The aims of the studies described in this thesis were to investigate the pharmacokinetics of and cellular resistance to chemotherapy as causes of treatment failure in childhood acute lymphoblastic leukemia (ALL). Leukemic cells from 370 children with newly diagnosed ALL were tested by the Fluorometric Microculture Cytotoxicity Assay to measure their resistance to each of ten standard cytotoxic drugs. In the high-risk group, increased in vitro resistance to each of the drugs dexamethasone, etoposide and doxorubicin was associated with a worse clinical outcome. Combining the results for these drugs yielded a drug resistance score, showing a relative risk of relapse in the most resistant group that was 9.8 times higher than in the most sensitive group. In the standard-risk and intermediate-risk groups, final evaluation must await longer follow-up. The new cytotoxic agent CHS 828 was equally active in vitro in samples from children with acute myeloblastic leukemia (AML) and ALL, with 50% cell kill at concentrations achievable in vivo. In AML samples CHS 828 also displayed high frequencies of synergistic interactions with four standard drugs. The well-known differences in clinical outcome between Down´s syndrome (DS) and non-DS children with acute leukemia may partly be explained by our finding of differences in drug resistance at the cellular level. Pharmacokinetic studies were performed at the start of induction treatment of ALL. Doxorubicin was assayed by reversed-phase liquid chromatography with fluorometric detection, and vincristine by high performance liquid chromatography with electrochemical detection. Plasma doxorubicin concentrations were measured in 107 children after 23 h of a 24-h infusion. The median steady-state concentration in children 4-6 years old, a group known to have a favorable outcome of treatment, was about 50% higher than in those 1-2 and >6 years old Vincristine pharmacokinetics was evaluated in 98 children. There was no correlation between age and total body clearance or any other pharmacokinetic parameters. In vitro testing of cellular drug resistance might be useful in predicting the outcome in high-risk ALL. The further exploration of CHS 828 in childhood leukemia seems warranted. There is no pharmacokinetic rationale for the common practice of administering relatively lower doses of vincristine to adolescents than to younger children.

Despite major advances in the understanding of the biology of chronic lymphocytic leukaemia (CLL), progress in improving its treatment has been limited and it still remains an incurable disorder. In the present research, we have performed in vitro drug sensitivity testing of primary CLL cells for preclinical evaluation of cytotoxic drugs, using the fluorometric microculture cytotoxicity assay (FMCA). The tumour type-specific activities of 14 standard drugs, evaluated in vitro on tumour cells from patients with CLL and acute leukaemias, were in good agreement with their known clinical activities. A correlation between drug treatment and development of cellular drug resistance was demonstrated in CLL, but not in the acute leukaemias. Moreover, the nucleoside analogues fludarabine, cladribine, cytarabine and gemcitabine, as well as the anthracycline idarubicin, were highly active in CLL cells. A new cytotoxic drug candidate, CHS 828, was evaluated in primary cell cultures from a broad spectrum of tumours. CHS 828 was highly active against haematological malignancies in vitro, especially CLL, but also against some solid tumours. The drug appeared to be non cross-resistant with standard drugs. In addition, the relationship between drug sensitivity in vitro and a recently described prognostic factor in CLL, the mutational status of the immunoglobulin variable heavy chain (IgVH) gene, was evaluated. Interestingly, cells with unmutated IgVH genes were more chemosensitive than the mutated cells. In summary, our results indicate that in vitro studies on tumour cellsfrom leukaemia patients may yield considerable information regarding the activity, mechanisms of action and cross-resistance of cytotoxic drugs, as well as concerning the relationship between drug sensitivity and prognostic factors, which can be useful in the preclinical evaluation of new cytotoxic drugs. Furthermore, the results suggest that the pyrimidine analogues cytarabine and gemcitabine, as well as the anthracycline idarubicin, may have a role in the treatment of CLL. The new cyanoguanidine CHS 828 is highly active in CLL cells and appears to be non cross-resistant with standard drugs. The poorer prognosis in patients with CLL cells with unmutated IgVH genes can not be explained by increased chemoresistance.

Treatment results and clinical characteristics in adult acute lymphoblastic leukaemia (ALL) were evaluated regarding three issues: a new treatment with cytarabine up-front, stem cell transplantation and a comparison between adult and paediatric treatment protocols. All studies were conducted on a national basis. Furthermore, activity of imatinib was investigated by in vitro cytotoxicity assay. The national protocol was evaluated in 153 adult ALL patients. A high complete remission rate, 86%, was achieved with 29% overall survival at 3-years. Favourable outcome was identified in patients < 40 years with precursor B phenotype and continuous complete remission was higher for precursor B compared to T-ALL. Stem cell transplantation was evaluated in 187 patients. No differences in outcome between allogeneic and autologous transplantation were found, with the exception of Philadelphia-positive ALL, in which allogeneic transplantation was preferable. Limited chronic graft-versus-host disease (compared to none) resulted in superior disease free survival. The paediatric NOPHO-92 and the Adult protocols were evaluated for 243 ALL-patients. Superior remission rate and survival were achieved for 10-18 year-olds treated according to the Paediatric protocol compared to both 15-25 and 25-40 year-olds treated according to the Adult protocol. Treatment protocol was a significant prognostic factor for patients aged 15-20 years. Fluorometric Microculture Cytotoxicity Assey was used to analyze 15 tumour cell samples from ALL patients. High concordance was determined between in vitro sensitivity to imatinib and presence of BCR-ABL. Daunorubicin, prednisolone and cytarabine had the greatest benefit from a combination with imatinib. The national adult treatment protocol’s results were consistent with international trials regarding precursor B ALL but may be under performing for T-ALL. Adolescents may benefit from treatment according to the Paediatric protocol. No difference in outcome between allogeneic and autologous stem cell transplantation was determined except for Philadelphia-positive patients, despite the indication of a graft-versus-leukaemia effect.

The development of a malaria vaccine is necessary for disease eradication. Successful vaccine candidates to date have targeted the asymptomatic, pre-erythrocytic stage of the disease, however even the most efficacious vaccines are only partially protective. Research undertaken in our laboratory has demonstrated that one such regimen, using an 8 week prime-boost viral vector approach of ChAd63 ME-TRAP and MVA ME-TRAP, induces sterile efficacy in 21% of vaccinees, with a key role identified for TRAP-specific CD8⁺ T cells. The work described in this thesis explores the most immunogenic regimen by which to administer these two pre-erythrocytic malaria vaccines. A shortening of the prime-boost interval from 8 to 4 weeks, and the addition of an extra ChAd63 ME-TRAP priming vaccination, both demonstrated improved T cell immunogenicity over the standard 8 week regimen. Further to this, novel assays were developed to aid the evaluation of vaccine-induced immune responses. Adaptations of the existing methodology for ELISpot analysis and to whole blood flow cytometry techniques, enabled more detailed analyses of paediatric vaccine-induced T cell responses in The Gambia. This work also permitted the comparison of vaccine immunogenicity in this paediatric population, with malaria-naïve and malaria-exposed adult vaccinees. The results suggest that vaccine-induced T cell responses in infants of 8 weeks and older are comparable to that of adults. A second approach involved the development of a novel functional assay. This assay quantitatively measured the in vitro inhibition of intrahepatic Plasmodium parasite development using T cells from ChAd63.MVA ME-TRAP vaccinated volunteers. The assay demonstrated the ability of CD8⁺ T cells to inhibit parasite development in a TRAP-specific manner, and provides a platform with which to further explore pre-erythrocytic immune responses.

Made available in DSpace on 2016-12-23T13:56:12Z (GMT). No. of bitstreams: 1 Renata Monti Rocha.pdf: 1272962 bytes, checksum: 4d1bebef0b65d090fb6d7fe64d1b0b74 (MD5) Previous issue date: 2012-04-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A miltefosina tem sido utilizada com sucesso para o tratamento de LV na Índia, com um índice de cura de 94%. Entretanto, um ensaio clínico realizado pela primeira vez no Brasil demonstrou que cerca de 50% dos pacientes com LV apresentou falha após o tratamento. Uma hipótese para justificar a ocorrência de falha terapêutica em pacientes tratados no Brasil seria a ocorrência de sensibilidade variada à miltefosina entre os isolados de L. chagasi. A variação na sensibilidade à miltefosina tem sido relacionada a diferenças na capacidade de internalização da droga como resultado direto de um defeito na maquinaria de translocação presente na membrana celular da Leishmania, composta por pelo menos duas proteínas, LdMT, e sua subunidade β, LdRos3. Sendo assim, os objetivos desse estudo foram avaliar se há correlação entre a sensibilidade in vitro à miltefosina de isolados de L. chagasi com a resposta ao tratamento, e correlacionar a suscetibilidade à miltefosina com a expressão de proteínas relacionadas à sua maquinaria de translocação. Utilizando ensaios de infecção de macrófagos, os resultados obtidos mostraram que todos os isolados de pacientes curados após o tratamento foram sensíveis in vitro à miltefosina (CI50 2,6 - 7,94 μM), enquanto 10 dos 12 isolados de pacientes que apresentaram falha ao tratamento mostraram-se resistentes (CI50 > 15 μM). Os dois isolados que mostraram-se sensíveis antes do tratamento tornaram-se resistentes, mostrando a aquisição de resistência durante o tratamento. Esses dados sugerem que a falha de tratamento observada em pacientes portadores de LV no Brasil está relacionada a resistência do parasita à droga. Por outro lado, a análise da expressão gênica de proteínas responsáveis pela captação da miltefosina não mostrou diferença entre os isolados sensíveis e resistentes. Diante destes resultados acreditamos que o monitoramento da sensibilidade de isolados clínicos de Leishmania à miltefosina seja de grande relevância para predizer falha de tratamento. Além disso, considerando que testes in vitro de infecção de macrófagos são laboriosos e demorados, a busca de marcadores de resistência utilizando metodologias mais simples e rápidas é importante para facilitar esse monitoramento / The miltefosine has been used successfully for treatment of VL in India, with a cure rate of 94%. However, a clinical trial demonstrated that, in Brazil, about 50% of patients with VL failed after treatment. One hypothesis to explain the occurrence of therapeutic failure in patients treated in Brazil would be the occurrence of variable sensitivity to miltefosine among L. chagasi isolate. The variation in the sensitivity to miltefosine has been related to differences in the ability to internalize the drug as a direct result of a defect in the translocation machinery present in the cell membrane of Leishmania, comprising at least two proteins, LdMT and its β subunit, LdRos3. Therefore, the objectives of this study were to evaluate the correlation of in vitro sensitivity to miltefosine in L. chagasi isolates with the response to treatment, and correlate miltefosine susceptibility with the expression of proteins related to their translocation machinery. Using macrophage infection assays, our results showed that all strains obtained from patients cured after treatment were susceptible to miltefosine in vitro (IC50 2.6 - 7.94 mM), whereas 10 of 12 samples of patients who presented treatment failure were resistant (IC50> 15 mM). Those two isolates that were sensitive pre-treatment have become resistant, indicating the acquisition of resistance during the treatment. Therefore, these data suggest that the treatment failure observed in patients with VL in Brazil is related to parasite drug resistance. On the other hand, the analysis of gene expression of proteins responsible for miltefosine uptake showed no difference between sensitive and resistant isolates. Considering these results, we believe that monitoring the sensitivity of clinical isolates of Leishmania to miltefosine is highly relevant for predicting treatment failure. Moreover, since in vitro infection of macrophages used to evaluate the parasite sensitivity to the drug is laborious and time consuming, the search for drug resistance markers using simple and rapid methods is important to facilitate this monitoring.

In laboratory bioassays, the efficacy of the entomopathogenic fungus Beauveria bassiana against the yellow mealworm (Tenebrio molitor) was tested under various temperature conditions. Six different strains of fungus B. bassiana was investigated. The evaluation was based on vitality bioassays including germination and growth index assessment and the bioassay of virulence based on target organism T. molitor was also assessed growth and yield of conidia different strains of fungus B. bassiana on natural substrates and artificial nutrient substrates.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Empresa Brasileira de Pesquisa AgropecuÃria / O objetivo desta pesquisa foi desenvolver formulaÃÃes de sucos tropicais mistos, baseando-se em dados da Dieta MediterrÃnea (DM), utilizando ferramentas de otimizaÃÃo de processos, para avaliar e quantificar possÃveis efeitos aditivo, sinergÃstico e antagÃnico entre as variÃveis, e avaliar o perfil funcional in vitro e in vivo dos sucos. Foi utilizado um planejamento estatÃstico do tipo fracionado para seleÃÃo das variÃveis (P<0,10), seguido de um delineamento composto central rotacional (DCCR) 25 com P<0,05. As variÃveis independentes foram Ãs concentraÃÃes das polpas de frutas (%) das seis espÃcies de frutas tropicais (camu-camu, acerola, caju, cajÃ, aÃaà e manga) e como variÃveis dependentes a capacidade antioxidante total (TAC) atravÃs do mÃtodo ABTS, polifenÃis totais (TP), Ãcido ascÃrbico e aceitaÃÃo sensorial. Para os ensaios in vivo foram utilizadas duas formulaÃÃes de suco tropical misto: formulaÃÃo A (suco tropical misto de acerola, abacaxi, aÃaÃ, caju, cajà e camu-camu) e formulaÃÃo B (suco tropical misto de acerola, abacaxi, aÃaà e cajÃ), com suas diferentes porcentagens de polpa (%). Ratos machos da linhagem Wistar, recÃm-desmamados foram distribuÃdos em 7 grupos, sendo controle (Ãgua), e seis grupos de animais tratados por gavagem com a reconstituiÃÃo em Ãgua das formulaÃÃes dos sucos liofilizadas nas concentraÃÃes: 100, 200 ou 400mg/kg de peso corpÃreo, durante 30 dias. Foram avaliados os Ãndices nutricionais de consumo de raÃÃo e ganho de peso; anÃlises bioquÃmicas: glicose, triglicerÃdeos, colesterol total, HDL (High Density Lipoprotein), alanina aminotransferase (ALT) e aspartato aminotransferase (AST); peroxidaÃÃo lipÃdica do soro e fÃgado, pelo mÃtodo TBARS (substÃncias reativas ao Ãcido tiobarbitÃrico) e atividade das enzimas antioxidantes, catalase (CAT), superÃxido dismutase (SOD) e glutationa peroxidase (GSH-Px), nos eritrÃcitos, e fÃgado. A partir da anÃlise dos planejamentos estatÃsticos, o camu-camu, a acerola e o aÃaà foram os principais fatores que influenciaram o potencial antioxidante das formulaÃÃes, e o cajà mostrou um efeito positivo sobre a aceitaÃÃo sensorial dos sucos tropicais. Observou-se um efeito antagÃnico entre acerola e camu-camu para a resposta TAC. A formulaÃÃo otimizada foi composta por 20% acerola, 10% de camu-camu, 10% de cajÃ, 10% caju e 10% de aÃaÃ, que correspondeu a um resultado de 155,46 mg.100 g-1 de Ãcido ascÃrbico, 103,01 mg de GAE.100 g-1 para TP, 10,27 ÂM Trolox g-1 para TAC e aproximadamente 6,1 de aceitaÃÃo sensorial. Os grupos tratados com as formulaÃÃes de sucos mistos nÃo apresentaram diferenÃa significativa em relaÃÃo aos Ãndices nutricionais e parÃmetros bioquÃmicos, incluindo a atividade das enzimas ALT e AST, indicando que as formulaÃÃes nÃo ocasionaram danos hepÃticos aos animais. Os resultados demonstraram que a atividade das enzimas SOD e CAT no fÃgado (FA200), e GSH-Px nos eritrÃcitos (FB400), e TBARS no soro e fÃgado (FB100, FA400, FB200, FB400) foi significantemente reduzida nos grupos tratados com os sucos de frutas, quando comparados com o grupo controle, enquanto que o HDL-c aumentou (FB400). Os resultados in vitro e in vivo sugerem que o consumo dos sucos tropicais mistos desenvolvidos neste trabalho foi eficaz na defesa antioxidante endÃgena, sugerindo efetivamente que os sucos de frutas tropicais podem ter significativa relevÃncia para efeitos benÃficos a saÃde. / The aim of this research was to optimize the formulation of mixed tropical juices, based on research into the Mediterranean Dietâ (MD), using a statistical design of fractional type for variable selection (P<0.10), followed by a planning type DCCR (Delineation central composite rotational) 25 with P<0.05, and response surface methodology (RSM), which it was possible to assess. Moreover this investigation proposed to quantify possible additive effects, synergisms and antagonisms between variables, and to evaluate in vitro and in vivo profile of functional the juices. We used six species of tropical fruits (camu-camu, acerola, cashew, yellow mombin, acai and mango). The dependent variables were analyzed: total antioxidant capacity (TAC) using ABTS method, total polyphenols (TP), ascorbic acid and sensory acceptance. The independent variables were the concentrations of fruit pulp (%). For evaluate the in vivo assays were used two formulations of optimized mixed tropical fruit: The formulation (mixed tropical acerola juice, pineapple, acai, cashew, yellow mombin and camu-camu) and formulation B (mixed tropical acerola juice, pineapple, acai and yellow mombin), with different pulp proportions (%) and weaned rats that were divided in 7 groups: control (water), six groups of animals treated by gavage in water to reconstitute lyophilized juice formulations at concentrations of 100, 200 or 400mg/kg for 30 days. The followed analyzes were performed: The nutritional indices of feed intake and weight gain; biochemical analyzes of glucose, triglycerides, total cholesterol, HDL (High Density Lipoprotein), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), serum lipid peroxidation and liver method TBARS (thiobarbituric acid reactive substances) and activities of antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in erythrocytes and liver. Concerning to the statistical planning and MSR, camu-camu, acerola and acai were the main factors that influenced the antioxidant potential of the juice, and yellow mombin showed a positive effect on sensory acceptability of tropical juice. There was an antagonistic effect between acerola and camu-camu in regarding to TAC. The optimal formulation was composed of 20% acerola, 10% camu-camu, 10% yellow mombin, 10% cashew and 10% acai, which corresponding a result of 155,46 mg.100g-1 ascorbic acid, 103,01 mg GAE. 100 g-1 TP, 10,27 ÂM Trolox g-1 for TAC and sensory acceptance of approximately 6.1. The groups treated with the formulations of mixed juices showed no statistical significant difference in relation to nutritional indices and biochemical parameters, including the activity of the enzymes ALT and AST, indicating that the formulations did not cause liver damage these animals. The results showed that the SOD activity and CAT in the liver (FA200) and GSH-Px in erythrocytes (FB400) and in serum and liver TBARS (FB100, FA400, FB200, FB400) were efficiently reduced in the groups treated with the fruit juices, when compared with the control group, while HDL-c increased (FB400). In conclusion, daily consumption of 200mL of optimized formulation is responsible for approximately 50% of the recommended amount of antioxidants in the Mediterranean diet pattern, therefore, a rich source for these bioactive compounds. The results of in vitro and in vivo studies suggest that consumption of tropical juices mixed evaluated was effective in endogenous antioxidant defense, and effectively suggest that the tropical fruit juices may have significant relevance to the health beneficial effects.

Eukaryontische Zellen besitzen ein Zytoskelett, ein zelluläres Netzwerk aus Biopolymeren. Unter diesen Biopolymeren sind die Mikrotubuli weitgehend konserviert. Diese aus Tubulin aufgebauten Filamente sind dynamisch und wechseln zwischen Phasen des Wachstums und der Schrumpfung. Die genauen Mechanismen, die die dynamische Instabilität der Mikrotubuli bestimmen, werden noch erforscht. Die Allgegenwart von Mikrotubuli wirft die Frage auf, wie sie in verschiedenen thermischen Umgebungen konservierte Funktionen ausführen können. Um dieser Fragestellung nachzugehen, habe ich verwandte Froscharten mit unterschiedlich temperierten Lebensräumen untersucht: Xenopus laevis (16-22 °C), Xenopus borealis (19-23 °C) und Xenopus tropicalis (22-30 °C). Um zu untersuchen, ob sich die biochemischen Eigenschaften von Tubulin und die Dynamik der Mikrotubuli bei den drei Arten an die Temperatur angepasst hat, habe ich die Methoden der Tubulin-Affinitätsreinigung und die temperaturgesteuerte TIRF-Mikroskopie zur Rekonstitution der Mikrotubuli-Dynamik kombiniert. Dabei habe ich festgestellt, dass bei einer Temperatur von 25°C die Wachstumsgeschwindigkeit der Mikrotubuli im Bezug zur thermischen Nische der einzelnen Arten negativ korreliert. Die Verwendung der Arrhenius-Gleichung zum Vergleich der Aktivierungsenergie der Mikrotubuli-Polymerisation für jede Spezies ergab, dass die freie Energie des Tubulins umso höher ist, je kälter die thermische Nische der Spezies ist. Die Mikrotubuli von X. laevis und X. borealis zeigten eine längere Lebensdauer und wurden häufiger zerstört als die von X. tropicalis. Die Tubuline von X. laevis und X. borealis sind phosphoryliert, im Gegensatz zu X. tropicalis. Die Ergebnisse zeigen, dass sich Xenopus Tubulin und die Dynamik der Mikrotubuli an die Temperatur angepasst haben. Kalt lebende Arten kommen mit der niedrigeren Energie des Milieus zurecht, durch verbessertes Wachstum und Stabilität. / Eukaryotic cells hold a cytoskeleton, a cellular network of biopolymers. Among the filaments of the cytoskeleton, microtubules are widely conserved. Built from tubulin, those filaments are dynamic, alternating between phases of growth and shrinkage. The biochemical properties of tubulin shape the dynamic behavior of microtubules, which is crucial for many cellular processes. The precise mechanisms determining microtubule dynamic instability are still under investigation. The ubiquity of microtubules raises the question of how they can perform conserved functions within various thermal environments. To address this, I turned to closely related frog species living at different temperatures, Xenopus laevis (niche: 16-22°C), Xenopus borealis (19-23°C) and Xenopus tropicalis (22-30°C). To probe whether the biochemical properties of tubulin and microtubule dynamics adapted to temperature across those three species, I combined tubulin affinity purification and temperature-controlled TIRF microscopy of in vitro reconstitution of microtubule dynamics. I found that at 25°C, the microtubule growth velocity inversely correlates with the thermal niche of each species. Adjusting temperature to each species’ endogenous condition modulates the growth rate differences across species. Using the Arrhenius equation to compare the activation energy of microtubule polymerization for each species suggested that the colder the thermal niche of the species, the higher the free energy of its tubulin. Microtubules from the cold-adapted species X. laevis and X. borealis have longer lifetimes and rescue more often than those of X. tropicalis, both at 25°C and at each species’ endogenous condition. X. laevis and X. borealis tubulins are phosphorylated, contrary to X. tropicalis. My results show that Xenopus tubulin and microtubule dynamics have adapted to temperature. Cold-living species cope with the lower energy of the milieu by facilitating growth and stability.

PEREIRA, Ana Carolina da Silva. Desenvolvimento de sucos tropicais mistos com elevada capacidade antioxidante e avaliação in vivo. 2014. 121 f. : Tese (Doutorado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Tecnologia de Alimentos, Fortaleza-CE, 2014 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-07T14:40:27Z No. of bitstreams: 1 2014_tese_acspereira.pdf: 2190995 bytes, checksum: 682be49075557742cf2cb621f52b139f (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-07T14:40:44Z (GMT) No. of bitstreams: 1 2014_tese_acspereira.pdf: 2190995 bytes, checksum: 682be49075557742cf2cb621f52b139f (MD5) / Made available in DSpace on 2016-07-07T14:40:44Z (GMT). No. of bitstreams: 1 2014_tese_acspereira.pdf: 2190995 bytes, checksum: 682be49075557742cf2cb621f52b139f (MD5) Previous issue date: 2014 / The aim of this research was to optimize the formulation of mixed tropical juices, based on research into the Mediterranean Diet” (MD), using a statistical design of fractional type for variable selection (P<0.10), followed by a planning type DCCR (Delineation central composite rotational) 25 with P<0.05, and response surface methodology (RSM), which it was possible to assess. Moreover this investigation proposed to quantify possible additive effects, synergisms and antagonisms between variables, and to evaluate in vitro and in vivo profile of functional the juices. We used six species of tropical fruits (camu-camu, acerola, cashew, yellow mombin, acai and mango). The dependent variables were analyzed: total antioxidant capacity (TAC) using ABTS method, total polyphenols (TP), ascorbic acid and sensory acceptance. The independent variables were the concentrations of fruit pulp (%). For evaluate the in vivo assays were used two formulations of optimized mixed tropical fruit: The formulation (mixed tropical acerola juice, pineapple, acai, cashew, yellow mombin and camu-camu) and formulation B (mixed tropical acerola juice, pineapple, acai and yellow mombin), with different pulp proportions (%) and weaned rats that were divided in 7 groups: control (water), six groups of animals treated by gavage in water to reconstitute lyophilized juice formulations at concentrations of 100, 200 or 400mg/kg for 30 days. The followed analyzes were performed: The nutritional indices of feed intake and weight gain; biochemical analyzes of glucose, triglycerides, total cholesterol, HDL (High Density Lipoprotein), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), serum lipid peroxidation and liver method TBARS (thiobarbituric acid reactive substances) and activities of antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in erythrocytes and liver. Concerning to the statistical planning and MSR, camu-camu, acerola and acai were the main factors that influenced the antioxidant potential of the juice, and yellow mombin showed a positive effect on sensory acceptability of tropical juice. There was an antagonistic effect between acerola and camu-camu in regarding to TAC. The optimal formulation was composed of 20% acerola, 10% camu-camu, 10% yellow mombin, 10% cashew and 10% acai, which corresponding a result of 155,46 mg.100g-1 ascorbic acid, 103,01 mg GAE. 100 g-1 TP, 10,27 µM Trolox g-1 for TAC and sensory acceptance of approximately 6.1. The groups treated with the formulations of mixed juices showed no statistical significant difference in relation to nutritional indices and biochemical parameters, including the activity of the enzymes ALT and AST, indicating that the formulations did not cause liver damage these animals. The results showed that the SOD activity and CAT in the liver (FA200) and GSH-Px in erythrocytes (FB400) and in serum and liver TBARS (FB100, FA400, FB200, FB400) were efficiently reduced in the groups treated with the fruit juices, when compared with the control group, while HDL-c increased (FB400). In conclusion, daily consumption of 200mL of optimized formulation is responsible for approximately 50% of the recommended amount of antioxidants in the Mediterranean diet pattern, therefore, a rich source for these bioactive compounds. The results of in vitro and in vivo studies suggest that consumption of tropical juices mixed evaluated was effective in endogenous antioxidant defense, and effectively suggest that the tropical fruit juices may have significant relevance to the health beneficial effects. / O objetivo desta pesquisa foi desenvolver formulações de sucos tropicais mistos, baseando-se em dados da Dieta Mediterrânea (DM), utilizando ferramentas de otimização de processos, para avaliar e quantificar possíveis efeitos aditivo, sinergístico e antagônico entre as variáveis, e avaliar o perfil funcional in vitro e in vivo dos sucos. Foi utilizado um planejamento estatístico do tipo fracionado para seleção das variáveis (P<0,10), seguido de um delineamento composto central rotacional (DCCR) 25 com P<0,05. As variáveis independentes foram às concentrações das polpas de frutas (%) das seis espécies de frutas tropicais (camu-camu, acerola, caju, cajá, açaí e manga) e como variáveis dependentes a capacidade antioxidante total (TAC) através do método ABTS, polifenóis totais (TP), ácido ascórbico e aceitação sensorial. Para os ensaios in vivo foram utilizadas duas formulações de suco tropical misto: formulação A (suco tropical misto de acerola, abacaxi, açaí, caju, cajá e camu-camu) e formulação B (suco tropical misto de acerola, abacaxi, açaí e cajá), com suas diferentes porcentagens de polpa (%). Ratos machos da linhagem Wistar, recém-desmamados foram distribuídos em 7 grupos, sendo controle (água), e seis grupos de animais tratados por gavagem com a reconstituição em água das formulações dos sucos liofilizadas nas concentrações: 100, 200 ou 400mg/kg de peso corpóreo, durante 30 dias. Foram avaliados os índices nutricionais de consumo de ração e ganho de peso; análises bioquímicas: glicose, triglicerídeos, colesterol total, HDL (High Density Lipoprotein), alanina aminotransferase (ALT) e aspartato aminotransferase (AST); peroxidação lipídica do soro e fígado, pelo método TBARS (substâncias reativas ao ácido tiobarbitúrico) e atividade das enzimas antioxidantes, catalase (CAT), superóxido dismutase (SOD) e glutationa peroxidase (GSH-Px), nos eritrócitos, e fígado. A partir da análise dos planejamentos estatísticos, o camu-camu, a acerola e o açaí foram os principais fatores que influenciaram o potencial antioxidante das formulações, e o cajá mostrou um efeito positivo sobre a aceitação sensorial dos sucos tropicais. Observou-se um efeito antagônico entre acerola e camu-camu para a resposta TAC. A formulação otimizada foi composta por 20% acerola, 10% de camu-camu, 10% de cajá, 10% caju e 10% de açaí, que correspondeu a um resultado de 155,46 mg.100 g-1 de ácido ascórbico, 103,01 mg de GAE.100 g-1 para TP, 10,27 µM Trolox g-1 para TAC e aproximadamente 6,1 de aceitação sensorial. Os grupos tratados com as formulações de sucos mistos não apresentaram diferença significativa em relação aos índices nutricionais e parâmetros bioquímicos, incluindo a atividade das enzimas ALT e AST, indicando que as formulações não ocasionaram danos hepáticos aos animais. Os resultados demonstraram que a atividade das enzimas SOD e CAT no fígado (FA200), e GSH-Px nos eritrócitos (FB400), e TBARS no soro e fígado (FB100, FA400, FB200, FB400) foi significantemente reduzida nos grupos tratados com os sucos de frutas, quando comparados com o grupo controle, enquanto que o HDL-c aumentou (FB400). Os resultados in vitro e in vivo sugerem que o consumo dos sucos tropicais mistos desenvolvidos neste trabalho foi eficaz na defesa antioxidante endógena, sugerindo efetivamente que os sucos de frutas tropicais podem ter significativa relevância para efeitos benéficos a saúde.

Tumor malignancies are the second leading cause of death worldwide. One of the reasons for the failure of oncological treatment are the uniformly set clinical guidelines, which neglect the effect of high intertumoral heterogeneity. The in vitro chemosensitivity and resistance (CSRA) assays allow for the stratification of patients prior to therapy. Therefore, the CSRA are a long-considered method for personalization of components of chemotherapy regime. Nevertheless, none of them is being routinely used in clinical practice. Certain chemotherapeutics used for their cytotoxic and cytostatic effect are also able to induce so-called immunogenic cell death (ICD) of tumor cells and activate an anti-tumor immune response. Monitoring of changes in the expression of molecules associated with the regulation of the innate immune system on the surface of dying tumor cells would enable to predict the patient's ability to respond to treatment involving modern immunotherapeutics. The feasibility of CSRA using flow cytometry and microscopy is critically evaluated in this thesis on a model of bladder cancer. Simultaneously, the correlation of the immunogenic phenotype of tumor cells and their sensitivity to selected chemotherapeutics is discussed.