The aim of this investigation was to determine the effect of a nicotine-conditioned context on locomotor hyperactivity in an animal model of D2-priming, and whether conditioned hyperactivity could be blocked by the D2 antagonist eticlopride or the D3 antagonist nafadotride. D2-primed male rats showed enhanced nicotine sensitization as evidenced by statistically significant differences in horizontal activity. D2-primed female rats administered nicotine demonstrated an increased hypoactive response after initial sensitization and increased stereotypy. Eticlopride and nafadotride blocked sensitization to nicotine in both D2-primed and non D2-primed males and females. Eticlopride blocked conditioned hyperactivity in females but not in males. D2-primed female rats administered nicotine demonstrated significantly higher conditioned-hyperactivity as compared to non D2-primed females and controls, and this increase was more effectively blocked by nafadotride as compared to eticlopride. These results suggest differential roles of the dopamine D2 and D3 receptors in both adolescent nicotine sensitization and conditioned activating effects of nicotine.

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Estrogen is the natural agonist of the estrogen receptor (ER). However, certain plant-derived compounds or phytoestrogens have been identified that mimic estrogens and act as agonists and/or antagonists of ERs, depending on subtype and target tissue. Understanding how phytoestrogens interact with ERs, and therefore effect the estrogenic response, may prove beneficial in hormone replacement therapy and in the prevention and treatment of hormone-related diseases. Using Thin Layer Chromatography, gas chromatography/mass spectrometry (GC/MS), and proton nuclear nagnetic resonance (HNMR), I identified 4-ethoxymethylphenol (4EM) found in Maclura pomifera. While most phytoestrogens are heterocyclic compounds, 4EM is a simple phenol that acts as an agonist of ER-alpha and -beta in HeLa and MCF-7 cells. To study the effect of 4EM on ER-alpha and -beta activity, I performed transient transfection assays and showed that 4EM activates ER dependent gene transcription in a dose dependent manner in both ER subtypes. Further, 4EM- mediated transcription in ER-alpha, like estrogen, was enhance in the presense of co-activators, SRC-1 (steroid receptor coactivator-1), CBP (CREB binding proteins), and E6-AP (E6-associated protein) and inhibited by trans-4- hydroxytamoxifen (4HT). I found that 4EM was specific for ER and did not activate transcription of the progesterone receptor in HeLa cells.

There is significant sequence homology and anatomical co-distribution between cannabinoid (CB1) and sphingosine-1-phosphate (S1P) receptors in the CNS, but potential functional relationships between these lysolipid receptors have not been examined. Therefore, to investigate possible relationships between these two systems at the level of G-protein activation, agonist-stimulated [35S]GTPγS binding and autoradiography were conducted. Autoradiographic studies were first performed to localize receptor-mediated G-protein activation in mouse brain. Coronal brain slices were processed for stimulation of [35S]GTPγS binding using the synthetic cannabinoid agonist WIN 55,212-2 (WIN) or SIP. High levels of WIN- and S1P-stimulated [35S]GTPγS binding were observed in the caudate putamen, hippocampus, substantia nigra, and cerebellum. To further characterize the relationship between S1P-and CB1-mediated G-protein activation, spinal cords from adult male CB1 receptor knockout mice, CNS-deleted S1Pl receptor knockout mice and wild type C57 mice were collected, and assessed using agonist-stimulated [35S]GTPγS binding. Results from this experiment revealed that the S1Pl receptor is predominant in mouse spinal cord. To further investigate potential CBl and SIP receptor interactions spinal cords were collected from adult male ICR mice. Additivity studies were preformed using agonist-stimulated [35S]GTPγs binding. Results showed significantly less than additive stimulation when spinal cord tissue was treated with both WIN and SIP. These results suggest an interaction between the CB1 and S1P receptors in the mouse spinal cord. The effect of cannabinoid antagonists, SR141716A (CB1) and SR144528 (CB2) on S1P-and WIN-stimulated [35S]GTPγS binding were also examined in mouse spinal cord homogenates. These results showed that there was no significant difference between S1P-stimulated [35S]GTPγS binding in the presence of SR141716A or SR144528 compared to vehicle control. This shows that S1P produced stimulation independent of the CBl or CB2receptor. In addition WIN-stimulated [35S]GTPγS binding was not affected by SR144528, but was inhibited by SR141716A, confirming that this action is due to the CB1 receptor. The combined results of this project demonstrate an interaction between CB1 and S1P receptors in certain CNS regions where they are co-distributed, such as the caudate putamen, hippocampus, substantia nigra, cerebellum and spinal cord. These results may be due to convergence on a common pool of G-proteins via dimerization or co-localization in lipid rafts, or a possible direct ligand-receptor interaction.

Objetivos: Comparar os efeitos dos tratamentos com montelucaste e dexametasona no recrutamento eosinofílico e na avaliação de células positivas para eotaxina, RANTES, fibronectina, IGF-I e NF-B tanto no parênquima pulmonar distal, quanto nas vias aéreas de cobaias com inflamação alérgica crônica. Métodos: As cobaias receberam inalação com ovoalbumina (grupo OVA- 2 vezes semanais, durante 4 semanas, totalizando 7 inalações). Após a quarta inalação, as cobaias foram tratadas com montelucaste (grupo OVA-M: 10mg/Kg/VO/dia) ou dexametasona ( grupo OVA-D: 5mg/Kg/IP/dia). Após 72 horas da sétima inalação, as cobaias foram anestesiadas e os pulmões foram removidos e submetidos a avaliação histopatológica. Resultados: Os tratamentos com montelucaste e dexametasona reduziram o número de eosinófilos tanto no parênquima pulmonar distal quanto nas vias aéreas, quando comparados ao grupo OVA (p<0.05). No parênquima pulmonary distal, ambos os tratamentos foram efetivos na redução de células positivas para RANTES, NF-B e fibronectina, quando comparados ao grupo OVA (p<0.001). O tratamento com montelucaste mostrou melhor eficácia na redução de células positivas para eotaxina, quando comparado ao tratamento com dexametasona (p<0.001), por outro lado, o tratamento com dexametasona mostrou-se mais significativo na redução de células positivas para IGF-I, quando comparado ao tratamento com montelucaste (p<0.001). Nas vias aéreas, ambos os tratamentos foram efetivos na redução de células positivas para IGF-I, RANTES e fibronectina, quando comparados ao grupo OVA (p<0.05). O tratamento com dexametasona foi mais efetivo na redução de células positivas para eotaxina e NF-B, quando comparado ao tratamento com montelucaste (p<0.05). Conclusões: Neste modelo animal, ambos os tratamentos foram efetivos no controle da resposta inflamatória, tanto no parênquima pulmonar distal, quanto nas vias aéreas / Aims: Compare the effects of montelukast or dexamethasone treatments on eosinophilic recruitment, eotaxin, RANTES, fibronectin, IGF-I and NF-B positive cells of distal lung parenchyma and also in airway walls of guinea pigs (GP) with chronic allergic inflammation. Methods: GP were inhaled with ovalbumin (OVA group-2x/week/4weeks). After 4th inhalation, GP were treated with montelukast (M group: 10mg/Kg/PO/day) or dexamethasone (D group: 5mg/Kg/IP/day). After 72 hrs of 7th inhalation, GP were anesthetised, lungs were removed and submitted to histopathological evaluation. Results: Montelukast and dexamethasone treatments reduced the number of eosinophils both in airway wall as well as in distal lung parenchyma compared to OVA group (p<0.05). On distal parenchyma both montelukast and dexamethasone were effective in reducing RANTES, NF-B and fibronectin positive cells compared to OVA group (p<0.001). Montelukast was more effective in reducing the eotaxin positive cells on distal parenchyma compared to dexamethasone treatment (p<0.001), while there was a more expressive reduction of IGF-I positive cells in OVA-D group (p<0.001). On airway walls, both montelukast and dexamethasone were effective in reducing IGF-I, RANTES and fibronectin positive cells compared to OVA group (p<0.05). Dexamethasone was more effective reducing the number of eotaxin and NF-kB positive cells than Montelukast (p<0.05). Conclusions: In this animal model, both treatments were effective in modulating the eosinophilic response in distal lung parenchyma and in airway wall, contributing to a better control of the inflammatory response in distal lung parenchyma as well as in airway walls. Dexamethasone treatment induced a greater reduction of NF-B expression in airway walls which suggests one of the mechanisms that explains the higher efficacy of this therapeutic approach

O uso de fármacos cardiovasculares tem aumentado consideravelmente nos últimos anos, incluindo o uso de beta-bloqueadores como o metoprolol. E embora estudos experimentais e clínicos tenham demonstrado benefícios na utilização desta classe de fármacos em pacientes sépticos, o bloqueio de receptores beta-adrenérgicos continua sendo contraditório, especialmente no choque séptico. Vinte fêmeas suínas nas quais o choque séptico foi induzido através da infusão intravenosa de Escherichia coli (6x109 UFC/kg em duas horas), foram randomicamente distribuídas (n=10 por grupo) em um grupo Metoprolol (GM; 214.2 ?g/kg de metoprol infundido em 45 minutos) ou grupo Controle (GC), o qual recebeu um volume correspondente de solução salina. Os parâmetros hemodinâmicos e de oxigenação foram avaliados no momento basal (TB), T30, T60, T120, T240 e T360 minutos. Amostras de sangue foram colhidas ainda em TB, T120 e T360 e armazenadas para posterior mensuração da concentração sérica de citocinas e marcadores cardíacos. Durante este período utilizou-se um protocolo de ressuscitação com Ringer lactato, norepinefrina (NE) e dobutamina para manutenção da pressão arterial média (PAM) > 65 mmHg, da pressão venosa central (PVC) em 8-12 mmHg e da saturação venosa mista de oxigênio (SvO2) > 65%. Os dados paramétricos foram analisados utilizando ANOVA de duas vias para medidas repetidas, seguidas por Tukey quando necessário, enquanto para os dados não paramétricos utilizaram-se os testes de Kruskall-Wallis e Mann-Whitney. A quantidade de fluido, NE e dobutamina foi analisada pelo teste t de Student, e a análise de sobrevivência foi realizada utilizando o Kaplan-Meier log-rank test. Exceto por um valor mais elevado do índice de resistência vascular sistêmica (IRVS) em T240 (p=0,02) nos animais tratados com metoprolol, todos os parâmetros analisados neste estudo apresentaram um comportamento similar em ambos os grupos. Nenhuma diferença foi observada também em relação ao volume total de fluido (p=0,914), à quantidade total de NE (p=0,069) e dobutamina (p=0,560) administrada, e ainda em relação à mortalidade. Embora estudos tenham demonstrado diversos benefícios na utilização de beta-bloqueadores em pacientes sépticos, e que estes tenham demonstrado resultados promissores, a administração de metoprolol não apresentou benefícios em nenhum dos parâmetros analisados em nosso modelo experimental. Porém, em um cenário onde a administração de beta-bloqueadores está em constante crescimento, é de grande importância saber que sua administração não apresenta efeitos deletérios nestes pacientes / The use of cardiovascular drugs considerably increased in recent years, including the use of ?-blockers, such as metoprolol. And although experimental and clinical studies had demonstrated benefits on the administration of these drugs in septic patients, the ?-blockade is still contradictory, especially in the septic shock. Twenty female pigs in which septic shock was induced through intravenous E. coli infusion (6x109 c.f.u/kg in 2h) were randomly assigned (n=10 per group) to the Metoprolol group (214.2 ?g kg-1 of metoprol infused in 45 minutes) or Control group, which received a correspondent volume of normal saline. Hemodynamic and oxygenation parameters were then evaluated at baseline (TB), T30, T60, T120, T240 and T360 minutes. Blood samples were collected at TB, T120 and T360 for posterior mensuration of serum cytokines and cardiac markers. During this period, a resuscitation protocol with lactated Ringer\'s solution, norepinephrine and dobutamine was used to maintain the mean arterial pressure (MAP) > 65 mmHg, the central venous pressure (CVP) between 8-12 mmHg and the mixed venous oxygen saturation (SvO2) > 65%. Parametric data were compared using the two-way repeated measures ANOVA, followed by the Tukey test when necessary, while the nonparametric data were analyzed with the Kruskall-Wallis and the Mann-Whitney test. The total volume of fluid and total amount of norepinephrine and dobutamine infused were analyzed by the Student\'s t-test. Survival analysis was performed using Kaplan-Meier log-rank test. Except for the higher values of systemic vascular resistance index (SVRI) at T240 (p=0.02) in the animals that received metoprolol, all the parameters analyzed in this study showed a similar behavior in both groups. No difference was also observed between groups in relation to the total volume of fluid (p=0.914), the total amount of norepinephrine (p=0.069) and dobutamine (p=0.560) infused, and related to the survival rate. Although some studies have demonstrated several benefits with the use of beta-blockers in patients with sepsis and show promising results, the administration of metoprolol did not improve any of the parameters analyzed in our experimental model. However, in a scenario where the administration of ?-blockers is increasing constantly, it is important to know that its administration do not present deleterious effects in these patients

Introdução: Os pacientes com bronquiolite obliterante (BO) costumam apresentar acometimento importante da função pulmonar que resulta em hipoxemia crônica e limitação da atividade física. Não há terapêutica de grande eficácia na BO, a resposta aos broncodilatadores costuma ser pobre, entretanto, não se conhece a resposta broncodilatadora a um agente anticolinérgico de longa ação como o brometo de tiotrópio (BT). Já se demonstrou eficácia e segurança do BT em adultos com doença pulmonar obstrutiva crônica (DPOC) com resposta broncodilatadora significativa e sustentada em dose única diária. Objetivo: Avaliar se existe melhora do grau de obstrução brônquica e do aprisionamento aéreo, através de medidas funcionais, após o uso de dose única de brometo de tiotrópio por via inalatória comparado a placebo em crianças e adolescentes com BO. Métodos: Ensaio clínico prospectivo, duplo cego, randomizado, placebocontrolado e cruzado em pacientes com BO estáveis na faixa etária de 6 a 16 anos. Espirometrias e pletismografias foram realizadas antes e aos 30, 60, 120, 180 minutos e 24 horas após a inalação de 18 mcg de tiotrópio ou placebo. Após 7-14 dias, os medicamentos foram invertidos e os procedimentos repetidos. As mudanças nos parâmetros de função pulmonar em cada momento foram comparadas com o basal através da análise de variância (ANOVA) e pós-teste de Tukey e as diferenças entre todos os momentos versus o basal nos grupos tiotrópio versus placebo foram comparados usando o teste de Friedman. Resultados: Trinta pacientes participaram do estudo (23 do sexo masculino, 7 do feminino; idade 10,9±2,8a ), com valores basais de função pulmonar (% do previsto) de CVF, VEF1, VEF1/CVF, FEF25-75%, CI, CPT, VR, VR/CPT, resistência das vias aéreas (raw) e condutância (sGaw) de 75±15, 48±14, 59±11, 22±11, 64±23, 120±19, 281±101, 49±13, 250±65 e 23±9, respectivamente. Diferenças estatisticamente significantes foram observadas após inalação do tiotrópio nos seguintes parâmetros comparados ao tempo basal: CVF em 60/120/180 min/24h, VEF1 em 30/60/120/180min, VEF1/CVF em 60/120/180min, FEF25- 75% em 60/120/180min, VR em 30/60/120/180min, CPT em 30/120/180min, VR/CPT em 30/60/120/180min, raw em 30/60/120/180min/24h e sGaw em 30/60/120/180min/24h. Na fase placebo não houve diferença estatisticamente significante em nenhum parâmetro funcional em nenhum momento após a administração. As diferenças entre as medidas funcionais comparando o grupo tiotrópio versus o grupo placebo foram estatisticamente significantes. Conclusões: O brometo de tiotrópio, após dose única, diminuiu agudamente o grau de obstrução e de aprisionamento aéreo por até 24 horas em crianças com BO. Estudos de longo prazo são necessários para se avaliar o papel do BT na terapêutica desses pacientes / Introduction: Patients with bronchiolitis obliterans (BO) usually have severe airflow obstruction that results in chronic hypoxemia and limitation of physical activity. There is no efficient therapy for BO, bronchodilator response is usually poor, however, the bronchodilator response to a long action anticholinergic agent such as tiotropium bromide (TB) is not known. Efficacy and safety of TB with one daily administration has already been shown in chronic obstructive pulmonary disease (COPD) with significant and sustained bronchodilator response. Objective: Verify through functional measurements whether the level of bronchial obstruction and air trapping was improved by the administration of a single dose of TB by inhalation when compared to placebo in children and adolescents with BO. Methods: A randomized, double blind, placebo-controlled, crossover, prospective study in stable BO patients, 6 to 16 years of age. Spirometry and plethysmography were performed before and at 30, 60, 120 and 180 minutes and 24 hours after inhalation of 18 mcg of tiotropium or a placebo. After 7-14 days, the drugs were inverted, and the procedures were repeated. The changes in lung function parameters at each time point were compared to the baseline by analysis of variance (ANOVA) and Tukey\'s post-test and the differences in all time points assessment versus baseline in tiotropium versus placebo groups were compared using the Friedman test. Results: Thirty patients were enrolled in the study (23 male, 7 female; age 10.9±2.8 y) with baseline lung function values (% predicted) of FVC, FEV1, FEV1/FVC, FEF25-75%, IC, TLC, RV, RV/TLC, airway resistance (raw) and conductance (sGaw) of 75±15, 48±14, 59±11, 22±11, 64±23, 120±19, 281±101, 49±13, 250±65 and 23±9, respectively. Statistically significant differences were observed after tiotropium inhalation in the following parameters compared to baseline: FVC at 60/120/180min/24h, FEV1 at 30/60/120/180min, FEV1/FVC at 60/120/180min, FEF25-75% at 60/120/180min, RV at 30/60/120/180min, TLC at 30/120/180min, RV/TLC at 30/60/120/180min, raw at 30/60/120/180min/24h and sGaw at 30/60/120/180min/24h. For the placebo group, no significant differences were observed in any lung function parameters at any time. The differences between the functional measurements comparing the tiotropium versus placebo groups were statistically significant. Conclusions: Tiotropium bromide, after a single dose, acutely decreased airway obstruction and air trapping for up to 24 hours in children with BO. Long-term studies are necessary to evaluate the role of BT in the management of these patients

Brain glutamic acid decarboxylase 65 (GAD65) catalyzes the rate-limiting step in the biosynthesis of the major inhibitory neurotransmitter-amino butyric acid (GABA) from the substrate L-glutamic acid. Severe lapse in GABA neurotransmission is one of the etiologies documented in the manifestation of certain neurodegenerative diseases such as epilepsy, Parkinson's disease, Huntington's disease etc. Because GAD65 synthesizes GABA, any modulation of GAD65, therefore, has direct implications on the quanta of GABA released at the synapse. Hence, the major objective of this study was to focus on the regulation of GAD65, with special emphasis on investigating the proteolytic cleavage of fGAD65. Previously, we have shown in vitro that GAD65 was cleaved to form its truncated form (tGAD65), which was more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiologica l stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this study, we examined the cleavage of fGAD65 under a range of conditions encompassing both physiological and pathological aspects, including rats under ischemia/reperfusion insult, rat brain synaptosomes or primary neuronal cultures subjected to excitotoxic stimulation with KCl. It was observed that the formation of tGAD65 progressively increased with increasing stimulus concentration. More importantly, cleavage of synaptic vesicle (SV) - associated fGAD65 by calpain was demonstrated, and the resulting tGAD65 harboring the active site of the enzyme was detached from the SVs. Vesicular uptake of the newly synthesized GABA into the SVs was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate indica / d by in vivo micro dialysis. Based on these observations, we conclude that calpain cleavage of fGAD65 occurs under pathological conditions. / by Chandana Buddhala. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Huntington's disease (HD) is caused by an expanded plyglutamine repeat in the huntingtin protein. In this study, I focused on the effect of the mutant huntingtin protein (mhtt) on the subcellular localization of glutamic acid decarboxylase (GAD), the enzyme responsible for synthesizing gama-aminobutyric acid (GABA). Subcellular distribution of GAD65 is significantly altered in two neuronal cell lines that express either the N-terminus or full length mhtt. GAD65 is predominantly associated with the Golgi membrane in cells expressing normal huntingtin (Htt). However, it diffuses in the cytosol of cells expressing mhtt. Palmitoylation of GAD65 is required for GAD65 trafficking, and I demonstrated the palmitoylation of GAD65 is reduced in the HD model. Overexpression of huntingtin-interacting protein 14 (HIP14), the enzyme that palmitoylates GAD65, rescues GAD65 palmitoylation and vesicle-associated trafficking. This data suggests that impairment of GAD65 palmitoylation by mhtt may alter its localization and lead to altered inhibitory neurotransmission in HD. / by Daniel Rush. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

by Ip Wai-Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 213-233). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vi / 撮要 --- p.x / TABLE OF CONTENTS --- p.xiv / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- Introduction to Hematopoiesis --- p.1 / Chapter 1.1.2 --- Cytokines in Hematopoiesis --- p.4 / Chapter 1.2 --- Leukemia --- p.6 / Chapter 1.2.1 --- Leukemia: Abnormalities in Blood Cell Formation --- p.6 / Chapter 1.2.2 --- Classification of Leukemia --- p.8 / Chapter 1.2.3 --- The Causes and Molecular Basis of Leukemia --- p.8 / Chapter 1.2.4 --- Therapy of Leukemia --- p.11 / Chapter 1.2.5 --- Control of Leukemia by Hematopoietic Growth Factors and Other Compounds --- p.12 / Chapter 1.2.6 --- Molecular Control of Apoptosis and Cell Cycle in Leukemia --- p.13 / Chapter 1.2.6.1 --- Regulation of Cell Cycle and Apoptosis by Genes and Regulatory Proteins --- p.14 / Chapter 1.2.6.1.1 --- Cell Cycle --- p.14 / Chapter 1.2.6.1.2 --- Apoptosis --- p.15 / Chapter 1.2.6.2 --- Role of Apoptosis and Cell Cycle in the Development of Leukemia --- p.17 / Chapter 1.3 --- Green Tea --- p.19 / Chapter 1.3.1 --- Origin and Cultivation of Tea Plants --- p.19 / Chapter 1.3.2 --- Classification and Manufacturing of Tea --- p.21 / Chapter 1.3.3 --- The Chemistry of Tea --- p.22 / Chapter 1.3.3.1 --- Chemical Composition of Tea --- p.22 / Chapter 1.3.3.2 --- Separation and Purification of Green Tea Polyphenols --- p.27 / Chapter 1.3.3.3 --- The Chemical Properties of Green Tea Polyphenols --- p.28 / Chapter 1.3.4 --- Bioavailability and Pharmacokinetic of Green Tea Epicatechins --- p.28 / Chapter 1.3.4.1 --- Human Studies --- p.29 / Chapter 1.3.4.2 --- Animal Studies --- p.30 / Chapter 1.3.5 --- Physiological and Pharmacological Activities of Green Tea Catechins --- p.31 / Chapter 1.3.5.1 --- Anti-oxidative Activity --- p.32 / Chapter 1.3.5.2 --- Hypocholesterolemic and Hypolipidemic Activity --- p.33 / Chapter 1.3.5.3 --- Anti-inflammatory Activity --- p.34 / Chapter 1.3.5.4 --- Anti-microbial Activity --- p.35 / Chapter 1.3.5.5 --- Anti-mutagenic Activity --- p.36 / Chapter 1.3.5.6 --- Anti-carcinogenesis --- p.37 / Chapter 1.3.5.7 --- Direct Anti-tumor Activity --- p.41 / Chapter 1.3.5.8 --- Modulating Activity in Endocrine System --- p.43 / Chapter 1.3.5.9 --- Other Biological Activities --- p.43 / Chapter 1.3.6 --- Possible Anti-cancer Mechanisms of Green Tea Epicatechins --- p.44 / Chapter 1.3.6.1 --- Modulation of Anti-tumor Immunity --- p.44 / Chapter 1.3.6.2 --- Direct Growth Inhibition by Controlling the Signal Transduction Pathways --- p.45 / Chapter 1.3.6.3 --- Induction of Apoptosis and Cell Cycle Arrest --- p.46 / Chapter 1.3.6.4 --- Inhibition of Tumor Metastasis --- p.47 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.48 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.50 / Chapter 2.1.1 --- Animals --- p.50 / Chapter 2.1.2 --- Cell Lines --- p.50 / Chapter 2.1.3 --- Sheep Red Blood Cells (SRBC) --- p.52 / Chapter 2.1.4 --- "Cell Culture Medium, Buffers and Reagents" --- p.52 / Chapter 2.1.5 --- Tea Extracts and Green Tea Epicatechins --- p.56 / Chapter 2.1.6 --- Recombinant Cytokines --- p.57 / Chapter 2.1.7 --- Vitamin Analogs --- p.59 / Chapter 2.1.8 --- Taxol (Baccatin III N-benzoyl-β-phenyllisoserine ester) --- p.59 / Chapter 2.1.9 --- 18β-Glycyrrhetinic Acid (18β-GA) --- p.60 / Chapter 2.1.10 --- [methyl-3H] Thymidine (3H-TdR) --- p.60 / Chapter 2.1.11 --- Liquid Scintillation Cocktail --- p.60 / Chapter 2.1.12 --- Reagents and Buffers for Flow Cytometery --- p.61 / Chapter 2.1.13 --- Reagents for DNA Extraction --- p.62 / Chapter 2.1.14 --- Reagents for Total RNA Isolation --- p.63 / Chapter 2.1.15 --- Reagents and Buffers for RT-PCR Study --- p.64 / Chapter 2.1.16 --- Reagents and Buffers for Gel Electrophoresis --- p.67 / Chapter 2.1.17 --- Reagents and Buffers for Western Blot Analysis --- p.68 / Chapter 2.2 --- Methods --- p.77 / Chapter 2.2.1 --- Culture of the Leukemic Cell Lines --- p.77 / Chapter 2.2.2 --- "Isolation, Preparation and Culture of Primary Mouse Cells" --- p.77 / Chapter 2.2.3 --- Determination of Cell Viability --- p.78 / Chapter 2.2.4 --- [3H]-TdR Incorporation Assay --- p.79 / Chapter 2.2.5 --- Cell Morphology Study --- p.79 / Chapter 2.2.6 --- Apoptosis Study --- p.80 / Chapter 2.2.7 --- Animal Studies --- p.81 / Chapter 2.2.8 --- Gene Expression Study --- p.82 / Chapter 2.2.9 --- Protein Expression Study --- p.85 / Chapter 2.2.10 --- Statistical Analysis --- p.88 / Chapter CHAPTER 3: --- THE ANTI-TUMOR ACTIVITIES OF TEA EXTRACTS AND PURIFIED GREEN TEA EPICATECHIN ISOMERS ON VARIOUS LEUKEMIC CELL LINES / Chapter 3.1 --- Introduction --- p.89 / Chapter 3.2 --- Results --- p.91 / Chapter 3.2.1 --- The Effects of Tea Extracts on Various Leukemia Cells --- p.91 / Chapter 3.2.1.1 --- Differential Anti-proliferative Effect of Different Tea Extracts on Various Leukemic Cell Lines In Vitro --- p.91 / Chapter 3.2.1.2 --- Differential Cytotoxic Effect of Different Tea Extracts on the Murine Lymphocytic Leukemia L1210 Cells In Vitro --- p.92 / Chapter 3.2.1.3 --- Induction of Apoptosis in HL-60 Cells by Different Tea Extracts In Vitro --- p.92 / Chapter 3.2.2 --- The Effects of Purified Green Tea Epicatechin Isomers on Various Leukemic Cell Lines --- p.101 / Chapter 3.2.2.1 --- In Vitro Anti-proliferative Effect of Green Tea Epicatechin Isomers on Various Human and Murine Leukemic Cell Lines --- p.101 / Chapter 3.2.2.2 --- In Vitro Cytotoxic Effect of Green Tea Epicatechin Isomers on Various Human and Murine Leukemic Cell Lines --- p.117 / Chapter 3.2.2.3 --- Effects of Green Tea Epicatechin Isomers on the Differentiation of Myeloid Leukemia Cells --- p.131 / Chapter 3.2.2.4 --- Apoptosis-Inducing Effect of Different Green Tea Epicatechin Isomers on HL-60 and JCS Cells --- p.134 / Chapter 3.2.2.5 --- Effect of EGCG on the In Vivo Tumorigenicity of Leukemia JCS and L1210 Cells --- p.142 / Chapter 3.3 --- Discussion --- p.144 / Chapter CHAPTER 4: --- MECHANISTIC STUDIES ON THE ANTI PROLIFERATIVE AND APOPTOSIS-INDUCING ACTIVITIES OF GREEN TEA EPICATECHIN ISOMERS ON LEUKEMIA CELLS / Chapter 4.1 --- Introduction --- p.149 / Chapter 4.2 --- Results --- p.152 / Chapter 4.2.1 --- Combining Effect of EGCG and Physiological Differentiation Inducers on the Proliferation of HL-60 and JCS Cells --- p.152 / Chapter 4.2.2 --- Combining Effect of EGCG and Cytokines on the Proliferation of JCS Cells --- p.155 / Chapter 4.2.3 --- Combining Effect ofEGCG and Other Phytochemicals on the Proliferation of HL-60 and JCS Cells --- p.161 / Chapter 4.2.4 --- Modulatory Effect of EGCG on the Expression of Apoptosis-regulatory Genes in HL-60 Cells --- p.168 / Chapter 4.2.5 --- Modulatory Effect of EGCG on the Expression of Growth-related and Apoptosis-regulatory Proteins in HL-60 Cells --- p.170 / Chapter 4.3 --- Discussion --- p.177 / Chapter CHAPTER 5: --- EFFECTS OF GREEN TEA EPICATECHIN ISOMERS ON THE GROWTH AND DIFFERENTIATION OF MURINE HEMATOPOIETIC CELLS / Chapter 5.1 --- Introduction --- p.184 / Chapter 5.2 --- Results --- p.186 / Chapter 5.2.1 --- In Vitro Effects of EGCG on Murine Lymphocytes --- p.186 / Chapter 5.2.1.1 --- In Vitro Effect of EGCG on the Proliferation of Murine Splenocytes --- p.186 / Chapter 5.2.1.2 --- In Vitro Effect of EGCG on the Mitogen-induced Proliferation of Murine Splenocytes --- p.186 / Chapter 5.2.1.3 --- Cytotoxic Effect of EGCG on Murine Lymphocytes --- p.189 / Chapter 5.2.2 --- Primary Humoral Immune Response to SRBCin EGCG-treated Mice --- p.191 / Chapter 5.2.3 --- In Vitro Studies of the Effects of EGCG on Murine Bone Marrow Cells --- p.192 / Chapter 5.2.3.1 --- Effects of EGCG on the In Vitro Proliferation of Murine Bone Marrow Cells --- p.192 / Chapter 5.2.3.2 --- The Combining Effect of EGCG and Growth Factors on the In Vitro Proliferation of Murine Bone Marrow Cells --- p.192 / Chapter 5.2.3.3 --- In Vitro Cytotoxic Effect of EGCG on Murine Bone Marrow Cells --- p.196 / Chapter 5.2.4 --- Effect of EGCG on the Differentiation of Murine Bone Marrow Cells --- p.199 / Chapter 5.2.5 --- Combining Effects of EGCG and Growth Factors on the Morphology of Murine Bone Marrow Cells --- p.199 / Chapter 5.3 --- Discussion --- p.204 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.207 / REFERENCES --- p.213