Introdução e Justificativa: O câncer colorretal é a 3ª neoplasia maligna mais incidente no mundo, e apresenta comportamento bastante heterogêneo aos tratamentos instituídos. Conseguir diferenciar os pacientes de acordo com seu prognóstico e comportamento tumoral é um desafio. A biologia molecular, através dos microRNA (miRNA), pode contribuir na identificação do perfil tumoral e ajudar a predizer os desfechos clínicos. Objetivos: Comparar a expressão tecidual de miRNA-92a, miRNA-133a e miRNA-145 de pacientes com câncer de cólon, correlacionando o perfil de expressão de miRNA com aspectos clínicopatológico e prognóstico. Casuística e Métodos: Quantificação de miRNA-92a, miRNA-133a e miRNA-145 pela técnica de reação em cadeia de polimerase em tempo real (RTPCR) em amostras de tecido tumoral em pacientes com câncer esporádico de cólon (grupo de estudo) operados entre janeiro de 2010 a dezembro de 2011 e em amostras de tecido colônico sadio de pacientes saudáveis (grupo controle) submetidos à colonoscopia de rotina para rastreio de câncer colorretal. Os resultados das dosagens de miRNA foram correlacionados ao valor do antígeno cárcinoembrionário (CEA), idade, estadiamento, invasão linfovascular, sobrevida livre de doença e sobrevida global. Resultados: A idade média dos pacientes foi de 66 anos e 60% eram do sexo masculino. No momento do diagnóstico inicial 22% apresentavam metástase à distância, e em 80% dos pacientes foi atingida ressecção R0. A sobrevida global foi de 46 meses e a sobrevida livre de doença foi de 32 meses. Apenas o miRNA-133a esteve hiopoexpresso no grupo de estudo (p=0,0007). Ao se correlacionar os perfis de expressão dos miRNA às variáveis clínico-patológica e prognóstica, a hiperexpressão do miRNA-92a esteve associada a maior sobrevida global (p= 0,044). Discussão: O tempo de espera entre o encaminhamento para hospitais terciários e o início do tratamento oncológico pode contribuir para o elevado índice de pacientes com doença metastática ao diagnóstico. Melhores desfechos têm sido associados a hospitais de maior volume, aparentemente devido à maior expertise de que esses hospitais possuem. O miRNA-133a, um supressor tumoral, tem sido hipoexpresso em amostras tumorais quando comparadas a tecidos sadios. Por sua vez, a hiperexpressão do promotor tumoral miRNA-92a que esteve associada a melhor prognóstico, pode ser explicada por uma desregulação dos genes alvo desse miRNA ou por diferenças geográficas na expressão dos miRNA. Conclusão: o miRNA-133a encontra-se hipoexpresso em amostras tumorais, enquanto que a hiperexpressão do miRNA-92a esteve associada a melhor sobrevida global. / Introduction: Colorectal cancer is the 3rd most common malignancy in the world, and their incidence has been increase in the last decades. Although patients diagnosed with early stage tumors have more than 65% of overall survival, behavior of colorectal tumors and their response to established treatments is quite heterogeneous. Gap: In this context, becomes interesting differentiate patients according to their prognosis and tumor behavior. Molecular biology through the miRNA quantification can improve the identification of tumor profile and predict clinical outcomes. Propose: Evaluate tumor profile tissue expression of miRNA- 92a, miRNA- 133a and miRNA-145 in patients with non-hereditary colon cancer, correlating with clinicopathological variables and oncological outcomes. Assessing the ability of the expression of these miRNA predict the prognosis. Methods: A dosage of miRNA-92a, miRNA-133a and miRNA-145 was performed in tumor tissue samples from patients with colon cancer (called study group) and non-tumoral colonic tissue samples from healthy patients (called the control group) by real-time polymerase chain reaction. The results of miRNA levels were correlated with clinicopathological data and oncological outcomes. Results: Mean age was 66 years and 60% was male. At initial diagnosis, 22% of lesions had distant metastasis. R0 resection was achieved in 80% of patients. Disease-free survival was 32 months, and overall survival was 46 months. The MiRNA-133a showed higher values in the control group when compared to the study group (p = 0.0007), and miRNA-92a was associated with higher overall survival (p = 0.044). Discussion: Better outcomes in highly specialized centers could be explained by advances in surgical technique and perioperative care. MiRNA-133a was underexpressed in colonic tumoral tissue, likely others tumors. MiRNA-92a overexpression was associated with improve overall survival. It could be explained through deregulation of target genes or geographic patterns of miRNA expression. Conclusion: MiRNA-133a was underexpressed in study group, and over expression of miRNA-92a was associated with better overall survival.

Liu Bonnie Pui-ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 134-163). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abbreviations --- p.ii / Abstract / English --- p.1 / Chinese --- p.3 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Colorectal Cancer Statistics --- p.5 / Chapter 1.2 --- Colon Cancer --- p.5 / Chapter 1.3 --- Treatment --- p.6 / Chapter 1.4 --- Effects of Cytotoxic Drug Treatment --- p.7 / Chapter 1.5 --- Cell Cycle --- p.8 / Chapter 1.6 --- Oxidases --- p.9 / Chapter 1.7 --- Chemistry of Novel β-carboline: Tangutorine --- p.11 / Chapter 1.8 --- Aim of Study --- p.14 / Chapter Chapter 2 --- Cytotoxicity / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Materials and Methods --- p.18 / Chapter 2.3 --- Results --- p.23 / Chapter 2.4 --- Discussion --- p.44 / Chapter Chapter 3 --- Oxidase Activity and Protein Oxidation / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Materials and Methods --- p.54 / Chapter 3.3 --- Results --- p.60 / Chapter 3.4 --- Discussion --- p.80 / Chapter Chapter 4 --- Cell Cycle / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.2 --- Materials and Methods --- p.93 / Chapter 4.3 --- Results --- p.96 / Chapter 4.4 --- Discussion --- p.118 / Chapter Chapter 5 --- General Discussion --- p.126 / References --- p.134

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 95-113). Also available on the Internet.

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

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 49-53.

Thesis (Ph. D.)--University of Texas Health Science Center at Houston, School of Public Health, 1995. / Includes bibliographical references (leaves 100-107).

Orientador: Jose Barreto Campello Carvalheira / Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T00:26:02Z (GMT). No. of bitstreams: 1 Flores_MarceloBeneditodaSilva_D.pdf: 2957448 bytes, checksum: f168cee98307f1e264e0d77d10a83423 (MD5) Previous issue date: 2012 / Resumo: O câncer colorretal é um dos maiores problemas de saúde em todo o mundo acometendo 1 milhão de pessoas por ano. O índice de morte associado a essa doença é de aproximadamente 33% no mundo desenvolvido. A associação entre a obesidade e o risco para o desenvolvimento desse câncer é observada tanto em homens quanto em mulheres. A inflamação e a hiperinsulinemia, condições verificadas na obesidade podem contribuir, a princípio, para o risco de desenvolvimento do câncer colorretal. O fator de necrose tumoral alfa (TNF-alfa) é um dos mais importantes mediadores da resposta inflamatória e sua alta expressão pelo tecido adiposo é verificada nas condições de obesidade tanto em modelos animais quanto em humanos. O TNF-alfa contribui para a desregulação da via da sinalização insulínica através da fosforilação em serina dos substratos do receptor desse hormônio (IRS) mediada pela ativação de quinases como c-jun N terminal quinase (JNK) e da quinase inibidora do fator nuclear NF-kB (IKK). A ativação dessas quinases induz a fosforilação inibitória do substrato 1 do receptor de insulina (IRS-1) através da serina 307 (Ser307). Esse mecanismo reduz a ativação da via da fosfatidilinositol 3-quinase (PI3K)/Akt e da proteína alvo da rapamicina em mamíferos (mTOR) mediada pela insulina. TNF-alfa foi, a princípio, identificado como um agente antitumoral. Atualmente essa citoquina é reconhecida como uma promotora da tumorigênese e que associa a inflamação ao câncer. A importância do TNF-alfa e de seus mediadores intracelulares, JNK e IKK, como promotores do câncer de cólon é corroborada por estudos farmacológicos que utilizaram o azoximetano (AOM) ou AOM associado ao dextran sulfato de sódio (DSS) como indutores do câncer colorretal. Ademais, o aumento das concentrações do TNF-alfa associado à obesidade é um mecanismo comprovado de aumento do câncer de fígado em modelos animais. Embora as evidências de que a inflamação e a hiperinsulinemia tenham um envolvimento em potencial na gênese tumoral mediada pela obesidade, à avaliação sistemática da contribuição independente desses fatores para o desenvolvimento do câncer colorretal ainda é inconsistente. Neste trabalho, foi avaliado se a obesidade modulou a sinalização insulínica e a inflamação em tecido colônico e nos tumores colorretais. Foi mostrado que a resposta inflamatória anormal induzida pela obesidade promoveu de forma contundente o câncer colorretal / Abstract: Colorectal cancer (CRC) remains a major health burden with more than 1 million cases worldwide and a disease-specific mortality of approximately 33% in the developed world. The association between obesity and the risk for CRC development is observed in both men and women. In addition to its association with obesity, inflammation and hyperinsulinemia also primarily may contribute to the risk for development of CRC. Among the major mediators of the inflammatory response is tumor necrosis factor (TNF-alfa), whose overexpression in adipose tissue is a common feature in human and animal models of obesity. TNF-alfa contributes to the deregulation of the insulin-signaling pathway, including serine phosphorylation of insulin-receptor substrate (IRS) proteins by kinases such as c-jun N terminal kinase (JNK) and inhibitor of nuclear factor-kB kinase (IKK). JNK and IKK activation induce inhibitory serine 307(Ser307) phosphorylation of IRS-1, which decreases insulin-mediated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activation. TNF-alfa, first identified as an antitumor agent, now also is recognized as a tumor-promoting cytokine that links inflammation and cancer. The importance of TNF-alfa and its intracellular mediators, such as JNK and IKK, as colonic tumorigenic promoters is strengthened by knockout and pharmacologic studies, using azoxymethane (AOM) or AOM combined with dextran sulfate sodium (DSS) as inducers of colorectal carcinogenesis. Furthermore, the increased TNF-alfa levels associated with obesity are a potent liver tumor promoter in mice. Although the evidence for the potential involvement of inflammation and hyperinsulinemia in the development of obesity-mediated cancer is quite extensive, a systematic evaluation of the independent contribution of these factors to CRC development is lacking. Here, we examined whether obesity modulates insulin signaling and inflammation in the colon and CRC. We show that the obesity induced abnormal inflammatory response strongly promotes CRC / Doutorado / Clinica Medica / Doutor em Clínica Médica

Colonic epithelial glycoproteins were histochemically studied in rats during the induction of colorectal cancer with 1,2,-dimethylhydrazine-diHCl (DMH). 310 male Wistar rats were separated into 3 groups: 10 control rats were sacrificed without treatment, 150 rats were given weekly subcutaneous injections of the carcinogen and 150 rats were given sham Injections on the same schedule. Groups of rats (10 control and 10 treated) were sacrificed at various time intervals in an effort to obtain a large number of pre-neoplastic rat colons to be used to determine whether histochemical changes could be detected prior to morphologic change. Three histochemically distinct regions in the Wistar rat distal colon have been described using recently developed techniques. Two major histochemical changes have been shown to occur prior to malignancy. Change 'A' represented a decrease or loss of sulphate from the glycoproteins; this was the earliest and predominant change and has been shown to occur prior to any identifiable morphologic change. Change 'B' represented a decrease or loss of sulphate occurring in the same cells as a decrease or loss of side chain 0-acetylation of sialic acid residues; this change occured later and to a lesser extent than change 'A'. The data suggests that histochemical change may be a premalignant marker, however, further investigations are necessary to firmly establish this conclusion. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

強心苷是一類多年普遍用於心力衰竭治療的化合物,包括蟾蜍靈和地高辛。鈉泵（也可稱為鈉鉀ATP酶）是強心苷的受體。最近流行病學研究，體外實驗，動物實驗和臨床試驗表明，強心苷具有癌症治療的強大潛力。 / 大腸癌是全球第三大殺手，約有一半的大腸癌患者需要手術切除後的輔助治療。因此，通過化療殺死腫瘤細胞，是一個可行的辦法來治療大腸癌患者。在本課題的研究中，強心苷抗人結腸癌的作用在HT-29和Caco-2細胞上進行了評價與闡釋。在結腸癌細胞研究模型中，蟾蜍靈誘導caspase非依賴性的細胞死亡，伴隨沒有早期凋亡，沒有聚（ADP-核糖）聚合酶(PARP)與caspase-3裂解，這些發現與強心苷誘發其它類腫瘤細胞凋亡的機製完全不同。相反，蟾蜍靈激活自噬途徑，促進LC3-II積累和自噬流動。此外，其它強心苷如地高辛與烏本苷也促使LC3-II在HT-29細胞內聚集。沉默ATG5和Beclin-1顯著降低蟾蜍靈誘導的LC3- II積累和細胞死亡。蟾蜍靈誘導的自噬與活性氧（ROS）產生和JNK活化相關。我們的研究結果揭示了蟾蜍靈藥物對抗結腸癌細胞的一種新的機制，開闢了強心苷通過自噬途徑來治療大腸癌的可能性。 / 最近的研究表明，強心苷誘導多種癌細胞系的細胞包括促使凋亡與自噬的細胞週期阻滯在G2／M期。然而，沒有詳細的信息闡述強心苷如何阻滯細胞週期進展。在本課題研究中，我們研究了強心苷介導的細胞週期阻滯的分子機制。蟾蜍靈處理的HeLa H2B-YFP細胞被阻滯在前中期，伴隨姐妹染色單體凝聚，染色體未排列在赤道板，未退出有絲分裂期。這一結果被蟾蜍靈誘導的四倍DNA含量細胞既不在四倍體G1期也不在胞質分裂期進一步證明。此後，我們檢測了紡錘體組裝和染色體分離所需的Aurora激酶和Polo-like kinase 1 (Plk1)。結果發現，在HT-29和HeLa細胞上，蟾蜍靈和其它強心苷能顯著降低總蛋白質和磷酸化的Aurora激酶與Plk1。此外，我們還發現，蟾蜍靈通過PI3K下調有絲分裂酶的活性。這些結果已經通過沉默鈉泵α做了驗證。總之，我們的結果表明, 蟾蜍靈和其它強心苷鈉鉀泵抑製劑強有力的抑制細胞在前中期是通過PI3K/HIF-1α/NF-κB途徑下調Aurora激酶的蛋白質和磷酸化水平和Plk1的蛋白質水平。我們的研究發現在了解如何利用強心苷的潛能治療癌症以及認知鈉泵在細胞週期中的功能方面提供了有用的信息。 / The sodium pump (also known as Na+/K+-ATPase) is the receptor for cardiac glycosides, a group of compounds including bufalin and digoxin which have been commonly used for heart failure treatment for many years. Recent epidemiological studies, in vitro studies, animal studies and clinical trials have shown that cardiac glycosides have potential applications for cancer treatment. / Colorectal cancer is the third leading cause of cancer death worldwide and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treat patients with colorectal cancer. In this study, the effects of cardiac glycosides were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to their well documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis, as well as poly(ADP-ribose) polymerase (PARP) and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. Moreover, other cardiac glycosides digoxin and ouabain could also induce the accumulation of LC3-II in HT-29 cells. The silencing of ATG5 and Beclin-1 significantly reduced bufalin-induced LC3-II accumulation and cell death. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS) and JNK activation. My findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer by cardiac glycosides through an autophagy pathway. / Recent studies have revealed that cardiac glycosides induce G2/M phase arrest in many cancer cells, which include apoptosis- and autophagy-promoting cells. However, no detailed information is available on how cardiac glycosides arrest cell cycle progression. In this study, I studied the molecular mechanisms of cell cycle arrest mediated by cardiac glycosides. Bufalin-treated HeLa H2B-YFP cells were arrested at prometaphase, as characterized by the presence of sister chromatid cohesion, absence of chromosomes alignment on the metaphase plate, and failure to exit mitosis. This result was further confirmed by bufalin-induced cells with 4N DNA content in neither tetraploid G1 phase nor cytokinesis. Thereafter, I detected the Aurora kinases and Polo-like kinase 1 (Plk1), which are required for both spindle assembly and chromosome segregation. It was found that bufalin and other cardiac glycosides could significantly reduce the total protein and phosphorylation of Aurora kinases and Plk1 in HT-29 and HeLa cells. In addition, I found that PI3K was responsible for the bufalin-induced downregulation of the activities of mitotic kinases. This result was validated by silencing of sodium pump alpha. Taken together, my results demonstrate that bufalin and other cardiac glycoside inhibitors of the sodium pump potently arrest cancer cells at prometaphase by downregulating the total protein and phosphorylation of Aurora kinases and the total protein of Plk1 through the PI3K/HIF-1α/NF-κB pathway. My findings provide useful information in understanding how cardiac glycosides could be exploited for their potentials in treating cancer and in identifying the function of sodium pump in cell cycle progression. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xie, Chuanming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 133-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Declaration of Originality --- p.i / Acknowledgements --- p.iii / Abstract --- p.vi / Abstract (in Chinese) --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvi / List of Tables --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.2 --- The chemical structure of cardiac glycosides --- p.2 / Chapter 1.3 --- The traditional use of cardiac glycosides in cardiology --- p.4 / Chapter 1.4 --- The role of cardiac glycosides in cancer treatment --- p.4 / Chapter 1.5 --- The mechanisms of action by cardiac glycosides in cancer --- p.5 / Chapter 1.5.1 --- The structure and functions of cardiac glycosides receptor sodium pump --- p.5 / Chapter 1.5.2 --- Sodium pump as anticancer target --- p.6 / Chapter 1.5.3 --- The signal pathways involved in anticancer effect of cardiac glycosides --- p.7 / Chapter 1.6 --- The role of cardiac glycosides in apoptosis and autophagy --- p.8 / Chapter 1.7 --- Objectives of this project --- p.12 / Chapter Chapter 2 --- Bufalin induces autophagy but not apoptosis in human colon cancer cells --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.19 / Chapter 2.2.1 --- Reagents and antibodies --- p.19 / Chapter 2.2.2 --- Cell culture --- p.19 / Chapter 2.2.3 --- Cell viability and cell death assay --- p.20 / Chapter 2.2.4 --- Annexin V and PI staining --- p.20 / Chapter 2.2.5 --- Cell cycle analysis --- p.21 / Chapter 2.2.6 --- Analysis of cleaved caspase-3-positive cells by flow cytometry --- p.21 / Chapter 2.2.7 --- Western blot analysis --- p.21 / Chapter 2.2.8 --- Immunofluorescence analysis of LC3 distribution --- p.22 / Chapter 2.2.9 --- RNA isolation and RT-PCR --- p.22 / Chapter 2.2.10 --- siRNAs transfection and treatments --- p.23 / Chapter 2.2.11 --- Transmission electron microscopy --- p.23 / Chapter 2.2.12 --- Statistical analysis --- p.24 / Chapter 2.3 --- Results --- p.24 / Chapter 2.3.1 --- Bufalin induces cell death and cell cycle arrest at G2/M phase in colon cancer cells --- p.24 / Chapter 2.3.2 --- Bufalin induces caspase-independent cell death in colon cancer cells --- p.28 / Chapter 2.3.3 --- Bufalin induces autophagy in colon cancer cells --- p.30 / Chapter 2.3.4 --- Bufalin-induced autophagy is dependent on ATG5 and Beclin-1 --- p.37 / Chapter 2.3.5 --- Increased autophagy is responsible for bufalin-induced cell death --- p.40 / Chapter 2.4 --- Discussion --- p.42 / Chapter Chapter 3 --- Bufalin mediates autophagic cell death through ROS generation and JNK activation --- p.44 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.46 / Chapter 3.2.1 --- Reagents and antibodies --- p.46 / Chapter 3.2.2 --- Cell culture --- p.47 / Chapter 3.2.3 --- Cell viability and cell death assay --- p.47 / Chapter 3.2.4 --- Western blot analysis --- p.47 / Chapter 3.2.5 --- Quantification of cells with > 5 LC3 punctate staining --- p.47 / Chapter 3.2.6 --- siRNAs transfection and treatments --- p.48 / Chapter 3.2.7 --- RNA isolation and RT-PCR --- p.48 / Chapter 3.2.8 --- ROS analysis --- p.48 / Chapter 3.2.9 --- JC-1 staining --- p.49 / Chapter 3.2.10 --- Statistical analysis --- p.49 / Chapter 3.3 --- Results --- p.50 / Chapter 3.3.1 --- Bufalin induces autophagy-mediated cell death via ROS generation --- p.50 / Chapter 3.3.2 --- Activation of JNK is required for the upregulation of ATG5 and Beclin-1, and subsequent autophagy-mediated cell death in response to bufalin --- p.54 / Chapter 3.3.3 --- ROS generation is upstream of JNK activation in bufalin-induced cell death --- p.59 / Chapter 3.3.4 --- Bufalin-induced ROS generation is derived from mitochondria --- p.62 / Chapter 3.4 --- Discussion --- p.66 / Chapter Chapter 4 --- Bufalin arrests cells at prometaphase --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Materials and Methods --- p.70 / Chapter 4.2.1 --- Reagents and antibodies --- p.70 / Chapter 4.2.2 --- Cell synchronization --- p.70 / Chapter 4.2.3 --- Mitotic index analysis of phosphorylation of MPM2 --- p.71 / Chapter 4.2.4 --- Cell cycle analysis --- p.71 / Chapter 4.2.5 --- Time-lapse experiments --- p.71 / Chapter 4.2.6 --- Immunofluorescence analysis of phospho-histone H3 (Ser10) --- p.72 / Chapter 4.2.7 --- Western blot analysis --- p.73 / Chapter 4.3 --- Results --- p.73 / Chapter 4.3.1 --- Bufalin reduces mitotic marker phosphorylation of histone H3 and MPM2 and increases cells with 4N DNA content --- p.73 / Chapter 4.3.2 --- Increased cells with 4N DNA content after bufalin treatment are in neither a tetraploid G1 phase nor a cytokinesis arrest --- p.77 / Chapter 4.3.3 --- Bufalin-treated cells can enter prophase, but fail to pass through metaphase --- p.80 / Chapter 4.4 --- Discussion --- p.83 / Chapter Chapter 5 --- Bufalin induces prometaphase arrest through downregulating mitotic kinases --- p.87 / Chapter 5.1 --- Introduction --- p.87 / Chapter 5.2 --- Materials and Methods --- p.89 / Chapter 5.2.1 --- Reagents and antibodies --- p.89 / Chapter 5.2.2 --- Cell synchronization --- p.90 / Chapter 5.2.3 --- Immunofluorescence staining --- p.90 / Chapter 5.2.4 --- siRNAs transfection and treatments --- p.91 / Chapter 5.2.5 --- Western blot analysis --- p.91 / Chapter 5.2.6 --- Statistic analysis --- p.91 / Chapter 5.3 --- Results --- p.92 / Chapter 5.3.1 --- Bufalin downregulates Aurora A and B in protein and phosphorylation levels --- p.92 / Chapter 5.3.2 --- Bufalin prevents Aurora A recruitment to mitotic centrosomes and Aurora B recruitment to unattached kinetochores --- p.97 / Chapter 5.3.3 --- Bufalin prevents Plk1 recruitment to mitotic centrosomes and unattached kinetochores through downregulation of protein levels of Plk1 --- p.101 / Chapter 5.3.4 --- Bufalin decreases the activities of Aurora A, Aurora B and Plk1 through PI3K pathway --- p.105 / Chapter 5.3.5 --- HIF-1α and NF-κB pathways are involved in sodium pump-mediated the regulation of mitotic kinases --- p.109 / Chapter 5.4 --- Discussion --- p.112 / Chapter Chapter 6 --- General discussion --- p.115 / Chapter 6.1 --- Potential toxicity of bufalin --- p.115 / Chapter 6.2 --- Cardiac glycosides induced programmed cell death --- p.115 / Chapter 6.3 --- Signal pathways involved in cardiac glycosides-mediated autophagy --- p.117 / Chapter 6.4 --- The relationship between ROS and JNK in cardiac glycosides-induced autophagy --- p.120 / Chapter 6.5 --- The role of ROS in apoptosis and autophagy --- p.121 / Chapter 6.6 --- The role of cardiac glycosides in cell cycle arrest --- p.122 / Chapter 6.7 --- Application of cardiac glycosides in combination with chemotherapy and radiotherapy --- p.125 / Chapter Chapter 7 --- Conclusions and future perspectives --- p.127 / References --- p.133 / Appendices --- p.153 / Publication --- p.153

Thesis (Ph.D. in Toxicology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 156-176). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;