Glioblastoma multiforme (GBM) is the most malignant form of brain cancer. Therapies targeting glioblastoma have not consistently been able to give those diagnosed the best prognosis. Treatments that directly infiltrate into the tumor are highly sought after. Indirubins have been used to treat various types of cancers and are a promising avenue for future glioma research. In the current study, we further researched several key GSK-3 inhibitors, BIO (an indirubin) and CHIR99021, in addition to LiCl, to see their effects on the translocation of β-catenin to the nucleus, and the invasion and migration of cells in both a sphere assay and an aortic ring assay. Here we studied anti-invasive therapies that may have a future role in GBM treatment. It is thought that combining conventional treatments with anti-invasive therapies will create cytotoxicity in and reduce migration of the tumor. Three types of cells were used throughout the experiments: HBMEC, HUVEC, and U251 glioma cells. We reported that GSK-3 inhibitors might have a valuable role in the treatment of GBM. The selected inhibitors (BIO, CHIR99021, and LiCl) all were shown to lessen cell migration and invasion in vitro in a range of assays and in all cell lines tested. All inhibitors tested cause a dose-dependent, reversible inhibition of glioma cell invasion in spheroid assays. BIO was shown to cause a rapid upregulation of total and nuclear β-catenin. BIO, at higher concentrations, also created a toxic environment for cells, sometimes killing them. This shows that a more in-depth experiment involving different BIO concentrations is needed to test the optimal concentration for treatment. Each of the experimented GSK-3 inhibitors also showed a change in the junctions between cells. NaCl as a control showed normal, spikey, junctions, while CHIR99021 and BIO caused the junctions to become more smooth. This suggests that GSK-3 inhibition has a role in either maintaining the ECM and/or in communication between cells. Also in this assay, there was a heterogeneity between cells treated with the same inhibitor and in the same dish, indicating that not all cells respond to each drug the same way. The reasons for this are not known and further investigation is required. A new construct was also made to report β-catenin transcriptional coactivation using luciferase expression as the reporter in response to these selected GSK-3 inhibitors.With the combined results of these experiments, we concluded that GSK-3 inhibitors may be a promising approach to the treatment of GBM. Further investigation is required before any treatments can be administered to those diagnosed.

A homeostase do cálcio possui um papel central no funcionamento dos tecidos e células animais e torna-se, então, fundamental o entendimento dos mecanismos celulares de transporte de Ca2+ transmembrânico, e para isso, os crustáceos têm sido utilizados como modelo de estudo. Caranguejos terrestres e semi-terrestres, devido ao acesso limitado ao meio líquido desenvolveram mecanismos fisiológicos para a conservação de Ca2+ (em gastrólitos, hemolinfa e hepatopâncreas), sendo que os crustáceos apresentam quatro tecidos especializados para troca bi-direcional específica de Ca2+: (i) brânquias, (ii) epitélio do sistema digestivo, (iii) glândulas antenais e (iv) a camada da hipoderme da cutícula (não analizado no presente projeto). Nesses tecidos, a entrada de Ca2+ através de membranas apicais envolve o trocador de Ca2+/nNa+ (nH+) e um canal de Ca2+; os trocadores basolaterais, por outro lado, envolvem (i) o trocador de Ca2+/Na+ (ii) uma bomba de Ca2+- ATPase, e (iii) um canal de cálcio inibido por verapamil. Estudou-se aqui a ação conjugada de inibidores (amiloride e verapamil) e a ação indireta da ouabaína (inibidor da Na+/K+ ATPase) sobre a tomada de Ca2+, visando testar a necessidade de um gradiente de Na+ para o transporte secundário de Ca2+ em células isoladas de brânquias, glândulas antenais e hepatopâncreas preparadas a partir de animais em fase de inter-muda e pós-muda. Nos caranguejos utilizados no presente projeto (Superfamília Grapsoidea), os quais apresentam diferentes níveis de terrestrialidade: (A) Casmagnathus granulatus T2, (B) Sesarma rectum T3 e (C) Goniopsis cruentata T3., os inibidores amiloride (12,7 mM), verapamil (40 mM), e amiloride + verapamil conjugados, promoveram tendências à redução no transporte de Ca2+. O uso do inibidor ouabaína (17,1 mM), sugere a necessidade de um gradiente de Na+ para o transporte secundário de Ca2+. Os resultados obtidos comprovaram também a relação entre o nível de terrestrialidade e as adaptações fisiológicas em relação à homeostase do Ca2+ somente em hepatopâncreas, enquanto que a glândula antenal parece não exercer papel relevante na homeostase do Ca2+ em caranguejos semi-terrestres. / Calcium homeostasis possess a central role on animal cells and tissues and the understanding of transepithelial Ca2+ transport mechanisms becomes fundamental, and crustaceans have been used as study model. Since terrestrial and semi-terrestrial crabs have limited access to water, they have evolved physiological mechanisms in order to conserve Ca2+ (in gastroliths, hemolymph and hepatopancreas), and crustacean have shown four specialized tissues for specific bidirectional Ca2+ exchange: (i) gills, (ii) the gut epithelia, (iii) antennal glands e (iv) the layer of cuticle hypodermis (not analyzed in this project). In these tissues, the apical Ca2+ uptake involves a Ca2+/nNa+ (nH+) exchanger and a Ca2+ channel; in contrast, basolateral exchangers may involve: (i) a Na+/Ca2+ exchanger (ii) a Ca2+ ATPase, and (iii) a verapamil-inhibited Ca2+ channel. The objective of this project was to study the action of conjugated inhibitors (amiloride and verapamil) and ouabain (Na+/K + ATPase inhibitor) on Ca2+ uptake, to show the necessity of a Na+ gradient for the secondary Ca2+ transport in isolated gills, antennal glands and hepatopancreatic cells from intermoult and postmoult animals. The crabs used in this project (Grapsoidea Superfamily), present different levels of terrestrial adaptations: (a) Casmagnathus granulatus T2, (b) Sesarma rectum T3 and (c) Goniopsis cruentata T3. The inhibitors amiloride (12,7 mM), verapamil (40 mM), and amiloride + verapamil used in this project promoted a tendency to reduce Ca2+ transport. The results on ouabain inhibitor (17.1 mM) suggests the necessity of a Na+ gradient for secondary Ca2+ transport,. The results also suggest a relationship between level of terrestrial adaptation and physiological responses related to Ca2+ homeostasis in hepatopancreas, while the antennal gland does not seem to have a relevant role on Ca2+ homeostasis in semi-terrestrial crabs.

A homeostase do cálcio possui um papel central no funcionamento dos tecidos e células animais e torna-se, então, fundamental o entendimento dos mecanismos celulares de transporte de Ca2+ transmembrânico, e para isso, os crustáceos têm sido utilizados como modelo de estudo. Caranguejos terrestres e semi-terrestres, devido ao acesso limitado ao meio líquido desenvolveram mecanismos fisiológicos para a conservação de Ca2+ (em gastrólitos, hemolinfa e hepatopâncreas), sendo que os crustáceos apresentam quatro tecidos especializados para troca bi-direcional específica de Ca2+: (i) brânquias, (ii) epitélio do sistema digestivo, (iii) glândulas antenais e (iv) a camada da hipoderme da cutícula (não analizado no presente projeto). Nesses tecidos, a entrada de Ca2+ através de membranas apicais envolve o trocador de Ca2+/nNa+ (nH+) e um canal de Ca2+; os trocadores basolaterais, por outro lado, envolvem (i) o trocador de Ca2+/Na+ (ii) uma bomba de Ca2+- ATPase, e (iii) um canal de cálcio inibido por verapamil. Estudou-se aqui a ação conjugada de inibidores (amiloride e verapamil) e a ação indireta da ouabaína (inibidor da Na+/K+ ATPase) sobre a tomada de Ca2+, visando testar a necessidade de um gradiente de Na+ para o transporte secundário de Ca2+ em células isoladas de brânquias, glândulas antenais e hepatopâncreas preparadas a partir de animais em fase de inter-muda e pós-muda. Nos caranguejos utilizados no presente projeto (Superfamília Grapsoidea), os quais apresentam diferentes níveis de terrestrialidade: (A) Casmagnathus granulatus T2, (B) Sesarma rectum T3 e (C) Goniopsis cruentata T3., os inibidores amiloride (12,7 mM), verapamil (40 mM), e amiloride + verapamil conjugados, promoveram tendências à redução no transporte de Ca2+. O uso do inibidor ouabaína (17,1 mM), sugere a necessidade de um gradiente de Na+ para o transporte secundário de Ca2+. Os resultados obtidos comprovaram também a relação entre o nível de terrestrialidade e as adaptações fisiológicas em relação à homeostase do Ca2+ somente em hepatopâncreas, enquanto que a glândula antenal parece não exercer papel relevante na homeostase do Ca2+ em caranguejos semi-terrestres. / Calcium homeostasis possess a central role on animal cells and tissues and the understanding of transepithelial Ca2+ transport mechanisms becomes fundamental, and crustaceans have been used as study model. Since terrestrial and semi-terrestrial crabs have limited access to water, they have evolved physiological mechanisms in order to conserve Ca2+ (in gastroliths, hemolymph and hepatopancreas), and crustacean have shown four specialized tissues for specific bidirectional Ca2+ exchange: (i) gills, (ii) the gut epithelia, (iii) antennal glands e (iv) the layer of cuticle hypodermis (not analyzed in this project). In these tissues, the apical Ca2+ uptake involves a Ca2+/nNa+ (nH+) exchanger and a Ca2+ channel; in contrast, basolateral exchangers may involve: (i) a Na+/Ca2+ exchanger (ii) a Ca2+ ATPase, and (iii) a verapamil-inhibited Ca2+ channel. The objective of this project was to study the action of conjugated inhibitors (amiloride and verapamil) and ouabain (Na+/K + ATPase inhibitor) on Ca2+ uptake, to show the necessity of a Na+ gradient for the secondary Ca2+ transport in isolated gills, antennal glands and hepatopancreatic cells from intermoult and postmoult animals. The crabs used in this project (Grapsoidea Superfamily), present different levels of terrestrial adaptations: (a) Casmagnathus granulatus T2, (b) Sesarma rectum T3 and (c) Goniopsis cruentata T3. The inhibitors amiloride (12,7 mM), verapamil (40 mM), and amiloride + verapamil used in this project promoted a tendency to reduce Ca2+ transport. The results on ouabain inhibitor (17.1 mM) suggests the necessity of a Na+ gradient for secondary Ca2+ transport,. The results also suggest a relationship between level of terrestrial adaptation and physiological responses related to Ca2+ homeostasis in hepatopancreas, while the antennal gland does not seem to have a relevant role on Ca2+ homeostasis in semi-terrestrial crabs.

The current increased use of antifungal agents has resulted in the development of resistance to these drugs. Search for new antifungals with different mechanisms of action overcoming the multidrug resistance is thus underway. Surface-active antifungals have the advantages of minimizing host toxicity and the emergence of drug resistance. We have developed a fluorescence method based on the use of the potentiometric fluorescent probe diS-C3(3), substrate of two major S. cerevisiae MDR pumps, Pdr5p and Snq2p. It allows us to monitor with high sensitivity and in real time changes in the activities of both pumps and also in membrane potential. We present here an efficient strategy for identifying pump inhibitors with minimal side effects on membrane integrity, and compare the potencies of different inhibitors towards MDR pumps. New efficient inhibitors of MDR pumps could potentially be used in conjunction with current antimicrobials that are MDR pump substrates. The method can be also used to determine the mechanism of action of surface-active drugs and their lowest effective concentrations.

by Wong Kin Kwan Kelvin. / Thesis submitted in: September 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 91-101). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Abbreviations --- p.x / List of Figures --- p.xiii / List of Tables --- p.xiv / Chapter Chapter 1 --- Intorduction --- p.1 / Chapter 1.1 --- Alzheimer，s disease --- p.1 / Chapter 1.2 --- Histopathological features --- p.1 / Chapter 1.3 --- Tau protein pathology and AD --- p.4 / Chapter 1.4 --- Tau protein kinase I (TPKI)- GSK-3β --- p.6 / Chapter 1.5 --- Tau protein kinase II (TPKII)- Cyclin dependent kinase 5 (Cdk5) --- p.8 / Chapter 1.6 --- Available treatment --- p.9 / Chapter 1.7 --- Objectives of the present study --- p.12 / Chapter Chapter 2 --- Screening for GSK-3p inhibitors from Traditional Chinese Medicine (TCM) --- p.13 / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.1.1 --- Phosphorylation of tau in AD --- p.13 / Chapter 2.1.2 --- Gsk-3p inhibitors --- p.14 / Chapter 2.1.3 --- Screening of GSK-3β inhibitor from TCM --- p.16 / Chapter 2.2 --- Material and Methods --- p.18 / Chapter 2.2.1 --- Preparation of extracts and fractions (AOF1-5) --- p.18 / Chapter 2.2.2 --- General cell culture techniques --- p.21 / Chapter 2.2.3 --- "3-(4,5-dimethyltiazoI-2-yl)-2, 5-diphenyl-tetrazolium (MTT) assay of AOF" --- p.23 / Chapter 2.2.4 --- Recombinant DNA techniques --- p.23 / Chapter 2.2.5 --- Transfection of GSK-3β and tau cDNA into COS7 cells --- p.28 / Chapter 2.2.6 --- Extraction of total proteins from culture cells --- p.28 / Chapter 2.2.7 --- Quantitation of protein by the Bradford method --- p.29 / Chapter 2.2.8 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29 / Chapter 2.2.9 --- Western blot analysis --- p.31 / Chapter 2.2.10 --- GSK-3β kinase assay --- p.32 / Chapter 2.2.11 --- Determination of lithium content by atomic adsorption spectrophotometry --- p.34 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- Establishment of a co-transfected cell model for GSK-3β induced tau hyperphosphorylation --- p.35 / Chapter 2.3.2 --- Preliminary screening results of aqueous and ethanol extracts (AOF1 and AOF2) --- p.37 / Chapter 2.3.3 --- Ethanol extract of AOF inhibits GSK-3p induced tau phosphorylation in COS-7 cells --- p.40 / Chapter 2.3.5 --- Effect of the essential oils of AOF on GSK-3P induced tau phosphorylation --- p.46 / Chapter 2.3.6 --- The effect of AOF essential oil on GSK-3P activity in COS7 --- p.50 / Chapter 2.3.7 --- Lithium content of AOF extracts --- p.52 / Chapter 2.4 --- Discussion --- p.54 / Chapter Chapter 4 --- Evaluation of the in vivo efficacy of cryptotenshinone (CT) in Morris Water Maze Task (WMT) --- p.59 / Chapter 4.1 --- Introduction --- p.59 / Chapter 4.1.1 --- Involvement of Cholinergic system in cognitive dysfunction in AD --- p.59 / Chapter 4.1.2 --- Animal model for Alzheimer's disease --- p.60 / Chapter 4.1.3 --- Morris Watermaze Task (WMT) --- p.61 / Chapter 4.2 --- MATERIAL AND METHODS --- p.64 / Chapter 4.2.1 --- Morris Water maze setup --- p.64 / Chapter 4.2.2 --- Animal model --- p.66 / Chapter 4.2.3 --- Drug preparation --- p.67 / Chapter 4.2.4 --- Toxicity test of CT --- p.67 / Chapter 4.2.5 --- Water maze task (WMT) --- p.68 / Chapter 4.2.6 --- Visual acuity test --- p.73 / Chapter 4.3 --- RESULTS --- p.74 / Chapter 4.3.1 --- Chronic crytotanshinone treatment does not cause hepatic damages to the mice --- p.74 / Chapter 4.3.2 --- Training Session --- p.76 / Chapter 4.4 --- DISCUSSION --- p.85 / Chapter Chapter 5 --- General Discussion and Future Directions --- p.87 / Chapter 5.1 --- "AOF, the potential GSK-3 inhibitor" --- p.87 / Chapter 5.2 --- CT´ؤthe AChEI --- p.88 / References --- p.91 / Appendix --- p.102 / Chapter A1 --- Reagents for SDS-PAGE --- p.103 / Chapter A3 --- Solution components provided by QIAGEN Plasmid Maxipreps kit --- p.108 / Chapter A4 --- Reagents and medium for cell culture --- p.109 / Chapter A5 --- Reagents for kinase assay --- p.110 / Chapter A6 --- Raw data of figures --- p.112 / Chapter A7 --- Plasmid map of PCI-neo --- p.119