Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 132-154). Also available in electronic version. Access restricted to campus users.

Butyrylcholinesterase (BuChE) is a serine hydrolase enzyme that, along with acetylcholinesterase (AChE), catalyzes the hydrolysis of acetylcholine. These enzymes are associated with the pathology of neurologic disorders such as Alzheimer's disease (AD) and multiple sclerosis (MS). In particular, AChE and BuChE accumulate in B- amyloid (AB) plaques and tau neurofibrillary tangles in the AD brain. Thus, imaging cholinesterase activity associated with plaques and tangles in the brain has the potential to provide definitive diagnosis of AD during life. This would be advantageous since, at present, confirmation of AD relies on detecting pathology through post-mortem examination of the brain. In a similar respect, BuChE is associated with the characteristic lesions in MS brain and thus, is a promising target for diagnosis and monitoring of pathology in this disease. It is hypothesized that cholinesterase-binding radiopharmaceuticals can be used in SPECT or PET imaging to visualize these enzymes associated with AD and MS pathology in the living brain. Several classes of cholinesterase ligands were synthesized and exhibited potent binding and specificity towards AChE and BuChE using enzyme kinetic analysis. These compounds were rapidly radiolabelled with 123I and purified. Radiolabelled molecules accumulated in vitro in areas known to contain cholinesterase activity in transgenic AD mice and post-mortem human AD brain tissues, using autoradiography. Furthermore, cholinesterase activity associated with AB plaques was visualized in human and transgenic mouse AD brain tissues. An enzyme kinetic approach was employed to determine critical residues in the BuChE active site gorge for ligand binding. In particular, residues pertaining to the peripheral site of the enzyme were identified and found to be involved in the binding of various ligands. These results are crucial for optimizing the enzyme binding properties of cholinesterase imaging agents. Finally, PET imaging of a transgenic mouse model of AD was performed as a vanguard for pre-clinical evaluation of cholinesterase imaging agents. PET imaging identified similar characteristics between this AD mouse model and the human condition. This is a promising approach for evaluation of cholinesterase imaging agents. Radioligands specific for cholinesterases have the potential to provide a noninvasive means for early diagnosis of neurological diseases using brain scanning.

Alzheimer’s disease (AD), a progressive age-related neurodegenerative disorder is characterized by impairments in memory and cognitive functions. The two main pathogenic hallmarks associated with the progression of this multifactorial disease include accumulation of amyloid-beta (Aβ) plaques and the deterioration of the cholinergic system in the brain. Using cost-effective synthetic procedures, mono-, di-, and tri- substituted sym-triazine derivatives incorporating acetylcholine substrate analogues and aromatic phenyl moieties were synthesized for the targeted modulation of Aβ aggregation and acetylcholinesterase (AChE) activity. A subset of these sym-triazines demonstrated dual inhibition of Aβ fibrillization and AChE hydrolytic activity in vitro studies. These highly effective compounds were also shown to be well tolerated by differentiated human neuronal cells in cell viability tests. These novel compounds have the potential to undergo future in vivo pharmaceutical analysis and have a positive impact on the quality of life of the people living with this devastating disease and their caretakers.

Alzheimer’s disease (AD), a progressive age-related neurodegenerative disorder is characterized by impairments in memory and cognitive functions. The two main pathogenic hallmarks associated with the progression of this multifactorial disease include accumulation of amyloid-beta (Aβ) plaques and the deterioration of the cholinergic system in the brain. Using cost-effective synthetic procedures, mono-, di-, and tri- substituted sym-triazine derivatives incorporating acetylcholine substrate analogues and aromatic phenyl moieties were synthesized for the targeted modulation of Aβ aggregation and acetylcholinesterase (AChE) activity. A subset of these sym-triazines demonstrated dual inhibition of Aβ fibrillization and AChE hydrolytic activity in vitro studies. These highly effective compounds were also shown to be well tolerated by differentiated human neuronal cells in cell viability tests. These novel compounds have the potential to undergo future in vivo pharmaceutical analysis and have a positive impact on the quality of life of the people living with this devastating disease and their caretakers.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Stuttgart, Univ., Diss., 2002.

Stuttgart, Univ., Diss., 2003.

Universiẗat, Diss., 1999--Stuttgart.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O presente trabalho descreve o estudo da espÃcie botÃnica Cissus verticillata (Vitaceae) conhecida como âinsulina vegetalâ que à utilizada popularmente como anti-diabÃtica. AtravÃs de cromatografia gravitacional utilizando gel de sÃlica como fase estacionÃria foi isolado um constituinte esterÃidal, β-Sitosterol a partir do extrato hexÃnico dos talos e do extrato hexÃnico das folhas foi isolado um Ãlcool primÃrio conhecido como n-tetracosanol e um triterpeno chamado 3β-taraxerol. Em adiÃÃo, foram efetuadas algumas reaÃÃes clÃssicas para obtenÃÃo de derivados, inclusive com o objetivo de comparar a atividade biolÃgica desses derivados com a do constituinte original. Assim, foram realizados testes de atividade anticolinesterÃsica do constituinte original e dos produtos das reaÃÃes de acetilaÃÃo, formilaÃÃo e oxidaÃÃo. O estudo do extrato hexÃnico dos talos e das folhas de C. verticillata L. apÃs saponificaÃÃo com KOH/MeOH seguida de metilaÃÃo, forneceu uma mistura de Ãsteres metÃlicos que foram identificados por CG/EM. Os principais componentes foram o Ãcido hexadecanÃico, Ãcido 9,12 octadecadienÃico, Ãcido octadecanÃico, Ãcido eicosanÃico, Ãcido docosanÃico. Na caracterizaÃÃo dos metabÃlitos secundÃrios isolados, assim como, de seus produtos reacionais, foram empregadas tÃcnicas espectromÃtricas de anÃlise de ressonÃncia magnÃtica nuclear (RMN) de 1H e 13C, infravermelho (IV) e espectrometria de massa (EM). / The presente work describe the results of chemical study of Cissus verticillata (Vitaceae), plant known as insulin and commonly used as anti-diabetic. Using column chromatography on silica gel from the hexane extract of the stems was isolated a steroid (β-sitosterol), while the hexane extract of leaves provided an alcohol (tetracosonol) and a pentaciclic triterpene (3β-taraxerol). In addition, from the original constituents, using classical chemical reactions (acetylation, oxidation and formulation), derivatives were prepared in order to compare the original constituents with the derivatives with respect to anticholinesterase activity. We also studied the composition of fatty acids present in the hexane extract of the stems through the process of saponification followed by methylation and analised using gas chromatography coupled to mass spectrometry. All compounds original constituents and derivatives) were characterized by spectrometric methods, nuclear magnetic resonance (NMR) 1H and 13C, infra-red and mass spectroscopic.

The effects of temperature upon acetylcholinesterase (AChE) from the nervous system of fish were studied to determine if such compensatory phenomena as thermal accommodation, thermal acclimation and evolutionary adaptation to temperature as displayed by this physiological system could be observed and interpreted at the level of enzyme function. At probable physiological substrate concentrations the rate of acetylcholine (ACh) hydrolysis by AChE from rainbow trout (Salmo gairdnerii) and electric eel remains relatively unaffected by assay temperature over the temperature ranges normally experienced by these animals. Plots of Km versus temperature for these enzymes yield U shaped curves with minimum Km values occurring at temperatures close to the minimum habitat temperature. It is proposed that thermal accommodation of reaction rate is achieved throughout the habitat temperature range by temperature directed changes in enzyme-substrate affinity. Thermal acclimation in rainbow trout, and probably in speckled trout (Salvelinus fontinalis) and lake trout (Salvelinus namaychus) is accompanied by alterations in the relative proportions of two electrophoretically distinct AChE variants displaying different and adaptive Km-temperature relationships. Since the minimum Km values and energies of activation of the two rainbow trout enzymes are similar, and the specific activities of the enzymes are essentially identical following acclimation of fish to 2° and 17°C, it is suggested that rate compensation of AChE activity may not occur at different acclimation temperatures. However, the possibility remains that changes in such factors as pH, ionic environment and membrane lipids which accompany the acclimation process may act to stabilize reaction rates. Comparisons of AChE enzymes from rainbow trout, electric eel and the Antarctic fish Trematomus borchgrevinki indicate that the evolutionary adaptation of AChE function in species inhabiting different thermal environments is based upon selection for a Km-temperature relationship that will allow thermal accommodation of reaction rate over the temperature range normally encountered. Shifts in the Km-temperature relationship during speciation are interpreted in terms of changes in enzyme conformation following the accumulation of amino acid substitutions. Possible mechanisms by which two AChE enzymes could be incorporated into the trout central nervous system were considered and a hypothesis involving hybridization between fish populations was tested with trout inter-species crosses. It was observed that hybrids formed between speckled and lake trout contained a greater number of electrophoretically distinct AChE variants than did either parent and further, the presence of similar thermally controlled AChE complexes in rainbow, speckled and lake trout indicated that the original incorporation of multiple AChE enzymes into the rainbow trout probably occurred prior to the evolutionary divergence of these three species. It is concluded from this study that changes in enzyme-substrate affinity with temperature, and the temperature directed production of enzyme variants displaying adaptive Km-temperature relationships, are both important mechanisms for controlling catalytic activity in an enzyme system which functions over a wide range of temperatures. / Science, Faculty of / Zoology, Department of / Graduate