (1,6-5,3) ÂM) em relaÃÃo as cÃlulas nÃo tumorais. Nenhum dos compostos estudados induziu efeitos hemolÃticos. Para estudo do mecanismo de aÃÃo foi escolhida a linhagem HL-60 como modelo experimental. Culturas de HL60 foram tratadas com os derivados (1 e 2 ÂM) por 24 h. Todos os derivados foram capazes de reduzir o nÃmero de cÃlulas viÃveis, avaliado pelo ensaio de exclusÃo do azul de tripan, na maior concentraÃÃo testada, sem induzir aumento na incidÃncia de cÃlulas nÃo viÃveis. A aÃÃo antiproliferativa esta relacionada com a capacidade de inibir a sÃntese de DNA. ApÃs o tratamento, os derivados foram capazes de induzir apoptose, como observado pelo padrÃo de morfologia celular: presenÃa de condensaÃÃo de cromatina e fragmentaÃÃo nuclear, bem como por citometria de fluxo (manutenÃÃo da integridade de membrana plasmÃtica, fragmentaÃÃo do DNA, externalizaÃÃo da fosfatidilserina e ativaÃÃo de caspases 3 e 7). Nenhum dos derivados causou despolarizaÃÃo da membrana mitocondrial, sugerindo a participaÃÃo da via extrÃnseca no processo apoptÃtico. Os compostos 3 e 4 foram capazes de causar acÃmulo de cÃlulas na fase G2/M do ciclo celular, indicando um mecanismo de aÃÃo diferenciado em relaÃÃo ao composto 2. Esses dados sugerem que os derivados da α-santonina avaliados no presente estudo apresentam um potencial anticÃncer, em especial o composto 4 pela moderada toxicidade em PBMC e por ser capaz de induzir uma maior taxa de morte celular via apoptose quando comparado aos demais derivados estudados. / The sesquiterpene lactones have different chemical structures, and a variety of biological activities, among which stand out the cytotoxic and antitumour activities. The aim of the present study was to determine the cytotoxic effects of three new α-Santonin (1) derivatives: 3-oxo-7αH,6H-eudesma-1,4,11-trien-6,12-olide (2), 11,13-dehydrolumissantonin (3) and 10α-acetoxi-3-oxo-1,7αH,6H-guai-4,11-dien-6,12-olide (4) and study your effects on cell proliferation, cell cycle, and apoptosis events. All new derivatives inhibited the proliferation of tumor cells, by MTT assay, except the prototype (α-santonina) after 72 h of incubation. The cell lines HL60 (leukemia) and HCT-8 (colon) showed greater sensitivity to treatment with these derivatives, with values of IC50 for HL60 equal to 1.14 (0.23-2.77); 2.30 (1.87-2.84) and 1.60 (1.09-2.35) ÂM and for HCT-8 equal to 2.92(0.98-4.86); 1.96 (1.64-2.29) and 0.36 (0.16-0.79) ÂM for compounds 2, 3 and 4, respectively.. Two derivatives were less cytotoxic to peripheral blood mononuclear cells (PBMC): IC50 equal to 10.75 (4.6-23.3) ÂM (3) and IC50 equal to 16.77 (7.3-36.8) ÂM (4). However, compound 2 showed lower selectivity (IC50 equal to 3.24 (1.6-5.3) ÂM) on PBMC. None of the studied compounds induced hemolytic effects. To evaluate the mechanism of action promoted by these derivatives, HL60 cells was chosen as an experimental model, since this linage was one of the most sensitive to treatment. HL60 cultures were treated with α-santonin derivatives (1 and 2 ÂM) during 24 h. All compounds were able to reduce the number of viable cells evaluated by the trypan blue dye exclusion test at highest concentration, without increasing the number of non-viable cells. The antiproliferative action is related to the ability to inhibit the synthesis of DNA. After treatment, the derivatives were able to induce apoptosis, as observed by cell morphology pattern (chromatin condensation, and nuclear fragmentation), and by flow cytometry (membrane integrity, DNA fragmentation, anexin positive cells, and caspases 3 and 7 activation). None of all derivatives analyzed caused depolarization of mitochondrial membrane, suggesting the involvement of the extrinsic pathway in the apoptotic process. The compounds 3 and 4 induce G2/M cell cycle arrest, indicating a different mechanism of action in relation to compound 2. These data suggest that α-santonin derivatives evaluated in the present study showed a anticancer potential, especially the compound 4, which induced moderate toxicity on PBMC, in addition this compound induced a higher rate of cell death via apoptosis when compared to the other α-santonin derivatives evaluated.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A fenstatina, quimicamente designada de 4-metoxifenil-3,4,5-trimetoxifenilmetanona, denominada RR07, Ã uma bisarilcetona de esqueleto estilbenÃide que pode ser obtida a partir da combretastatina A4, com reconhecida atividade citotÃxica, por meio da oxidaÃÃo de Jacobsen. Dentre as muitas atividades desencadeadas pelos estilbenos destacam-se atividade antiangiogÃnica, citotÃxica e antitumoral. Para avaliar o seu potencial antineoplÃsico, um estudo farmacolÃgico de suas propriedades anticÃncer foi realizado em vÃrios modelos biolÃgicos. Utilizando o ensaio do MTT foi feito um estudo comparativo da citotoxicidade de molÃculas estilbenÃides com estruturas relacionadas ao composto RR07, onde foi observado que a presenÃa do anel A (3,4,5-trimetoxifenil) Ã essencial para a atividade citotÃxica da molÃcula. Determinou-se inicialmente a atividade citotÃxica, frente a 13 linhagens tumorais pelo ensaio de reduÃÃo do MTT sendo testados 11 compostos, (RR01, RR02, RR03, RR04, RR05, RR06, RR07, RR08, RR09, RR10, RR11), onde o estilbenÃide RR07 destacou-se como um dos mais citotÃxicos apresentando valores de CI50 que variaram de 0,009 a 17,49 M. Posteriormente, os estudos de mecanismo de aÃÃo com o RR07 nas concentraÃÃes de 0,25; 0,50; 1,00; 2,00 e 4,00 M, revelaram reduÃÃo, de forma concentraÃÃo-dependente, na viabilidade celular pelos mÃtodos do azul de tripan e de BrdU (bromodeoxiuridina). As anÃlises morfolÃgicas feitas por hematoxilina/eosina mostraram nÃcleos metafÃsicos, onde no ensaio de incorporaÃÃo por brometo de etÃdio/laranja de acridina evidenciou-se morte celular por apoptose nas concentraÃÃes de 0,25 e 0,50 M, com cÃlulas em necrose nas concentraÃÃes de 1,00; 2,00 e 4,00 M, destacando-se alteraÃÃes como desintegraÃÃo membranar e picnose nuclear, nas concentraÃÃes de 0,25 M e 1,00 M, respectivamente. Nos ensaios por citometria de fluxo foi observado fragmentaÃÃo do DNA com parada de ciclo na fase G2/M a partir da concentraÃÃo 0,25 M. A anÃlise pelo ensaio do cometa, para o RR07 revelou atividade genotÃxica do tipo concentraÃÃo-dependente entre cÃlulas mononucleadas de sangue perifÃrico (PBMC), principalmente em 2,00 e 4,00 M. A avaliaÃÃo do RR07 no ensaio com tubulina isolada revelou inibiÃÃo na polimerizaÃÃo da mesma em uma concentraÃÃo de 10 M. A anÃlise antitumoral evidenciou inibiÃÃo de 30,9% e 48,19%, respectivamente, para as doses de 20 e 40 mg/kg/dia de RR07 por via intraperitoneal (ip), e 55,68% de inibiÃÃo para a associaÃÃo de 10 mg/kg/dia de 5-fluorouracil com 20 mg/kg/dia de RR07 (ip), em camundongos transplantados com Sarcoma 180, onde se verificou reduÃÃo no crescimento tumoral e alteraÃÃes renais iniciais e reversÃveis. Desta forma, a fenstatina, RR07, apresenta-se como uma proposta de ferramenta farmacolÃgica Ãtil para a pesquisa de novos derivados. / The phenstatin, chemically known as 4-methoxyphenyl-3,4,5-trimetoxiphenylmethane, was called in the present study as RR07. This compound is a bisarylketone with stilbenoid skeleton obtained from combretastatin A4, with recognized cytotoxic and antineoplasic activities. Then, a detailed study of RR07 anticancer properties was conducted in several biological models. The cytotoxic activity of eleven compounds (RR01, RR02, RR03, RR04, RR05, RR06, RR07, RR08, RR09, RR10, RR11) was determined against thirteen tumor cell lines by MTT assay. Structure-activity relationship pointed out that the presence of ring A (3,4,5-trimethoxiphenyl) is essential for the cytotoxic potential. The RR07 was one of the most cytotoxic compounds, showing IC50 values ranging from 0.009 to 17.49 ÂM. Investigations on mechanism of action (0.25, 0.50, 1.00, 2.00 and 4.00 ÂM) showed a concentration-dependent manner cell viability reduction (trypan blue dye exclusion test) and proliferation decreasing (BrdU assay). Morphological alterations determined by hematoxylin/eosin and acridine orange/ethidium bromide fluorescent double staining methods indicated an increased number of apoptotic cells at lowest concentrations (0.25 and 0.50 ÂM) and necrotic cells at higher concentrations (1.00, 2.00 and 4.00 ÂM). Flow cytometry analyses showed that RR07 induced DNA fragmentation and cell cycle arrest at G2/M starting at 0.25 ÂM. In the comet assay performed with human peripheral blood mononuclear cells (PBMC), RR07 caused DNA strand breaks only at higher concentrations (2.00 and 4.00 ÂM). Experiments with isolated tubulin, RR07 also induced tubulin polymerization inhibition in a concentration of 10 μM. In vivo antitumor experiments showed that Sarcoma 180 transplanted-mice treated with RR07 intraperitoneally (ip) presented a tumor growth inhibition ratios of 30.9% and 48.19% (20 mg/kg/day and 40 mg/kg/day, respectively) and inhibition of 55.68% in associated treatment (5-Fluoruracil 10 mg/kg/day + RR07 20 mg/kg/day). Histopathological analyses of kidneys, spleen and livers revealed incipient and reversible alterations. In summary, RR07 can be considered as a pharmacological useful tool as well as a lead molecule to obtain novel compounds with low toxicity and promising antitumor properties.

Les lignanes sont des métabolites secondaires végétaux, dont la fonction biologique reste aujourd’hui inconnue in planta, mais qui présentent un intérêt en santé humaine. Cette étude vise à progresser dans la connaissance de la fonction des lignanes dans la plante en étudiant la régulation de la biosynthèse de ces molécules. Cette étude a été menée chez des lins : Linum usitatissimum (espèce cultivée) et Linum flavum (espèce sauvage). Chez Linum usitatissimum, la régulation spatio-temporelle de la biosynthèse des lignanes a été établie et il a été démontré que l’acide abscissique régulait la biosynthèse des lignanes dans la graine. Chez Linum flavum, un gène clé dans la production de lignanes a été isolé et la régulation spatiotemporelle de la biosynthèse des lignanes a été établie. Un protocole amélioré d’extraction de sécoisolaricirésinol utilisant une cellulase a été mis au point à partir de téguments de graines de Linum usitatissimum. Enfin de nouvelles sources de lignanes cytotoxiques parmi les Juniperus et Callitris ont été identifiées, et l’extraction de ces composés à partir de feuilles a été optimisée. / Lignans are plant secondary metabolites whose in planta biological function is still unknown but are of interest for human health. This work aims at understanding the function of plant lignans through the study of the lignans biosynthesis regulation. This study was conducted in flax: Linum usitatissimum (cultivated species) and Linum flavum (wild species). In Linum usitatissimum, spatial and temporal regulation of lignans biosynthesis has been established and it has been shown that abscisic acid regulates the lignan biosynthesis in seed coats. In Linum flavum, a key gene of lignans production has been isolated, spatial and temporal regulation of lignans biosynthesis has been established. An improved protocol for secoisolariciresinol extraction from Linum usitatissimum seed teguments involving the use of a cellulase was developed. Finally new sources of cytotoxic lignans were identified among Juniperus and Callitris species and extraction from leaves was optimized.