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

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains xiv, 118 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

O trabalho propõe a discussão sobre a construção do vocabulário político moderno utilizado na província do Rio Grande e na Banda Oriental durante o processo de formação das Cortes de Portugal, no processo de independência brasileiro, durante a elaboração da constituição nacional, na anexação da Província Cisplatina ao território brasileiro e no início da guerra de independência em relação ao Brasil. Termos como povo, nação, constituição e soberania, além de outros como opinião pública e autonomia política são considerados à luz dos eventos políticos no território brasileiro e oriental entre os anos de 1821 e 1825. Também a relação que se estabeleceu entre o império brasileiro e a condição da Província Cisplatina, principalmente sobre a relativa autonomia política, são consideradas nessa pesquisa. / The paper proposes a discussion on the construction of modern political vocabularies used in the province of Rio Grande and Eastern Band during the process of formation of the Cortes of Portugal, the Brazilian independence process during the drafting of the national constitution, the annexation of cisplatin province to Brazil and at the beginning of the war of independence in relation to Brazil. Terms such as people, nation, constitution and sovereignty, as well as opinion public and political autonomy are considered in the light of political events in Brazil and eastern territory between the years 1821 and 1825. Also the relationship established between the Brazilian Empire and the condition of cisplatin province manly on the relative political autonomy is considered in this research.

Testicular germ cell cancer (TGCC) has been increasing in incidence over recent decades, and is currently the most common malignancy amongst young men resulting in significant morbidity. These tumours are believed to arise from premalignant germ cell neoplasia in situ (GCNIS) cells, which originate from the aberrant germ cell differentiation from gonocyte to spermatogonia during fetal/early postnatal life. GCNIS cells remain dormant in the testis until puberty when they are activated to become tumours. Therefore, GCNIS cells remain in a pre-invasive stage during early childhood and early adulthood prior to the development of a seminoma or non-seminoma TGCC. GCNIS cells are phenotypically similar to gonocytes with expression of stem cell/early germ cell markers including OCT4, PLAP and LIN28. Furthermore, proteins which are expressed in more mature germ cells (spermatogonia) such as MAGE-A4 have also been shown to be expressed in GCNIS cells and these studies have indicated that GCNIS cells are a heterogeneous population in terms of protein expression profile. The relationship between the protein expression profile of individual GCNIS cells populations and their oncogenic potential has not been fully explored. GCNIS cells are located in the seminiferous tubules supported by somatic Sertoli cells. These cells have been previously reported to exhibit an immature protein expression profile in GCNIS tubules from patients with testis cancer, suggesting that the germ stem cell niche in GCNIS tubules resembles that of a fetal one. Associations between Sertoli cell maturation and GCNIS progression into tumour formation has not been fully investigated. Oncogenes are key players in the regulation of oncogenic potential of cancer cells. Gankyrin is an oncogene that has been shown to down-regulate OCT4, and interact with MAGE-A4 in hepatocellular carcinoma and colorectal cancer, where Gankyrin interaction with MAGE-A4 reduces the oncogenic potential of tumour cells. In this study I aimed to investigate the heterogeneity of GCNIS in relation to disease stage and Sertoli cell development. We also aimed to determine the role of Gankyrin in TGCC cell survival and invasion. The co-expression of early germ cells proteins such as OCT4, LIN28 and PLAP was characterized in GCNIS cells during childhood and adulthood pre-invasive TGCC and in invasive disease characterized by the presence of a testicular tumour. These results show that LIN28 was expressed in 95% of OCT4 GCNIS cells, whereas PLAP expression in GCNIS cells increased as the disease progressed from childhood pre-invasive disease to invasive seminoma (32.3% v 76%; p < 0.05). In contrast there was a reduction in the proportion of MAGE-A4 expressing GCNIS cells with disease progression. The MAGE-A4 expressing population was also less proliferative than the MAGE-A4 negative GCNIS population. The methylation status of GCNIS cells was then investigated. EZH2 a methyltransferase previously reported to be important for TGCC development, was expressed in GCNIS cells at all stages of disease, however the histone 3 modification H3K27me3 (mediated by EZH2) was expressed in a significantly higher percentage of the proliferative OCT4+/MAGE-A4- GCNIS cells compared with the OCT4+/MAGEA4+ population (11.7% v 1.1%; p < 0.01) which could indicate a repressive role for H3K27me3 over MAGE-A4 expression. Next, it was determined whether an association between Sertoli cell maturation status and progression of TGCC could be observed. The maturation status of Sertoli cells was studied using proteins indicative of immature (desmin, cytokeratin, fibronectin and AMH) and mature (vimentin and androgen receptor) Sertoli cells. These studies demonstrated heterogeneity of Sertoli cells maturation in GCNIS-containing tubules. Desmin, fibronectin, AMH and vimentin expression did not show any association with TGCC progression. Cytokeratin was expressed in Sertoli cells of human fetal testis up to second trimester of fetal life, absent in tubules with active spermatogenesis but heterogeneously present in GCNIS, demonstrating that cytokeratin expression is indicative of the presence of GCNIS. Androgen receptor was weakly present in Sertoli cells from human fetal testis and pre-pubertal pre-invasive TGCC testis whereas in GCNIS of adult pre-invasive testis and invasive samples, androgen receptor was abundantly expressed in Sertoli cells of GCNIS-containing tubules. These combined results for cytokeratin and androgen receptor suggest that Sertoli cells from GCNIS-containing tubules, in pre-invasive and invasive TGCC patients are partially differentiated. Gankyrin expression was characterised in fetal germ cells, GCNIS cells and TGCC tissue. In fetal testis nuclear Gankyrin was absent in OCT4+/MAGE-A4- (gonocyte) population whereas it was present in a subpopulation of OCT4-/MAGE-A4+ (spermatogonia) germ cells. In GCNIS cells from TGCC patients nuclear Gankyrin was expressed in 87%, 63.3%, 91.5% and 79% in childhood pre-invasive, adult pre-invasive, seminoma and non-seminoma GCNIS cells respectively. Finally, in seminoma cells, Gankyrin was expressed in the cytoplasm indicating a change in localisation as the GCNIS cells become invasive. We used siRNA to knockdown Gankyrin in NT2 (a TGCC cell line) cells in-vitro and demonstrated a decrease in cell number, suggesting that Gankyrin might play a role in TGCC progression and invasiveness. Gankyrin down-regulation also resulted in an increase in p53 and p21 mRNA level. Given the role of P53 and p21 in cisplatin cytotoxic effect in TGCC we went on to investigate the role of Gankyrin in cisplatin resistance using NT2 cells. We demonstrate that Gankyrin mediated cisplatin resistance through the p53/p21 pathway, upregulating apoptosis rates through BAX and FAS, whilst there was no effect on cell proliferation, cell cycle or cell migration. In conclusion, we have shown that GCNIS cells are heterogeneous and their phenotype can determine their oncogenic potential. We also show that Sertoli cells from GCNIS-containing tubules undergo partial differentiation displaying markers of immature and mature Sertoli cells, with a heterogeneous association of cytokeratin with GCNIS presence. We also demonstrate that the oncogene Gankyrin has a role in NT2 cells survival and cisplatin resistance indicating that manipulation of Gankyrin may have a role in the treatment of TGCC.

O urucum (Bixa Orellana L.), planta arbustiva da família Bixaceae, é uma cultura que vem conquistando cada vez mais importância econômica, uma vez que do pericarpo da semente se extrai um corante natural, constituído por carotenóides, com predominância da bixina. A presença na dieta de compostos como os carotenóides podem contribuir para minimizar os efeitos dos radicais livres produzidos no organismo. Alguns quimioterápicos como a cisplatina, produzem efeitos colaterais indesejáveis aos pacientes em tratamento, proporcionando queda nos níveis de antioxidantes séricos e aumento na atividade dos radicais livres que podem refletir na falência renal e hepática. A presença na dieta de compostos como os carotenóides podem contribuir para minimizar tais efeitos. Neste sentido, torna-se de grande importância à investigação dos efeitos benéficos de tais pigmentos frente ao estresse oxidativo, sendo o estudo com animais de laboratório uma opção viável. O presente trabalho avaliou a composição centesimal, a quantidade de bixina presente e ação terapêutica da semente de urucum e dos cristais de bixina na redução da toxidade da cisplatina sobre as desordens renais e hepáticas em ratos adultos, da linhagem wistar, machos. Os animais receberam o pré-tratamento com bixina (0,065 mg/kg de peso) e urucum (0,500mg/kg de peso) durante 28 dias, com controle de peso a cada dois dias, para ajustar as doses dos carotenóides utilizados. A cisplatina foi administrada intraperitonealmente (5mg/kg de peso) 48 horas antes do término do experimento. Após abate, o sangue foi coletado na artéria aorta ascendente e foram retirados o fígado para a extração da gordura, dosagens enzimáticas e índice de peroxidação lipídica e os rins para análise histológica. O resultado da composição centesimal mostrou que as sementes de urucum apresentaram características nutricionais desejáveis, com alto teor de proteínas e fibras. Os resultados das análises hepáticas mostraram que a aspartato aminotrasferase (AST), alanina aminotrasferase (ALT), uréia e creatinina apresentaram reduções significativas nos grupos que receberam o pré-tratamento com bixina e urucum. A cisplatina causou aumento da deposição de gordura hepática e da peroxidação lipídica. Na análise histológica observou-se que os animais do grupo cisplatina sem a presença de urucum ou bixina apresentaram alterações inflamatórias, caracterizadas por modificações glomerulares e aumento no número de polimorfonucleares (PMN). Os resultados deste estudo mostraram que houve proteção hepática e renal contra a injuria causada pela cisplatina, quando administrado o urucum ou a bixina na dieta dos animais, antes da injeção deste fármaco. / The annatto (Bixa orellana L.) is a Bixaceae family bushy plant, being a cultivation with a growing economic relevancy since that from seed's pericarp can be extracted a natural colorant with carotenoid constitution and bixin predominance. The presence of compounds like the carotenoids can cooperate to minimize the free radicals effects produced at the organism. Some chemotherapeutic agents as cisplatin can produce unwanted side effects to the patients under treatment, providing serum antioxidants levels decrease and free radicals activity increase, that may imply in hepatic and renal collapse. The presence of compounds like the carotenoids, in diet, can minimize such effects. For such reasons, the investigation of the benefits of these pigments takes great relevancy against the oxidant stress, while the analysis through laboratory animals was a viable option. This study evaluated the centesimal composition, the bixin quantity presence and the therapeutic action of annatto's seed and bixin crystals at cisplatin toxicity decrease on renal and hepatic disorders in wistar male rats. The animals received the pretreatment with bixin (0,065 mg/kg) and annatto (0,500 mg/kg) along twenty eight days, with weight control every two days to order of carotenoid dosage adjust. The cisplatin was intraperitoneally injected (5 mg/kg) forty four hours before the experiment ending. After animal euthanasia , the blood was collected in ascending aorta and the liver was removed to order of fat extraction, enzymatic dosages and lipid peroxidation tax. Also, the kidnyes was remover to to histological analysis. The centesimal composition results showed that annatto's seeds featured wanted nutritional traces with high protein and fiber content. The hepatic analysis results demonstrated that aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea and creatinine levels decreased significantly in the groups receiving pretreatment with bixin and annatto. The cisplatin caused increased deposition of hepatic fat and lipid peroxidation. In the histological analysis was noticed that the animals which was given the cisplatin without annatto or bixin presence were found inflammatory alterations, characterized by glomerular changes and increased number of polymorphonuclear (PMN). The results of this study show a hepatic and renal protection against the injury caused by cisplatin, when applying annatto and bixin in the animals’ diet before injecting this drug.

Introdução: O câncer de ovário é o mais prevalente e letal câncer ginecológico. A quimioterapia é um componente importante do tratamento sistêmico clássico com uma combinação de um agente platinado e um taxano, usualmente. Invariavelmente, câncer de ovário avançado torna-se resistente à quimioterapia. Objetivos: Com base em dados recentes que demonstram um possível papel das neurotrofinas na regulação de quimiosensibilidade, decidimos estudar o impacto do fator neurotrófico derivado de cérebro (BDNF) sobre a atividade antitumoral de diferentes classes de agentes antineoplásicos. Métodos: Para avaliar um possível efeito sinérgico entre BDNF e diferentes combinações de tratamento para câncer de ovário, as células foram expostas a cisplatina, etoposideo, doxorrubicina e paclitaxel concomitantemente com BDNF durante 48 horas. Administração sequencial de BDNF e quimioterapia foi realizada para avaliar o potencial de BDNF em modificar a resposta ao tratamento quimioterápico dependendo de qual agente é aplicado em primeiro lugar. Resultados: Houve uma redução da viabilidade de células OVCAR-3 quando expostas a cisplatina, doxorubicina e etoposideo concomitantemente com BDNF em 61,18% (SE±1.12, p=0.002), 38,96% (SE±1.08, p=0.001) e 49,63% (SE±1.17, p<0.001), respectivamente. BDNF também reduziu significativamente o efeito do paclitaxel e doxorrubicina quando usado antes da quimioterapia com uma redução de efeito de 53,46% (SE±3.48, p=0.001) e 48,25% (SE±1.25, p=0.018), respectivamente. Além disso, o BDNF utilizado sequencialmente à doxorrubicina foi capaz de reverter a quimiotoxicidade deste agente em 37,77% (SE±1.25, p=0.018). Conclusão: Utilizando a linhagem celular de câncer de ovário (OVCAR-3), BDNF exibiu um efeito sinérgico quando administrado concomitantemente com os agentes citotóxicos doxorrubicina, etoposideo e cisplatina. Observamos também um efeito protetor de BDNF quando aplicado 24 horas antes de doxorrubicina e paclitaxel. Notavelmente, quando BDNF foi administrado após a exposição a agentes antineoplásicos, uma reversão da citotoxicidade foi observada apenas para a doxorrubicina e não para os outros agentes. / Background: Ovarian cancer is the most prevalent and lethal of gynecological malignancies. Chemotherapy is an important component of the systemic treatment with a combination of a platinum complex and a taxane one of the classic treatments. Invariably, advanced ovarian cancer becomes resistant to chemotherapy. Objective: Based on recent data demonstrating a possible role of neurotrophins regulating chemosensitivity, we decided to study the impact of brain-derived neurotrophic factor (BDNF) on the antitumor activity of different classes of antineoplastic agents. Methods: Primarily, to evaluate a possible synergistic effect of BDNF and different ovarian cancer treatments combination, cells were exposed to cisplatin, etoposide, doxorubicin and paclitaxel concomitantly with BDNF for 48 hours. Sequential administration of BDNF and any of the agents was carried out to evaluate if BDNF has the potential of enhancing or protecting cells from the effects of treatment depending of each agent is applied first. Results: There were a reduction in viability of OVCAR-3 cells exposed to cisplatin, doxorubicin and etoposide when used concomitantly with BDNF in 61.18% (SE 1.12, p=0.002), 38.96% (SE 1.08, p=0.001) and 49.63% (SE 1.17, p<0.001) respectively. We also found that BDNF reduced significantly the effect of paclitaxel and doxorubicin when used before chemotherapy with a reduction of effect of 53.46% (SE±3.48, p=0.001) and 48.25% (SE±1.25, p=0.018), respectively. Furthermore, BDNF used sequentially to doxorubicin was able to reverse the chemotoxicity of this agent in 37.77% (SE 1.25, p=0.018). Conclusion: In conclusion, using the human ovarian carcinoma cell line OVCAR-3, BDNF exhibited a synergistic effect when administered concomitantly to the cytotoxic agents doxorubicin, etoposide and cisplatin. We have also observed a protective effect of BDNF when applied 24 hours before doxorubicin and paclitaxel. Notably, when BDNF was administered after the exposure to the antineoplastic agents, a reversal of cytotoxicity was observed only for doxorubicin and not for the other agents.

Platinum-based therapy is the most often used chemotherapeutic agent to treat advanced cases of head and neck cancers. However, only a small fraction of the patient population responds to cisplatin, with a median survival time of less than a year. Currently, there is a lack of clinically employable molecular characterization of the disease beyond HPV status to classify patients who would respond favorably to platinum-based therapy. In this regard, CD24 expression level appears to be a significant molecular phenotype of cisplatin resistance in laryngeal carcinoma. This study demonstrates that CD24 expression level in HNSCC has a linear relationship with cisplatin resistance, and it affects the transcription of critical apoptotic, stem, and drug resistance genes. The knockdown of the CD24 transcript reduces tumor growth rate and increases the overall cisplatin sensitivity in mice xenograft experiments. A retrospective analysis of a cohort of 25 HNSCC patient tumor samples suggests that CD24-high tumors go on to show an unfavorable response to cisplatin treatment. Overall, based on the strength of further clinical analysis, CD24 presents a strong rationale to be utilized as a predictive indicator to stratify head and neck cancer patients for platinum-based therapy. This study also provides a rationale for using CD24 as a therapeutic adjuvant target along with standard cisplatin therapy in head and neck cancers.

xix, 111 p. : ill. (some col.) / Since discovery of its biological effects in the late 1960's, cisplatin (cis-diamminedichloroplatinum( II)) has become one of the most broadly-prescribed cancer drugs in use today. A majority of efforts to understand the metallobiochemistry of this drug have focused on describing the interactions of cisplatin-derived Pt(II) complexes with DNA. Drug binding to this "high value" cellular target is believed to trigger the apoptotic pathways that underlie cisplatin's cytotoxic effects. Although RNA is chemically similar to DNA and responsible for accurately transferring, regulating, and transforming the same genetic information that is stored within the DNA genome, surprisingly little is known about platinum(II) drug binding to RNA. Accordingly, the first three chapters of this dissertation describe efforts to address questions regarding cisplatin coordination to RNA on the molecular scale. Chapter I reviews fundamental aspects of how metal complexes interact with nucleic acids, highlighting the bioinorganic chemistry of platinum(II) antitumor drugs. This chapter also introduces the idea that drug binding to RNA may form an important part of how these complexes work in the cell. Chapter II describes cisplatin crosslinking between RNA nucleobases located on opposite sides of the internal loop of an RNA subdomain derived from the catalytic core of the spliceosome. Chapter III describes how platinum adducts disrupt the activity of RNA processing enzymes similar to those that are necessary for maturation, maintenance and recycling of the transcriptome. Chapter III also describes the reversal of RNA platination using thiourea. The chemistry of platinum(II) is also characterized by preferential coordination to sulfur ligands, or thiophilicity. Incorporating this property into RNA chemistry, Chapters IV and V describe the reaction of platinum(II) complexes with phosphorothioate-substituted RNAs. Chapter IV describes engineering platinum(II) crosslinks in the Hammerhead ribozyme through the targeting of a platinum(II) complex to a specific phosphorothioate substitution installed in the active site of this catalytic RNA. Chapter V outlines efforts to characterize the cleavage and isomerization reactions promoted by platinum(II) coordination to phosphorothioate-substituted RNAs. Finally, Chapter VI summarizes the insights gained throughout the course of our studies and provides an outlook on the future of platinum-RNA chemistry. This dissertation includes co-authored material and previously published results. / Committee in charge: Michael M. Haley, Chair; Victoria J. DeRose, Advisor; David R. Tyler; Andrew J. Berglund; Eric A. Johnson

[pt] A cisplatina é um dos mais importantes agentes quimioterápicos usados no tratamento de diversos tipos de câncer. Entretanto, seu uso ocasiona efeitos colaterais, como ototoxicidade, neurotoxicidade e, em especial, a nefrotoxicidade, que é um dos mais significativos. Isso tem sido relacionado à interação da cisplatina com biomoléculas sulfuradas, como proteínas e aminoácidos. De modo a reduzir esses efeitos, tem-se sugerido a co-administração de certos compostos químicos sulfurados, denominados agentes salvadores, junto à cisplatina, o que poderia impedir sua interação com as moléculas sulfuradas no organismo e, por sua vez, a redução dos efeitos colaterais. Dentre esses agentes, sugere-se o tripeptídeo glutationa e o aminoácido penicilamina. A glutationa é o principal agente desintoxicante e antioxidante do organismo, sendo indispensável à homeostase celular, e a penicilamina é utilizada como medicamento no tratamento da doença de Wilson e artrite reumatoide. Dessa forma, visou-se, com este trabalho, a síntese e caracterização de dois complexos entre a cisplatina e os mesmos. A partir das técnicas de caracterização, que foram análise elementar, análise termogravimétrica e espectroscopias no infravermelho e Raman, foi possível sugerir uma estrutura para cada um dos complexos em que os ligantes se comportam de forma monodentada, sendo o enxofre o átomo doador. Isso ratificou o que é predito pelo conceito de ácidos e bases duros e macios, que comenta que ácidos de Lewis macios, que é o caso de Pt(2+) presente na cisplatina, têm grande afinidade química por bases de Lewis macias, que é o caso do enxofre presente no grupo tiolato dos ligantes desprotonados. O uso de cálculos teóricos, baseados nas estruturas propostas, como uma técnica recurso complementar de caracterização, foi de grande utilidade, já que, através dos mesmos, foi possível atribuir com maior segurança certas bandas presentes nos espectros de infravermelho e Raman experimentais. / [en] Cisplatinum is considered an important chemotherapeutic drug used against some kinds of cancer. However, the use of this drug may cause side effects such as ototoxicity, neurotoxicity and, mainly, nephrotoxicity. This fact has been related to the interaction of cisplatinum with biomolecules containing sulfur, such as proteins and amino acids. To promote the reduction of these effects, the coadministration of some compounds, called rescue agents, has been suggested in an attempt to prevent the interaction between cisplatinum and those biomolecules. Glutathione, a tripeptide, and the amino acid penicillamine could be used for this purpose. The former is the most important antioxidant in the human body and acts in cell homeostasis, and the latter is used in the treatment of Wilson’s disease and rheumatoid arthritis. Thus, considering the possibilities of interaction between cisplatinum and these sulfur agents, this work intended to synthesize and characterize the compounds formed by them. Using characterization techniques such as elementary analysis, thermogravimetry, and infrared and Raman spectroscopies, it was possible to suggest the structure of each compound, in which the ligands behaved as monodentate, bounding through the sulfur atom. This agree with the theory of soft and hard acids and bases, which predicts the affinity between platinum (Lewis acid) and sulfur (Lewis base). The use of theoretical calculations based on the structures proposed was very useful because it was possible to attribute some bands in the infrared and Raman spectra with more certainty.