A utilização de plantas com fins medicinais, para o tratamento, a cura e a prevenção de doenças é uma das formas mais antigas de prática medicinal da humanidade. A espécie Croton cajucara BENTH (CcB) é uma planta nativa e endêmica da região Amazônica, onde é popularmente conhecida como “sacaca”. É muito utilizada na medicina popular, sob a forma de chás da casca e de folhas no tratamento de diversas doenças, como o diabetes. Estudos experimentais e clínicos sugerem que o estresse oxidativo esteja envolvido na patogênese e na progressão do diabetes. Este estudo tem como objetivos avaliar os efeitos do extrato aquoso da casca do CcB sobre as alterações hepáticas, os níveis plasmáticos de glicose, triglicerídeos (TG) e colesterol, os efeitos genotóxicos, o estresse oxidativo, a concentração de nitritos e nitratos e a ativação da subunidade p65 do fator de transcrição nuclear Kappa B em fígado de animais diabéticos induzidos por estreptozotocina (STZ). Foram utilizados ratos machos Wistar, divididos em seis grupos com dez animais cada: controle (CO); controle com tratamento durante cinco dias com CcB (CcB5D); controle com tratamento durante 20 dias com CcB (CcB20D); diabéticos (DM); diabéticos com tratamento durante cinco dias com CcB (DM5D); e diabéticos com tratamento durante 20 dias com CcB (DM20D). O DM foi induzido por administração intraperitonial de STZ na dose de 70mg/Kg. O extrato aquoso (EA) foi preparado com 5g da casca do CcB/100mL H2O e administrado na dose de 1,5mL intragástrica. Após 60 dias, foi coletado sangue do plexo retro-orbital para as análises das enzimas séricas: AST, ALT, FA e dosagem de glicemia, triglicerídeos e colesterol. Para avaliar a atividade genotóxica do extrato, foi utilizado o teste de micronúcleos (MN) em medula óssea. O homogeneizado do fígado foi utilizado para avaliação de lipoperoxidação (LPO) através das substâncias que reagem ao ácido tiobarbitúrico (TBARS) e para avaliação da atividade das enzimas antioxidantes SOD, CAT, GPx e GSH, avaliação dos metabólitos do óxido nítrico (nitritos e nitratos totais) e a expressão nuclear da subunidade p65 do NF-kB. A análise das enzimas séricas mostrou que houve aumento de ALT e FA nos animais DM em relação aos grupos CO, e que a ALT apresentou-se diminuida nos animas DM tratados durante cinco e 20 dias. O tratamento com CcB não diminuiu os níveis da glicemia e do colesterol, houve, porém, redução significativa nos níveis de TG nos animais diabéticos tratados durante cinco e 20 dias. O tratamento com CcB não diminuiu os níveis da glicemia e do colesterol, houve, porém, redução significativa nos níveis de TG nos animais diabéticos tratados durante cinco e 20 dias. O EA do CcB mostrou-se destituído de ação genotóxica. Houve aumento no TBARS e na SOD em animais DM e diminuição significativa nos animais diabéticos tratados durante cinco e 20 dias. Por outro lado, verificou-se aumento de TBARS e da atividade da SOD no grupo CO20D. A atividade da CAT e da GPx não apresentaram diferenças significativas entre os grupos estudados. A GSH apresentou-se diminuída nos animais diabéticos em relação aos controles e ao grupo DM5D. Os animais diabéticos apresentaram aumento nos níveis dos metabólitos do óxido nítrico em relação aos animais controles, e a administração do EA do CcB não reverteu essa situação. Houve ativação da expressão nuclear do p65 nos animais diabéticos que foi atenuada nos animais que receberam o EA do CcB. O tratamento dos ratos diabéticos com o EA de CcB parece melhorar os níveis de TG, não reduziu, no entanto, a glicemia e o colesterol, provavelmente por se tratar de um modelo de diabetes crônico. O tratamento diminuiu a LPO e a atividade da SOD, provavelmente devido à atividade antioxidante do EA do CcB como varredor de radicais ânion superóxido. Os resultados mostram que, em situações onde não há estresse oxidativo, o uso prolongado de CcB comporta-se como pró-oxidante e, em situações onde existe estresse oxidativo, o tratamento com o CcB pode possuir ação antioxidante varredora de radicais livres. Além disso, os resultados parecem sustentar a hipótese de que o estresse oxidativo presente do DM estimula a expressão do NF-kB e que administração do EA do CcB reduz essa expressão. / The use of plants for medical purposes in the treatment, cure and prevention of diseases is one of the oldest medical practices of mankind. Croton cajucara BENTH (CcB) is a vegetal species that is native and endemic in the Amazonian region, where it is popularly known as “sacaca”. Bark and leaf infusions of sacaca are widely used in folk medicine to treat many diseases, such as diabetes. Experimental and clinical studies suggest that oxidative stress is involved in the pathogenesis and progress of diabetes. The present study was designed to evaluate the effects CcB aqueous extracts (AE) on hepatic changes, plasma levels of glucose, triglycerides (TG) and cholesterol, genotoxic effects, oxidative stress, nitrites and nitrates levels, and on activation of the p65 subunit of nuclear Kappa B transcription factor (NF-B) in the livers of animals made diabetic by streptozotocin (STZ) administration. Six groups of 10 male Wistar rats each were used as follows: controls (CO); controls with 5-day treatment with CcB (CcB5D); controls with 20- day treatment with CcB (CcB20D); diabetics (DM); diabetics with 5-day treatment with CcB (DM5D); and diabetics with 20-day treatment with CcB (DM20D). DM was induced by intraperitoneal administration of STZ (70mg/Kg). The aqueous extract was obtained using 5g of CcB bark for 100ml H2O and intragastrically administered at a dose of 1.5mL. After 60 days retro orbital blood samples were obtained for analysis of serum enzymes AST, ALT, FA and glucose, triglyceride and cholesterol levels determination. The genotoxic activity was evaluated using the micronucleus assay (MN) in bone marrow. The liver homogenates were used for evaluation of lipoperoxidation (LPO) through thiobarbituric acid reactive substances (TBARS), antioxidant enzymes SOD, CAT, GPx and GSH, nitric oxide metabolites (total nitrites and nitrates) and nuclear expression of p65 of NF-kB. The analysis of serum enzymes showed that there was an increase in ALT and AP in DM animals as compared to CO groups, and ALT was decreased in DM animals treated for 5 and 20 days. The treatment with CcB did not reduce the glycemia and cholesterol level, but there was a significant reduction in the TG levels in DM animals treated for 5 and 20 days. The CcB aqueous extract failed to show any genotoxic action. TBARS and SOD were increased in DM animals and significantly decreased in diabetic animals treated for 5 and 20 days. On the other hand, TBARS and SOD activity were increased in the CcB20D group. CAT and GPx activities were not significantly different across the studied groups. GSH was decreased in the diabetic animals as compared to controls and the DM5D group. The diabetic animals presented an increase in the metabolite levels of nitric oxide as compared to controls, and the administration of CcB AE failed to reverse this situation. There was activation of p65 nuclear expression in the diabetic animals, which was attenuated in the animals receiving the CcB AE. The treatment of diabetic rats with CcB appears to improve TG levels, but it did not reduce glycemia and cholesterol levels, probably because the study dealt with a chronic diabetes model. The treatment decreased LPO and SOD activity, probably because of CcB’s antioxidant activity as scavenger of superoxide anion radicals. The results show that in situations where there is no oxidative stress the extended use of CcB behaves acts as a pro-oxidant and in situations where there is oxidative stress the treatment with CcB may be an antioxidant scavenger of free radicals. Furthermore, the results seem to support the hypothesis that the oxidative stress seen in DM stimulates NF-B expression and that CcB AE administration reduces such expression.

TNF defers apoptosis in macrophages undergoing spontaneous or pharmacologically (thapsigargin, ceramide, CCCP, etoposide or cisplatin)-induced apoptosis, as determined by measurements of caspase-3 activity and annexin-V staining (Chapter 2). The action requires TNF interaction with TNF-R1, not TNF-R2. Survival is uniquely reliant on the activity of the NF-B signaling pathway, and does not require activities arising from the PI3K/Akt, JNK, ERK, p38 MAP kinase or iNOS pathways (Chapter 3). Further, the general anti-apoptotic property of TNF and its specific antagonism of CCCP-induced apoptosis led to the finding that TNF action prevents cytochrome c release. This protection is likely mediated through effects on components of the MPTP itself, as TNF exhibited functional redundancy with the pore inhibitor cyclosporin A, and did not modify upstream events that promote MPTP opening during apoptosis, namely ROS production, cytosolic Ca2+ increase, or a reduction of total ATP (Chapter 4). Subsequent experiments with the mRNA synthesis inhibitor, actinomycin D, and the translation inhibitor, cycloheximide revealed that the protein(s) responsible for TNF-induced survival was transcribed and translated within 1 hr. However, western analyses provided no convincing evidence of the involvement of Mn-SOD, cIAP-1, XIAP, Bcl-2 or A1 in TNF cytoprotection (Chapter 5). Rather, microarray experiments identified the consistent induction of an early response gene, pim-1, within 30 min of TNF exposure (Chapter 6). This result was verified at the protein level with a specific Pim-1 antibody. Evidence was also found for induction of the anti-apoptotic protein A20, but only at mRNA level. Parthenolide, wortmannin, SP600125, PD98059, SB203580 or L-NAME1 acted against TNF-induced Pim-1 expression in a pattern that exactly matched the effects of these inhibitors on TNF-induced survival. That is, only parthenolide-mediated inactivation of NF-B abolished TNF-induced induction of Pim-1. TNF also stimulated the rapid phosphorylation (inactivation) of the pro-apoptotic BH3-only protein, Bad at Ser112 in a manner sensitive to NF-B inhibition, but not PI3K/Akt, JNK, ERK or p38 MAP kinase inhibition (Chapter 7). As Bad is a known substrate of Pim-1 and Bad 1 Parthenolide, wortmannin, SP600125, PD98059 and SB203580 are inhibitors of the NF-B, PI3K/Akt, JNK, ERK and p38 MAP kinase pathways, respectively. L-NAME inhibits iNOS. NF-B- and mitochondria-linked signaling events that contribute to TNF action in deferring physiological and chemotherapeutic drug-induced apoptosis in macrophages ii phosphorylation occurred coincident with Pim-1 upregulation, it is likely that Pim-1 kinase activity mediates the inactivation of Bad. The overall data therefore supports a model in which TNF ligation of TNF-R1 at the cell surface results in intracellular NF- B activation, leading to the induction of Pim-1 mRNA and protein, and the ensuing phosphorylation of Bad. Inactivation of pro-apoptotic Bad increases the resistance threshold of mitochondria to apoptotic insults, thereby reducing the occurrence of mitochondrial permeability transition, cytochrome c release and subsequent caspase-3 activation.

Thesis (Ph. D.)--University of Chicago, Committee on Immunology, June 2003. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 147-163).

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on December 29, 2009). Thesis advisor: Dr. Matthew C. Lucy and Rodney D. Geisert. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 97-114).

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 181-212). Also available on the Internet.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Ovarian cancer is one of the most deadly female malignancies. Despite advances in the treatment of ovarian cancer for the past decade, the cure rate of this disease is moderately improved. Emerging evidence suggests the molecular personalized therapeutic approach become popular for human cancer treatment. The nuclear factor-kappa B (NF-κB) signaling pathway has been shown to play multiple roles in cancer progression such as anti-apoptosis, cell cycle, angiogenesis and metastasis. This study attempted to characterize the functional roles of transforming growth factor (TGF)-β-activating kinase 1 (TAK1) in the activation of NF-κB signaling. Importantly, this study provided evidence showing the significance of TAK1-NF-κB signaling axis in ovarian cancer aggressiveness during omental metastasis. Using quantitative RT-PCR and immunohistochemical analyses, TAK1 was frequently up-regulated and was significantly associated with high-grade (P=0.001), lymph node and distant metastasis (P=0.025), as well as a tendency toward advanced stage ovarian cancers (P=0.08). Functionally, enforced expression of TAK1 could augment cell proliferation, colony formation, anchorage-independent growth ability and migration/invasion in ovarian cancer cells. Conversely, repression of TAK1 expression by genetically or pharmaceutical approach abrogated these tumorigenic capacities including tumor growth in vivo. Furthermore, co-treatment of (5Z) -7-Oxozeaenol could sensitize ovarian cancer cells to cisplatin-induced cell apoptosis, indicating TAK1 is also involved in chemoresistance. Mechanistically, using Western blotting and NF-κB -reporter luciferase analyses, the elevation of TAK1 phosphorylation at Ser412 but not Thr184/187 was found to associate with the activation of NF-κB in ovarian cancer cells solely. A series of functional studies with genetic and pharmaceutical alterations revealed that the increased TAK1 Ser412 phosphorylation was required for exerting the ovarian cancer cell oncogenesis. Omental metastasis is the common phenomenon observed in most of advanced-stage ovarian cancer. Using omentum conditioned medium (OCM), the findings of this study showed that the omentum tissue was able to secrete numerous factors including chemokines such as GRO-α and IL8 in activating TAK1-NF-κB signaling cascade, which thereby induced increased oncogenic capacities in cell growth, migration and invasion. Taken together, this study suggests that TAK1-NF-κB signaling axis is indispensable for promoting oncogenesis of ovarian cancer and targeting this pathway may be a promising personalized cancer therapeutic approach in ovarian cancer. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy