Cancer, despite significant advances in diagnosis and treatment, is the second most common cause of death in economically advanced countries. The main reason for the failure of anticancer therapy is the development of chemoresistance, which can be either internal or acquired, and is primarily mediated by the activation of various key regulators (eg MDR, PI3K/Akt, etc.). Genetic and epigenetic mechanisms are involved in activating these pathwa- ys. Significant epigenetic mechanisms that can participate in chemoresistance include regula- tion of gene expression by microRNA (miRNA) and long noncoding RNA (lncRNA). Dere- gulated expression of these non-coding RNAs has been observed in many diseases and their involvement in the initiation and progression of malignant tumors has been demonstrated. In this study, we investigated the expression of long non-coding RNA MIAT in hypoxia (1% O2) in chemosensitive and chemoresistant neuroblastoma cell lines (NBL), as hypoxia is a significant negative prognostic factor of many tumors and is involved in chemoresistance. Relative expression of MIAT was influenced by the number of cultured cells, where expression was increased by culturing more cells. MIAT expression was also significantly increased after 6 hours of NBL culture UKF-NB-4 in hypoxic conditions, and...

Neuroblastoma (NBL), the most common non-Central Nervous System (CNS) solid tumor of childhood, characteristically displays heterogeneous clinical presentation and biological behavior. Previous work has studied the genetic basis of the disease and revealed a low somatic mutation burden. In order to identify novel therapeutic targets and better understand the biology of high-risk NBLs, I investigated whole transcriptome profiles of two cohorts of metastatic NBLs using RNA sequencing. First, I studied changes in splicing pattern in a cohort of 29 patients. V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplified NBLs showed a distinct splicing pattern affecting multiple cancer hallmarks. Six splicing factors have altered expression patterns in MYCN-amplified tumors and cell lines, and binding motifs for these factors were significantly enriched in differentially-spliced genes. ChIP-seq analysis showed direct binding of MYCN to promoter regions of splicing factors PTBP1 and HNRNPA1, demonstrating that MYCN regulates splicing by directly regulating expression of key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage 4 NBL patients (p≤0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, a pro-tumor-growth isoform, resulted in repression of NBL cell growth. Second, I used whole transcriptome sequencing in a cohort of 150 patients to assess expressed mutations, fusion genes, and gene expression including long non-coding genes to provide clinically-relevant classification and to offer insights into NBL tumor biology. Twenty-four genes including ALK, ATRX and MYCN were recurrently mutated in NBL transcriptomes. In-frame FOXR1 fusions were detected in 4 samples, including 3 cases or 14% of stage 4S NBLs. Unsupervised gene expression analysis revealed four molecular subgroups. MYCN and tumor microenvironment were the primary discriminating signatures in these molecular subgroups. Fifty-eight percent of MYCN-not-amplified samples showed high MYCN signatures, which were potentially contributed by various genomic events such as MYCN activating mutations and FOXR1 fusions. High MYCN signature was significantly associated with poor overall survival in MYCN-not-amplified tumors (p=0.0017). In addition, the tumor microenvironment including stromal and immune cell infiltration significantly contributed to the NBL transcriptional landscape and tumor progression.

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Cancer is the second leading cause of death in America. In 2018, there were 9.5 million deaths due to cancer according to the International Agency for Research on Cancer (IARC), and this number is expected to grow to 16.3 million by 2040. Among the type of cancers, neuroblastoma and nerve tissue cancers have a 5-year survival rate of 33%, which is very low. One of the main issues linked to such situations is due to the lack of specificity in removing tumor cells. While clinical therapies work to reduce tumor mass as much as possible, they cannot always target all of them, and once some cancer cells are left behind, they regrow and spread. The work of this thesis seeks to enhance the treatment outcome by utilizing all-trans retinoic acid (ATRA), a metabolite of vitamin A, to induce differentiation of nerve tissue cancer cells and eliminate their ability to self-renew (reemerge). Differentiation therapy is currently utilized in select clinical applications but the utilization of ATRA is limited due to its poor solubility in the blood, low bioavailability, short half-life, and in vivo toxicity. In order to alleviate some of these issues, the ATRA molecule was engineered with a novel cell penetrating peptide and tested for its efficacy. Data and results presented herein report the differentiation induced by the CPP-conjugated ATRA may act as a viable method for neuroblastoma treatment. / Bioengineering

Introduction Advanced Kadish stage esthesioneuroblastoma requires more extensive resections and aggressive adjuvant therapy to obtain adequate disease-free control, which can lead to higher complication rates. We describe the case of a patient with Kadish D esthesioneuroblastoma who underwent multiple surgeries for infectious, neurologic, and wound complications, highlighting potential preventative and salvage techniques. Case Presentation A 61-year-old man who presented with a large left-sided esthesioneuroblastoma, extending into the orbit, frontal lobe, and parapharyngeal nodes. He underwent margin-free endoscopic-assisted craniofacial resection with adjuvant craniofacial and cervical radiotherapy and concomitant chemotherapy. He then returned with breakdown of his skull base reconstruction and subsequent frontal infections and ultimately received 10 surgical procedures with surgeries for infection-related issues including craniectomy and abscess evacuation. He also had surgeries for skull base reconstruction and CSF leak, repaired with vascularized and free autologous grafts and flaps, synthetic tissues, and CSF diversion. Discussion Extensive, high Kadish stage tumors necessitate radical surgical resection, radiation, and chemotherapy, which can lead to complications. Ultimately, there are several options available to surgeons, and although precautions should be taken whenever possible, risk of wound breakdown, leak, or infection should not preclude radical surgical resection and aggressive adjuvant therapies in the treatment of esthesioneuroblastoma.

O neuroblastoma é o tumor sólido extra-cranial mais comum e mortal da infância, sendo o tempo de sobrevida nos casos mais agressivos ainda muito curto. Uma das esperanças nesses casos é que os estudos moleculares possam fornecer informações sobre os genes ou as vias moleculares que governam a patogênese dos neuroblastomas. Pois, há poucos genes como o MYCN, que foi descrito por estar diretamente ligado ao neuroblastoma. A amplificação deste oncogene ocorre em pouco mais de 25% dos neuroblastomas e é considerada como o mais importante marcador de prognóstico nestes tumores, sendo fortemente relacionada aos estádios avançados da doença e falha no tratamento. Outros genes do amplicon do MYCN, incluindo o DDX1 \"DEAD box polypeptide 1 gene\" e o NAG \"neuroblastoma-amplified gene\", estão sendo observados por se apresentarem co-amplificados com o MYCN. Entretanto, a importância deste fenômeno no prognóstico ainda é desconhecida. Os objetivos deste trabalho foram determinar qual o melhor método para estudar a amplificação dos genes MYCN, DDX1 e NAG, além de esclarecer a importância da coamplificação dos genes DDX1 e NAG no prognóstico. Procedimento: O número de cópias dos genes MYCN, DDX1 e NAG foi determinado por PCR em Tempo Real e PCR convencional em 100 neuroblastomas primários. Os dados da PCR em Tempo Real foram analisados por quantificação absoluta e relativa. Os resultados da PCR convencional foram analisados por eletroforese em gel de agarose, medindo a intensidade das bandas formadas no gel no sistema Kodak. A relevância da amplificação gênica como marcador de prognóstico foi avaliada em 74 pacientes, dos quais nós obtivemos o acompanhamento clínico. Resultados: Nos 74 casos estudados, ambos os métodos demonstraram que a amplificação do MYCN estava associada com os estádios mais avançados da doença. A análise das curvas de sobrevida livre de progressão confirmou que pacientes com ausência de amplificação do MYCN apresentavam maior tempo de sobrevida. Nós também analisamos a amplificação do DDX1 nas mesmas amostras incluindo aquelas com ausência de amplificação de MYCN. Não foi encontrada nenhuma relação entre a co-amplificação com idade ao diagnóstico ou tempo de sobrevida. Conclusões: Os métodos aplicados para calcular o número de cópias dos genes na PCR em Tempo Real mostraram-se equivalentes. A PCR em Tempo Real apresentou maior acurácia nos resultados quando comparada à PCR convencional. A análise da sobrevida não demonstrou relação entre a amplificação dos genes DDX1 e/ou NAG com piora no prognóstico. / Neuroblastoma is the most common and deadly extra-cranial solid childhood tumor. Survival rates for aggressive neuroblastomas are still disappointingly low. One of the hopes is that molecular studies will provide insights into the genes and molecular pathways that govern neuroblastoma pathogenesis. However, at present only a few genes as MYCN have been directly linked to neuroblastoma. MYCN oncogene amplification, occurring in up to 25% of neuroblastomas, has been considered the most important prognostic factor, strongly correlating to advanced stage disease and treatment failure. Another genes in the MYCN amplicon, including the DEAD box polypeptide 1 (DDX1) gene, and neuroblastoma-amplified gene (NAG gene), have been found to be frequently co-amplified with MYCN in NB. But the prognostic significance of the coamplification remains unclear. The aims of this study were to evaluate which is the best method to study the gene amplification of those three genes MYCN, DDX1 and NAG, as well as clarify the prognostic significance of the co-amplification or DDX1 and NAG with MYCN. Procedure: The gene copy numbers of MYCN, DDX1, and NAG were determined by the real-time quantitative polymerase chain reaction and conventional polymerase chain reaction in 100 primary NBs. Real-Time data were analyzed by absolute and relative quantification. For conventional PCR, samples were electrophoresed on a 2% agarose gel and the intensity of each band evaluated by Kodak image software. To evaluate of the prognostic significance of the gene amplification we had only 74 cases in witch we could analyze the follow-up. Results: In all 74 cases, both methods demonstrated that MYCN amplification was associated mainly with advanced cancer stages, and the analysis of overall survival confirmed that patients without MYCN amplification had a cumulative survival significantly higher than patients with oncogene amplification. We also studied DDX1 and NAG amplification for all NB samples even that without MYCN amplification. No relationship between any gene co-amplification status and disease stage, age at diagnosis, or overall survival was found. Conclusions: The two methods used to calculate gene copy number for Real Time PCR assay shown to be equivalent. Real Time PCR assay shown to be more accurate to study gene amplification than conventional PCR assay. Survival analysis pointed out that DDX1 and/or NAG amplification has no additional adverse effect on prognosis.

Pouco se conhece a respeito da incidência ou prevalência da infecção pelo vírus da raiva em morcegos, ou ainda sobre a distribuição do vírus em tecidos e órgãos não nervosos. Os objetivos deste trabalho foram: i) verificar as espécies de morcegos mais freqüentemente envolvidas com a raiva no Estado de São Paulo, Sudeste do Brasil; ii) estudar a distribuição do vírus da raiva em tecidos e órgãos não nervosos de morcegos; iii) estudar os períodos de mortalidade das amostras de vírus da raiva encontradas nos cérebros e glândulas salivares de morcegos, após inoculação intracerebral em camundongos, e iv) comparação do isolamento do vírus da raiva no sistema camundongo e cultura de células de neuroblastoma (N2A). Entre abril de 2002 a novembro de 2003, 4.393 morcegos capturados de diferentes municípios do Estado de São Paulo foram enviados à Seção de Diagnóstico da Raiva do Instituto Pasteur de São Paulo. Destes, 82 (1,87%) foram positivos para raiva pela técnica de imunofluorescência aplicada aos materiais do cérebro e 33 morcegos pertenciam ao gênero Artibeus sp; 15 Myotis sp; 10 Epitesicus sp; 5 Lasiurus sp; 4 Nyctinomops sp; 4 Tadarida sp; 3 Histiotus sp; 1 Molossus sp; 1 Eumops sp e 6 vampiros Desmodus rotundus. A distribuição do vírus em diferentes órgãos foi examinada pela inoculação de camundongos e células N2A com suspensões a 20% preparadas a partir de fragmentos do cérebro, glândula salivar submandibular, pulmão, língua, coração, bexiga urinária, rins, gordura interescapular, músculo peitoral, trato genital (testículos ou ovários e útero) e estômago. O vírus foi prontamente recuperado de tecidos e órgãos não nervosos com diferentes graus de sensibilidade, tanto em camundongos como em células N2A, e os órgãos mais apropriados para o isolamento viral foram os cérebros e glândulas salivares. Os períodos máximos de mortalidade observados para os vírus presentes nos cérebros usualmente foram mais curtos que os das glândulas salivares, a média do período máximo &plusmn; desvio padrão calculado para os cérebros de morcegos hematófagos foi de 15,33 &plusmn; 2,08 dias e para as glândulas salivares, 11,33 &plusmn; 2,30 dias; para os morcegos insetívoros, 16,45 &plusmn; 4,48 dias para os cérebros e para as glândulas salivares, 18,91 &plusmn; 6,12 dias; e para os morcegos frugívoros, as suspensões cerebrais apresentaram período máximo médio 12,60 &plusmn; 2,13 dias e para as glândulas salivares, 15,67 &plusmn; 4,82 dias. O teste de ANOVA indicou existir diferenças significantes entre os períodos de mortalidade correspondentes às suspensões preparadas a partir dos cérebros de morcegos insetívoros (período mínimo) e glândulas salivares de morcegos insetívoros (período máximo) e entre glândulas salivares de morcegos insetívoros (período máximo) e cérebros de morcegos frugívoros (período mínimo), com p<0.001. O uso de células N2A para o primo-isolamento do vírus da raiva a partir de tecidos e órgãos não nervosos de morcegos, diferente de cérebros, não mostraram resultados consistentes, especialmente devido à contaminação bacteriana e fator toxicidade / Little is known about the incidence of infection or the prevalence rate of rabies in bats, or the distribution of virus in non-nervous tissues and organs. The aim of this work was to study: i) the most frequent species of bats involved with rabies virus infection in the State of São Paulo, Southeast Brazil; ii) the distribution of rabies virus in tissues and non-nervous organs of bats; iii) the mortality periods of virus found in brains and salivary glands of bats after intracerebral inoculation of mice, and iv) comparison of virus isolation in mice and N2A neuroblastoma cell culture. From April 2002 to November 2003, 4,393 bats captured from different municipalities of the State of São Paulo were sent to Rabies Diagnostic Section of the Instituto Pasteur de São Paulo - SP. Among these, 82 (1.87%) were found positive by the immunofluorescence technique applied to brain specimens and 33 bats were of the genus Artibeus sp; 15 Myotis sp; 10 Epitesicus sp, 5 Lasiurus sp, 4 Nyctinomops sp, 4 Tadarida sp, 3 Histiotus sp; 1 Molossus sp, 1 Eumops sp, and 6 vampires Desmodus rotundus. The distribution of virus in the organs was examined by inoculating mice and N2A cells with the 20% suspensions prepared from brain, submaxillary salivary gland, lungs, tongue, heart, urinary bladder, kidneys, brown fat, pectoral muscle, genital tract (testicles or ovaries and uterus), and stomach. The virus was promptly recovered from tissues and non-nervous organs at different degrees of sensitivity in both mice and N2A cells, and the most appropriate organs for the virus isolation were the brains and salivary glands. The maximum mortality periods found for the brain specimens usually were shorter than the salivary glands, the maximum mean perio &plusmn; standard deviation calculated for the brains taken from the vampire bats was 15.33 &plusmn; 2.08 days and for salivary glands, 11.33 &plusmn; 2.30 days; for the insectivorous bats the maximum for the brain suspensions was 16.45 &plusmn; 4.48 days and for the salivary glands, 18.91 &plusmn; 6.12; and for the frugivorous bats, the brain suspensions showed the maximum of 12.60 &plusmn; 2.13 days and the salivary glands, the mean maximum period of 15.67 &plusmn; 4.82 days. The ANOVA test indicated that the most significant differences in the mortality periods were between the suspensions prepared by the brains of insectivorous bats (minimum period) and salivary glands of insectivorous bats (maximum period); salivary glands of insectivorous bats (minimum period) and brains of frugivorous bats (minimum period) with p<0.001. The use of N2A cells for the prime isolation of rabies virus from tissues and non-nervous organs other than brains of bats did not show consistent results, especially due to bacterial contamination and toxicity factor

神經母細胞瘤是一種交感神經系統的腫瘤。它是最常見的兒童顱外實體瘤。神經母細胞瘤約佔兒童腫瘤的8-10%，佔15%的兒童腫瘤死亡率。目前神經母細胞瘤的治療方法包括外科手術， 化學藥物治療， 放射治療， 幹細胞移植， 誘導分化治療和免疫治療。 然而，這些治療方法通常會導致許多無法避免的嚴重的副作用。因此，開發能高效抑制神經母細胞瘤但對正常細胞無明顯副作用的新型藥物顯得至關重要。最近，用來源於天然產物或中藥的化合物治療癌症引起了科學家的廣泛關注。靛玉紅-3’-肟（Indirubin-3’-oxime, I3M）和色胺酮（tryptanthrin）分別是從板藍根中分離得到的靛藍生物鹼和吲哚喹唑啉類生物鹼。據研究報導，這兩種生物鹼具有多種生物學和藥理學作用，包括抗菌，抗炎症和抗腫瘤作用。它們對體外的多種人腫瘤細胞具有抗腫瘤作用。然而，它們對人神經母細胞瘤的調節作用和作用機理仍不太清楚。在我的博士研究課題中，我們對板藍根生物鹼包括靛玉紅-3’-肟和色胺酮對人神經母細胞瘤的抗腫瘤活性和作用機制進行了研究和探討。 / 首先，我們研究了靛玉紅-3’-肟對人神經母細胞瘤的抗腫瘤活性和作用機制。實驗結果表明，靛玉紅-3’-肟能夠抑制人神經母細胞瘤LA-N-1, SH-SY5Y 和 SK-N-DZ細胞系的生長，並且其抑制效果呈時間和濃度依賴性。然而，靛玉紅-3’-肟對正常細胞無顯著的細胞毒性作用。對其生長抑制作用機制的研究結果表明靛玉紅-3’-肟能夠特異性地減少LA-N-1細胞系中線粒體的調節子ERR和 PGC-1的表達，從而導致線粒體生成減少，線粒體膜電位降低以及線粒體活性氧（ROS）增加。靛玉紅-3’-肟還增加週期蛋白依賴性蛋白激酶（CDK）抑制蛋白p27{U+1D37}{U+2071}{U+1D56}¹的蛋白水平並降低週期蛋白依賴性蛋白激酶2（CDK2）和細胞週期蛋白E（Cyclin E）的表達，從而導致細胞週期阻滯在G0/G1期。 另外，我們發現靛玉紅-3’-肟能減少SH-SY5Y細胞系的線粒體生成，增加細胞內活性氧的水準從而導致細胞週期停滯在G0/G1期和細胞凋亡。以上結果表明靛玉紅-3’-肟可能通過破壞線粒體的功能從而導致LA-N-1和SH-SY5Y細胞的細胞週期阻滯和誘導SH-SY5Y細胞的細胞凋亡來發揮其抗腫瘤的作用。 / 接著，我們對色胺酮對人神經母細胞瘤的抗腫瘤活性和作用機制進行了探討。我們研究的結果表明，色胺酮可以時間和濃度依賴性地抑制人神經母細胞瘤LA-N-1, SH-SY5Y 和 SK-N-DZ細胞系的生長，而對正常的細胞無顯著的細胞毒性。對色胺酮抑制人神經母細胞瘤生長的機制研究表明，色胺酮能顯著地降低細胞週期蛋白（Cyclin D1和 Cyclin D3）和週期蛋白依賴性蛋白激酶（CDK4和CDK6）的蛋白水平從而導致細胞週期停滯在G0/G1期。色胺酮可以激活半胱氨酸天冬氨酸蛋白酶8,9和3/7（caspase 8, caspase 9 和 caspase 3/7）從而誘導LA-N-1細胞凋亡。色胺酮還可以誘導LA-N-1細胞分化，表現為神經細胞分化的細胞形態，乙醯膽鹼酯酶活性的增加和多種細胞分化的分子標記的表達上調。另外，色胺酮還能降低LA-N-1細胞中N-myc的表達。有趣的是，通過RNA干擾技術降低N-myc的表達能誘導LA-N-1細胞的分化。總的來說，以上結果顯示色胺酮通過誘導細胞週期阻滯，細胞凋亡和細胞分化從而發揮其抗腫瘤的作用。它可能被開發為治療有N-myc基因擴增的高危的人神經母細胞瘤的潛在藥物。 / 此外，我們還研究了靛玉紅-3’-肟和色胺酮是否具有抗血管生成的作用。體外實驗的研究結果表明，靛玉紅-3’-肟和色胺酮能夠濃度依賴性地抑制人微血管上皮細胞 （HMEC-1細胞）的增殖，遷徙和血管生成，但對HMEC-1細胞卻沒有顯著的細胞毒性作用。此外，靛玉紅-3’-肟和色胺酮能顯著地抑制小鼠體內的基質膠栓（Matrigel plug）的血管生成。對它們抑制血管生成的機制的研究表明，靛玉紅-3’-肟能下調血管生成素1（Ang-1）和基質金屬蛋白酶2（MMP2）的表達，上調血管生成素2（Ang-2）的表達。靛玉紅-3’-肟能結合到血管內皮生長因數受體2（VEGFR2） 的ATP結合位點上從而抑制血管內皮生長因數受體2的磷酸化和下游的MEK/ERK和PI3K/AKT/GSK信號轉導通路。色胺酮同樣可以抑制多種血管生成因子（Ang-1,PDGFB 和MMP2）的表達。此外，它可以結合到血管內皮生長因數受體2 的ATP結合位點上從而抑制血管內皮生長因數受體2的磷酸化和血管內皮生長因數受體2介導的ERK1/2信號通路。以上的體外和體內實驗研究結果表明靛玉紅-3’-肟和色胺酮通過靶向血管內皮生長因數受體2介導的信號通路來發揮其抗血管生成的作用。它們可能被開發為治療血管生成相關疾病的潛在藥物。 / 總而言之，我們的研究結果表明靛玉紅-3’-肟和色胺酮通過誘導人神經母細胞瘤細胞的細胞週期阻滯，細胞凋亡或誘導神經細胞分化從而抑制人神經母細胞瘤細胞的生長。然而，它們對正常細胞無顯著的細胞毒性作用。此外，靛玉紅-3’-肟和色胺酮通過靶向血管內皮生長因數受體2介導的信號通路來發揮其抗血管生成的作用。未來的研究將進一步探討靛玉紅-3’-肟和色胺酮對人神經母細胞瘤細胞的分子作用機理。另外，通過人神經母細胞瘤細胞的裸鼠移植瘤動物模型可進一步去了解這些板藍根生物鹼在體內的抗腫瘤效果。 / Neuroblastoma, a tumor of the sympathetic nervous system, is the most common extracranial solid cancer in childhood. It accounts for 8% to 10% of all childhood cancers and for approximately 15% of cancer deaths in children. Current treatment modalities consist of surgery, chemotherapy, radiation therapy, stem cell transplantation, differentiation therapy and immunotherapy. However, these treatments often cause severe and inevitable side effects. It is important to develop novel drugs with higher efficacy on neuroblastoma cells and minimal side effects on normal cells. The use of new promising therapeutic compounds derived from natural products or Chinese herbs have attracted much attention of scientist as an alternative strategy in cancer treatment. Indirubin-3’-oxime (I3M) is an indigo alkaloid and tryptanthrin is an indoloquinazoline alkaloid which can be isolated from the dried roots of medicinal indigo plants known as Banlangen. These two alkaloids have been reported to possess various biological and pharmacological activities, such as anti-microbial, anti-inflammatory, and anti-tumor effects. They were found to exhibit potent anti-tumor activities on various types of human cancer cells in vitro. However, their modulatory effects on human neuroblastoma and the underlying mechanisms remain poorly understood. In my PhD project, the possible anti-tumor activities and action mechanisms of Banlangen alkaloids, including I3M and tryptanthrin, on human neuroblastoma cells were investigated. / Firstly, the anti-cancer effects of I3M on human neuroblastoma cells and the underlying mechanisms were investigated. I3M was found to inhibit the growth of the human neuroblastoma LA-N-1, SH-SY5Y and SK-N-DZ cells in a concentration- and time-dependent manner, but exhibited little, if any, direct cytotoxicity on normal cells. Mechanistic studies showed that I3M specifically decreased the expression of mitochondrial regulators ERRγ and PGC-1βand resulted in decreased mitochondrial mass and altered mitochondrial function characterized by reduction in mitochondrial membrane potential and elevation of reactive oxygen species (ROS) level in LA-N-1 cells. I3M also increased the level of cyclin-dependent kinase (CDK) inhibitor p27{U+1D37}{U+2071}{U+1D56}¹ and reduced the levels of CDK2 and cyclin E in LA-N-1 cells, leading to cell cycle arrest at the G0/G1 phase. In addition, I3M was also found to reduce the mitochondrial mass and increase the ROS level leading to cell cycle arrest at G0/G1 phase and apoptosis in SH-SY5Y cells. These results, when taken together, suggest that I3M might exert its anti-tumor activity by causing mitochondrial dysfunction which led to cell cycle arrest in LA-N-1 cells and resulted in cycle arrest and apoptosis in SH-SY5Y cells. / The anti-tumor effects and action mechanisms of tryptanthrin on the human neuroblastoma cells were also examined. Our results showed that tryptanthrin inhibited the growth of the human neuroblastoma LA-N-1, SH-SY5Y and SK-N-DZ cells in a concentration- and time-dependent manner, but exhibited little, if any, direct cytotoxicity on normal cells. Mechanistic studies indicated that tryptanthrin significantly reduced the protein levels of cyclin D1, cyclin D3, CDK4 and CDK6 leading to cell cycle arrest at G0/G1 phase. In addition, tryptanthrin activated caspase 8, caspase 9 and caspase 3/7 resulting in apoptosis of the human neuroblastoma LA-N-1 cells. Moreover, tryptanthrin induced neuronal differentiation of LA-N-1 cells, as assessed by morphological criteria, enhancement of acetylcholine esterase activity and up-regulation of various differentiation markers. Tryptanthrin treatment also led to the significant reduction of N-myc expression in LA-N-1 cells. Interestingly, down-regulating N-myc expression using siRNA induced neuronal differentiation of LA-N-1 cells. Collectively, these results indicate that tryptanthrin might exert its anti-tumor activity on the human neuroblastoma LA-N-1 cells by inducing cell cycle arrest, apoptosis and neuronal differentiation. It might be exploited as a potential therapeutic candidate for the treatment of high-risk neuroblastomas with N-myc-amplification. / Moreover, the anti-angiogenic activities of I3M and tryptanthrin were studied. Our results showed that I3M and tryptanthrin inhibited the proliferation, migration, and tube formation of the human microvascular endothelial HMEC-1 cells in vitro in a concentration-dependent manner but exhibited no significant cytotoxicity on these cells. Moreover, I3M and tryptanthrin markedly suppressed the in vivo angiogenesis in Matrigel plugs in mice. Mechanistic studies indicated that I3M down-regulated the expression of Ang-1 and MMP2 and up-regulated the expression of Ang-2. It also bound to the ATP-binding site of VEGFR2 and inhibited the phosphorylation of VEGFR2 leading to suppression of the down-stream MEK/ERK and PI3K/AKT/GSK signaling pathways in HMEC-1 cells. Similarly, tryptanthrin also reduced the expression of several angiogenic factors (Ang-1, PDGFB and MMP2) in HMEC-1 cells. In addition, tryptanthrin also bound to the ATP-binding site of VEGFR2 and suppressed the phosphorylation of VEGFR2 and VEGFR2-mediated ERK1/2 signaling pathway in HMEC-1 cells. Collectively, our results demonstrated that I3M and tryptanthrin exhibited anti-angiogenic activity both in vitro and in vivo by specifically targeting the VEGFR2-mediated signaling pathways and might be exploited as potential therapeutic candidates for the treatment of angiogenesis-related diseases. / In conclusion, our findings indicate that I3M and tryptanthrin might exert their growth-inhibitory effect on the human neuroblastoma cells by causing cell cycle arrest, inducing apoptosis or inducing neuronal differentiation. However, they exhibited minimal cytotoxicity towards the normal cells. Moreover, I3M and tryptanthrin were found to possess anti-angiogenic activities by targeting the VEGFR2-mediated signaling pathways. In the future, investigations should be focused on further elucidation of the molecular action mechanisms of I3M and tryptanthrin on human neuroblastoma cells and to test the anti-tumor efficacy of I3M and tryptanthrin in animal models, using human neuroblastoma xenografts in nude mice. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liao, Xuemei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 206-229). / Abstract also in Chinese. / Acknowledgments --- p.i / Abbreviations --- p.ii / Publications --- p.vi / Abstract --- p.vii / 摘要 --- p.xii / Table of Contents --- p.xvi / Chapter One / General Introduction --- p.1 / Chapter 1.1 --- Neuroblastoma --- p.2 / Chapter 1.1.1 --- Epidemiology of neuroblastoma --- p.2 / Chapter 1.1.2 --- Classification of neuroblastoma --- p.6 / Chapter 1.1.3 --- Clinical symptoms and diagnosis of neuroblastoma --- p.10 / Chapter 1.1.4 --- Molecular pathogenesis of neuroblastoma --- p.13 / Chapter 1.1.4.1 --- Genetic alterations in neuroblastoma --- p.13 / Chapter 1.1.4.2 --- Disruption of cell division cycle, apoptotic and signaling pathways --- p.16 / Chapter 1.1.5 --- Treatment strategies --- p.19 / Chapter 1.1.5.1 --- Low-risk neuroblastoma treatment strategy --- p.19 / Chapter 1.1.5.2 --- Intermediate-risk neuroblastoma treatment strategy --- p.20 / Chapter 1.1.5.3 --- High-risk neuroblastoma treatment strategy --- p.21 / Chapter 1.1.5.4 --- Side effects of treatment --- p.23 / Chapter 1.2 --- Banlangen alkaloids --- p.23 / Chapter 1.2.1 --- Overview of Banlangen alkaloids --- p.23 / Chapter 1.2.2 --- Biological and pharmacological effects of Banlangen alkaloids --- p.28 / Chapter 1.2.2.1 --- Anti-inflammatory activity --- p.28 / Chapter 1.2.2.2 --- Anti-microbial activity --- p.29 / Chapter 1.2.2.3 --- Anti-tumor activity --- p.30 / Chapter 1.2.2.4 --- Other biological activities --- p.32 / Chapter 1.2.3 --- Bioavailability of Banlangen alkaloids --- p.33 / Chapter 1.2.4 --- Toxicity of Banlangen alkaloids --- p.34 / Chapter 1.3 --- Aims and scope of this project --- p.36 / Chapter Two / Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Animals --- p.39 / Chapter 2.1.2 --- Cell lines --- p.39 / Chapter 2.1.3 --- Cell culture media --- p.41 / Chapter 2.1.4 --- Drugs and chemicals --- p.42 / Chapter 2.1.5 --- Reagents and buffers for cell culture --- p.44 / Chapter 2.1.6 --- General staining solutions --- p.47 / Chapter 2.1.7 --- Reagents and buffers for cell growth assays --- p.48 / Chapter 2.1.8 --- Reagents and buffers for flow cytometry --- p.48 / Chapter 2.1.9 --- Reagents and buffers for acetylcholine esterase activity assay --- p.50 / Chapter 2.1.10 --- Reagents and buffers for immunocytochemistry --- p.51 / Chapter 2.1.11 --- Reagents and buffers for total RNA extraction --- p.53 / Chapter 2.1.12 --- Reagents and buffers for reverse transcription --- p.54 / Chapter 2.1.13 --- Reagents for quantitative real-time polymerase chain reaction (qRT-PCR) --- p.56 / Chapter 2.1.14 --- Reagents and buffers for Western blotting --- p.59 / Chapter 2.1.15 --- Assay kits --- p.65 / Chapter 2.2 --- Methods --- p.68 / Chapter 2.2.1 --- Culture of cells --- p.68 / Chapter 2.2.2 --- MTT assay --- p.69 / Chapter 2.2.3 --- Cell proliferation assay --- p.70 / Chapter 2.2.4 --- Trypan blue exclusion test --- p.70 / Chapter 2.2.5 --- Cytotoxicity assay --- p.71 / Chapter 2.2.6 --- Colony-forming assay --- p.72 / Chapter 2.2.7 --- Cell cycle analysis --- p.72 / Chapter 2.2.8 --- Assessment of apoptosis --- p.73 / Chapter 2.2.9 --- Caspase activity determination --- p.74 / Chapter 2.2.10 --- Mitochondrial mass assay --- p.75 / Chapter 2.2.11 --- Reactive oxygen species (ROS) assay --- p.75 / Chapter 2.2.12 --- Mitochondrial membrane potential determination --- p.76 / Chapter 2.2.13 --- Morphological detection of cell differentiation --- p.76 / Chapter 2.2.14 --- Acetylcholine esterase activity determination --- p.77 / Chapter 2.2.15 --- Immunocytochemistry --- p.77 / Chapter 2.2.16 --- RNA interference --- p.78 / Chapter 2.2.17 --- Wound healing assay --- p.79 / Chapter 2.2.18 --- Tube formation assay --- p.79 / Chapter 2.2.19 --- In vivo Matrigel plug assay --- p.80 / Chapter 2.2.20 --- Phospho-VEGFR2 Sandwich ELISA assay --- p.80 / Chapter 2.2.21 --- Isolation of total cellular RNA --- p.81 / Chapter 2.2.22 --- Reverse transcription (RT) --- p.82 / Chapter 2.2.23 --- Quantitative real-time PCR --- p.83 / Chapter 2.2.24 --- Total protein extraction --- p.84 / Chapter 2.2.25 --- Protein concentration determination --- p.84 / Chapter 2.2.26 --- Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) --- p.85 / Chapter 2.2.27 --- Semi-dry Western blotting --- p.85 / Chapter 2.2.28 --- Enhanced chemiluminescence (ECL) assay --- p.87 / Chapter 2.2.29 --- Molecular docking --- p.87 / Chapter 2.2.30 --- Statistical analysis --- p.88 / Chapter Three / Modulatory effects and action mechanisms of indirubin-3'-oxime on human neuroblastoma cells --- p.89 / Chapter 3.1 --- Introduction --- p.90 / Chapter 3.2 --- Results --- p.94 / Chapter 3.2.1 --- Indirubin-3’-oxime inhibited the growth and colony formation of human neuroblastoma cells in vitro --- p.94 / Chapter 3.2.2 --- Indirubin-3’-oxime exhibited no significant cytotoxicity on normal cells --- p.101 / Chapter 3.2.3 --- Indirubin-3’-oxime induced G0/G1 cell cycle arrest in LA-N-1 cells --- p.103 / Chapter 3.2.4 --- Indirubin-3’-oxime caused mitochondrial dysfunction in LA-N-1 cells --- p.106 / Chapter 3.2.5 --- Indirubin-3’-oxime selectively reduced ERR γ and PGC-1β protein and mRNA levels in LA-N-1 cells --- p.111 / Chapter 3.2.6 --- Indirubin-3’-oxime induced cell cycle arrest at G0/G1 phase and apoptosis of SH-SY5Y cells --- p.113 / Chapter 3.2.7 --- Indirubin-3’-oxime reduced mitochondrial mass and elevated mitochondrial ROS level in SH-SY5Y cells --- p.115 / Chapter 3.2.8 --- Indirubin-3’-oxime increased the caspase 8, caspase 9 and caspase 3/7 activities in SH-SY5Y cells --- p.117 / Chapter 3.3 --- Discussion --- p.119 / Chapter Four / Modulatory effects and action mechanisms of tryptanthrin on human neuroblastoma cells --- p.125 / Chapter 4.1 --- Introduction --- p.126 / Chapter 4.2 --- Results --- p.129 / Chapter 4.2.1 --- Tryptanthrin inhibited the cell growth and colony formation of human neuroblastoma cells --- p.129 / Chapter 4.2.2 --- Tryptanthrin exhibited no significant cytotoxicity on normal cells --- p.136 / Chapter 4.2.3 --- Tryptanthrin induced cell cycle arrest at G0/G1 phase --- p.138 / Chapter 4.2.4 --- Tryptanthrin induced apoptosis of LA-N-1 cells --- p.140 / Chapter 4.2.5 --- Tryptanthrin induced morphological neuronal differentiation in LA-N-1 cells --- p.143 / Chapter 4.2.6 --- Tryptanthrin induced the expression of neuronal differentiation markers --- p.146 / Chapter 4.2.7 --- Tryptanthrin down-regulated the expression of N-myc in LA-N-1 cells --- p.149 / Chapter 4.3 --- Discussion --- p.152 / Chapter Five / Anti-angiogenesis effects and action mechanisms of indirubin-3'-oxime and tryptanthrin --- p.158 / Chapter 5.1 --- Introduction --- p.159 / Chapter 5.2 --- Results --- p.163 / Chapter 5.2.1 --- Indirubin-3’-oxime and tryptanthrin inhibited the proliferation of endothelial cells --- p.163 / Chapter 5.2.3 --- Indirubin-3’-oxime and tryptanthrin reduced the tube formation of endothelial cells --- p.168 / Chapter 5.2.4 --- Indirubin-3’-oxime and tryptanthrin blocked angiogenesis in the in vivo Matrigel plug model --- p.171 / Chapter 5.2.5 --- Indirubin-3’-oxime and tryptanthrin reduced the angiogenic gene expression in endothelial cells --- p.174 / Chapter 5.2.6 --- Indirubin-3’-oxime and tryptanthrin attenuated VEGFR2-mediated signaling pathways in endothelial cells --- p.176 / Chapter 5.2.7 --- Indirubin-3’-oxime bound to the ATP-binding site of VEGFR2 kinase domain --- p.181 / Chapter 5.2.8 --- Tryptanthrin bound to the ATP-binding site of VEGFR2 kinase domain --- p.182 / Chapter 5.3 --- Discussion --- p.184 / Chapter Six / Conclusions and future perspectives --- p.191 / References --- p.206

Yip, Hon Yan Kelvin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 218-242). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vii / CHINESE ABSTRACT (摘要） --- p.xi / PUBLICATIONS --- p.xiv / TABLE OF CONTENTS --- p.xv / Chapter CHAPTER 1: --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Neuroblastoma --- p.2 / Chapter 1.1.1 --- An overview of neuroblastoma --- p.2 / Chapter 1.1.2 --- Epidemiology of neuroblastoma --- p.3 / Chapter 1.1.3 --- Clinical presentations of neuroblastoma --- p.5 / Chapter 1.1.4 --- Diagnosis and clinical assessment of neuroblastoma --- p.8 / Chapter 1.1.5 --- Staging of neuroblastoma --- p.10 / Chapter 1.1.6 --- Genetic aberrations of neuroblastoma --- p.12 / Chapter 1.1.7 --- Therapies of neuroblastoma --- p.15 / Chapter 1.2 --- Banlangen alkaloids --- p.20 / Chapter 1.2.1 --- An overview of Banlangen alkaloids --- p.20 / Chapter 1.2.2 --- "Pharmacokinetics of indirubin, tryptanthrin and their derivatives" --- p.24 / Chapter 1.2.2.1 --- Bioavailability of indirubin and its derivatives --- p.24 / Chapter 1.2.2.2 --- Toxicity of indirubin and its derivatives --- p.25 / Chapter 1.2.2.3 --- Bioavailability of tryptanthrin --- p.26 / Chapter 1.2.2.4 --- Toxicity of tryptanthrin --- p.27 / Chapter 1.2.3 --- "Pharmacological effects of indirubin, tryptanthrin and their derivatives" --- p.28 / Chapter 1.2.3.1 --- Selective inhibitor on kinases --- p.29 / Chapter 1.2.3.2 --- Anti-inflammatory activities --- p.31 / Chapter 1.2.3.3 --- Anti-tumour activities --- p.32 / Chapter 1.2.3.3.1 --- Anti-leukemic activity --- p.32 / Chapter 1.2.3.3.2 --- Apoptosis-inducing activity --- p.34 / Chapter 1.2.3.4 --- Anti-viral properties --- p.37 / Chapter 1.2.3.5 --- Anti-microbial properties --- p.37 / Chapter 1.3 --- Aims and Scope of This Study --- p.39 / Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.41 / Chapter 2.1 --- Materials --- p.42 / Chapter 2.1.1 --- Cell lines --- p.42 / Chapter 2.1.2 --- "Cell culture media, reagents and buffers" --- p.43 / Chapter 2.1.3 --- General staining solutions --- p.46 / Chapter 2.1.4 --- Drugs and chemicals --- p.47 / Chapter 2.1.5 --- Reagent for primary cultures preparation --- p.48 / Chapter 2.1.6 --- Reagents for cell proliferation assay --- p.48 / Chapter 2.1.7 --- Reagents for DNA extraction --- p.50 / Chapter 2.1.8 --- Reagents for gel electrophoresis of nucleic acids --- p.51 / Chapter 2.1.9 --- Reagents and buffers for flow cytometry --- p.53 / Chapter 2.1.10 --- Reagents and buffers for measuring caspase activity --- p.54 / Chapter 2.1.11 --- "Reagents, buffers and materials for Western blot analysis" --- p.58 / Chapter 2.1.12 --- Reagent for Hoechst staining --- p.68 / Chapter 2.2 --- Methods --- p.69 / Chapter 2.2.1 --- Culture of cell lines --- p.69 / Chapter 2.2.2 --- Determination of cell viability --- p.70 / Chapter 2.2.3 --- Determination of cell proliferation by tritiated thymidine ([ 3H]-TdR) incorporation assay --- p.72 / Chapter 2.2.4 --- "Isolation, culture and cytotoxicity test of murine peritoneal macrophages" --- p.73 / Chapter 2.2.5 --- "Isolation, culture and cytotoxicity test of murine bone marrow cells" --- p.74 / Chapter 2.2.6 --- Cytotoxicity test of primary cortical neurons from SD rats --- p.75 / Chapter 2.2.7 --- Determination of colony forming ability --- p.75 / Chapter 2.2.8 --- Analysis of cell cycle profile /DNA content by flow cytometry --- p.76 / Chapter 2.2.9 --- Detection of DNA fragmentation by agarose gel electrophoresis --- p.77 / Chapter 2.2.10 --- Quantitative detection of DNA fragmentation by Cell Death ELISAplus kit --- p.78 / Chapter 2.2.11 --- Detection of intracellular reactive oxygen species (ROS) generation --- p.80 / Chapter 2.2.12 --- Measurement of caspase activity --- p.81 / Chapter 2.2.13 --- Hoechst 33342 staining --- p.83 / Chapter 2.2.14 --- Cell morphological study --- p.83 / Chapter 2.2.15 --- Analysis of morphological changes by flow cytometry --- p.84 / Chapter 2.2.16 --- Assay for acetylcholine esterase (AChE) activity --- p.85 / Chapter 2.2.17 --- Protein expression study --- p.86 / Chapter 2.2.18 --- Statistical analysis --- p.89 / Chapter CHAPTER 3: --- IN VITRO STUDIES ON THE ANTI PROLIFERATIVE EFFECT OF INDIRUBIN-3'-OXIME AND TRYPTANTHRIN ON NEUROBLASTOMA CELLS --- p.90 / Chapter 3.1 --- Introduction --- p.91 / Chapter 3.2 --- Results --- p.95 / Chapter 3.2.1 --- Effects of indirubin-3'-oxime and tryptanthrin on the proliferation of human and the murine neuroblastoma cells --- p.95 / Chapter 3.2.2 --- Kinetic and reversibility studies of the anti-proliferative effect of indirubin-3'-oxime and tryptanthrin on the murine neuroblastoma Neuro-2a BU-1 cells --- p.107 / Chapter 3.2.3 --- Cytotoxic effect of indirubin-3'-oxime and tryptanthrin on the murine neuroblastoma Neuro-2a BU-1 cells --- p.115 / Chapter 3.2.4 --- Effects of indirubin-3'-oxime and tryptanthrin on the clonogenicity of the murine neuroblastoma Neuro-2a BU-1 cells --- p.120 / Chapter 3.2.5 --- Cytotoxicity of indirubin-3'-oxime and tryptanthrin on primary cells --- p.123 / Chapter 3.2.6 --- Effects of tryptanthrin on the cell cycle profile and expression of cyclins and cyclin-dependent kinases (CDKs) in the murine neuroblastoma Neuro-2a BU-1 cells --- p.132 / Chapter 3.2.7 --- Effect of indirubin-3'-oxime on the cell cycle profile in the murine neuroblastoma Neuro-2a BU-1 cells --- p.133 / Chapter 3.3 --- Discussion --- p.142 / Chapter CHAPTER 4: --- IN VITRO STUDIES ON THE APOPTOSIS-INDUCING EFFECT OF INDIRUBIN-3'-OXIME ON NEUROBLASTOMA CELLS --- p.150 / Chapter 4.1 --- Introduction --- p.151 / Chapter 4.2 --- Results --- p.156 / Chapter 4.2.1 --- Induction of DNA fragmentation in the indirubin-3'-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.156 / Chapter 4.2.2 --- Induction of chromatin condensation in the indirubin-3 '-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.160 / Chapter 4.2.3 --- Induction of caspase activities in the indirubin-3 '-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.162 / Chapter 4.2.4 --- Induction of Reactive Oxygen Species (ROS) in the indirubin-3' -oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.169 / Chapter 4.2.5 --- Expression of pro-apoptotic and anti-apoptotic proteins in the indirubin-3 '-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.173 / Chapter 4.3 --- Discussion --- p.177 / Chapter CHAPTER 5: --- STUDIES ON THE DIFFERENTIATION-INDUCING ACTIVITY OF TRYPTANTHRIN ON NEUROBLASTOMA CELLS --- p.188 / Chapter 5.1 --- Introduction --- p.189 / Chapter 5.2 --- Results --- p.193 / Chapter 5.2.1 --- Effects of tryptanthrin on the cell size and cellular complexity of the murine neuroblastoma Neuro-2a BU-1 cells --- p.193 / Chapter 5.2.2 --- Morphological studies on tryptanthrin-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.196 / Chapter 5.2.3 --- Effect of tryptanthrin on the acetylcholine esterase (AChE) activity in the murine neuroblastoma Neuro-2a BU-1 cells --- p.198 / Chapter 5.2.4 --- Effects of tryptanthrin on the expression of tau protein and other mediators involved in the differentiation pathway --- p.200 / Chapter 5.3 --- Discussion --- p.204 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.209 / REFERENCES --- p.218

Pan, Tak Yin. / Thesis submitted in: November 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iv / Content --- p.v / Abbreviations --- p.x / List of Figures --- p.xi / List of Tables --- p.xii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's Disease --- p.1 / Chapter 1.1.1 --- Cholinergic hypothesis --- p.2 / Chapter 1.1.2 --- p-amyloid hypothesis --- p.2 / Chapter 1.1.3 --- Taupathy hypothesis --- p.3 / Chapter 1.1.4 --- Current therapies --- p.4 / Chapter 1.2 --- Proteins Involved in Alzhemer's Disease --- p.5 / Chapter 1.2.1 --- Acetylcholinesterase (AChE) --- p.5 / Chapter 1.2.2 --- p-amyloid --- p.6 / Chapter 1.2.3 --- Paired helical filaments (PHF) --- p.7 / Chapter 1.2.4 --- Protein kinases --- p.8 / Chapter 1.2.4.1 --- Glycogen synthase kinase-3 (GSK-3) --- p.9 / Chapter 1.2.4.2 --- Cyclin-dependent kinase-5 (CDK-5) --- p.9 / Chapter 1.2.5 --- Protein phosphatase (PP) --- p.10 / Chapter 1.2.5.1 --- Protein phosphatase 1 (PP-1) --- p.11 / Chapter 1.2.5.2 --- Protein phosphatise 2A (PP-2A) --- p.12 / Chapter 1.2.5.3 --- Protein phosphatise 2B (PP-2B) --- p.13 / Chapter 1.2.6 --- Apoptotic and Anti-apoptotic proteins --- p.14 / Chapter 1.2.6.1 --- Caspase-3 --- p.15 / Chapter 1.2.6.2 --- Bcl-2 --- p.15 / Chapter 1.3 --- Flavonoids --- p.16 / Chapter 1.3.1 --- Biosynthesis of flavonoids --- p.17 / Chapter 1.3.2 --- Biological functions of flavonoids in plants --- p.18 / Chapter 1.3.3 --- Beneficial effects of flavonoids on human health --- p.19 / Chapter Chapter 2: --- Materials and Methods --- p.20 / Chapter 2.1 --- Differentiation of SHSY-5Y cells --- p.20 / Chapter 2.1.1 --- SHSY-5Y cell culture --- p.20 / Chapter 2.1.2 --- Counting cells --- p.20 / Chapter 2.1.3 --- Retinoic acid differentiation --- p.21 / Chapter 2.2 --- Western blot analysis --- p.21 / Chapter 2.2.1 --- Extraction of proteins from mammalian cells --- p.21 / Chapter 2.2.2 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.22 / Chapter 2.2.3 --- Semi-dry protein transfer to nitrocellulose membrane --- p.23 / Chapter 2.2.4. --- Membrane blocking and immunostaining --- p.24 / Chapter 2.3 --- MTT assay --- p.25 / Chapter 2.4 --- Hoechst 33342 Nuclei staining --- p.25 / Chapter 2.5 --- Cell cycle analysis --- p.25 / Chapter 2.5.1 --- Ethanol fixation --- p.25 / Chapter 2.5.2 --- Propidium iodide staining --- p.26 / Chapter 2.6 --- Annexin V-FITC & Propidium iodide staining --- p.26 / Chapter 2.7 --- DNA fragmentation analysis --- p.26 / Chapter 2.7.1 --- Phenol/Chloroform extraction of DNA --- p.26 / Chapter 2.7.2 --- Ethanol precipitation of DNA --- p.27 / Chapter 2.7.3 --- Agarose gel electrophoresis of DNA --- p.27 / Chapter 2.8 --- Proteomic analysis --- p.28 / Chapter 2.8.1 --- First dimension: isoelectric focusing --- p.28 / Chapter 2.8.2 --- Second dimension: SDS PAGE --- p.29 / Chapter 2.8.3 --- Gel staining --- p.30 / Chapter 2.8.3.1 --- Silver staining --- p.30 / Chapter 2.8.3.2 --- SYBRO Ruby staining --- p.31 / Chapter 2.8.4 --- Gel scanning and image analysis --- p.31 / Chapter 2.8.5 --- ln-gel digestion --- p.32 / Chapter 2.8.6 --- Zip Tip for desalting the digested sample --- p.33 / Chapter 2.8.7 --- Protein identification with mass spectrometry and database search --- p.33 / Chapter Chapter 3: --- Characterization of Okadaic acid-induced tail hyperphosphorylation in SHSY-5Y cells --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Objectives --- p.37 / Chapter 3.3 --- Results --- p.38 / Chapter 3.3.1 --- Differentiation of SH-SY5Y cell --- p.38 / Chapter 3.3.2 --- Changes of protein expression after okadaic acid treatment --- p.40 / Chapter 3.3.3 --- Neurite Retraction Induced by okadaic acid --- p.42 / Chapter 3.3.4 --- Okadaic acid-induced Cell Death measured by MTT assay --- p.44 / Chapter 3.3.5 --- Hoechst 33342 Nuclei Staining --- p.44 / Chapter 3.3.6 --- Cell cycle analysis by propidium iodide staining --- p.47 / Chapter 3.3.7 --- Early Apoptotic cells detection by Annexin V/PI staini --- p.49 / Chapter 3.3.8 --- DNA fragmentation --- p.51 / Chapter 3.4 --- Discussion --- p.53 / Chapter Chapter 4: --- Flavonoids screening for protecting neuronal death by preventing tau hyperphosphorylation --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Objectives --- p.58 / Chapter 4.3 --- Tested flavonoids --- p.59 / Chapter 4.4 --- Results --- p.60 / Chapter 4.4.1 --- Toxicity of flavonoids --- p.60 / Chapter 4.4.2 --- Effects of flavonoid pre-treatment on OA-induced neu retractions and cell death --- p.62 / Chapter 4.4.3 --- Western blot analysis --- p.65 / Chapter 4.4.4 --- The effect of different concentrations of hesperidin or OA treatment --- p.70 / Chapter 4.4.5 --- Proteomic analysis --- p.74 / Chapter 4.5 --- Discussion --- p.78 / Chapter Chapter 5: --- General Discussion --- p.82 / References