Background: In the United States, 30-60% of older adults fall each year; 10-20% of these falls result in injury, hospitalization, or even death. Better prevention of falls in this population may be facilitated by broader identification of risk factors. The use of statins has emerged as a potential risk factor, but the data provide conflicted results. Purpose: To examine the relationship between statin use and falls among community-dwelling older adults. Methods: We evaluated the patient registry of a Level 1 trauma center. All patients aged > 50 years who were admitted for falls in 2015 were included (n=615). Many of these patients had been previously admitted for falls and many were later readmitted for falls. We analyzed predictors of both prior admission and readmission with linear regressions. Independent variables were self-reported balance problems, diagnosis of dementia, and the use of statins. Results: On average, patients admitted for falls were 79.9 + 9.3 years old and 28% (n=173) were taking statins. Our collection of predictors explained 14.2% of the variance in the number of prior admissions (p<0.001). In this model, the use of statins significantly predicted the number of previous fall-related admissions (95% CI: 0.07–0.50, p=0.010). This same model maintained its significance when predicting admissions for future falls (p<0.001) and the use of statins continued to predict a greater number of readmissions (95% CI: 0.04–0.36, p=0.015). Conclusion: More than 25% of all Americans age > 40 years are taking cholesterol-lowering medication; 93% of those medications are statins. Although evidence is conflicted, these data support the finding that statin therapy increases the risk of falls in older adults. Incorporating exercise training as a prophylactic measure: enhancing lipid profiles and decreasing the need for statins while also improving balance, coordination, and mobility, may reduce fall-related injuries.

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Thesis (PhD (Biomedical Sciences. Medical Physiology))--Stellenbosch University, 2008. / The overall objective of this thesis is to provide additional data to assist clinicians and experimental neurologists alike in the quest for better understanding, more accurately diagnosing and more successfully treating patients suffering from Parkinson’s disease (PD). The general theme of the thesis is the interaction between certain environmental stimuli, including the exposure to adverse events during early central nervous system (CNS) development and the manifestation of elements of neurodegeneration, whether by means of neurochemical changes or expressed as a dysfunctional voluntary motor system. The first chapter provides a general introduction to the research theme of the thesis. This includes, in particular, a discussion on current understanding concerning the etiology and clinical profile of PD, the relative contribution made by genetic factors compared to environmental ones, and current treatment strategies for treating the disease. Mention is also made of the failure of these therapeutic applications for reversing or protecting against the disease, due to the side-effects associated with them. The material covered in chapter 1 provides the basis for the more complete discussion concerning these various aspects, contained in the chapters to follow. The overall aim was also to characterise the effects of commonly used toxin-induced animal models of PD, and the extent of vulnerability that the CNS displays towards them. The destruction of dopaminergic neurons following the administration of 6-OHDA at targeted points along the nigrostriatal tract is used extensively to model PD pathology in rats and is an established animal model of the disease. However, mature or even aged animals are mainly used in these studies, while the effects that the toxin might have on the developing CNS remain unclear. The study reported in chapter 4 aimed to elucidate some of 6-OHDA’s actions on the young adolescent (35 days-old) CNS by comparing the motor and biochemical effects of a unilateral infusion of the toxin into two anatomically distinct basal ganglia loci: The medial forebrain bundle (MFB) and the striatum. Animals were randomly assigned to receive either a direct delivery of 6-OHDA (12μg/4μl) into the MFB or an indirect injection, into the striatum. Although both lesion types were used, the MFB model is considered a more accurate portrayal of end-stage PD, while the striatum-model better reflects the long-term progressive pathology of the disease. The different lesions’ effects on motor function were determined by observing animal’s asymmetrical forelimb use to correct for weigh shifting during the vertical exploration of a cylindrical enclosure. Following the final behavioral assessment, the concentration of dopamine (DA) and DA metabolites remaining in the post-mortem brains were determined using 4 HPLC electrochemistry (HPLC-EC) and the levels compared between the two groups. The HPLC-EC results revealed a compensatory effect for DA production and DA turnover on the lesioned hemisphere side of the toxin-infused animal group. Thus, following 6-OHDA treatment, there appears to be extensive adaptive mechanisms in place within the remaining dopaminergic terminals that may be sufficient for maintaining relatively high extracellular and synaptic concentrations of DA. However, since substantial changes in motor-function were observed, it is suggested that the capacity of the remaining dopaminergic neurons to respond to increased functional demands may be limited. In addition, the behavioral results indicate that the distinct indices relating to different functional deficits depend on the lesioning of anatomically distinct structures along the nigrostrial tract. It has long been known that far fewer women are diagnosed with PD than men are. This seeming protection offered to females against degenerative disease of the CNS may relate to estrogen, although the hormone’s mechanism of action on the dopaminergic system is poorly defined. With an estimated 10-15 million women using oral contraceptives (OCs) in the United States alone, the aim of chapter 2 was to examine the evidence for a possible relationship between PD and the female reproductive hormone estrogen. A review of the current literature available on the topic was performed by consulting Medline, and by performing a search of the case-reports contained within the World Health Organization’s (WHO) International Drug Monitoring database, for possible PD-related symptoms that may arise from estrogen replacement therapy (ERT). The results, whilst conflicting, seem to suggest that estrogen protects women from obtaining the disease, or at least some features of it. Intensive research efforts are called for, with sufficient power to establish the relationship between ERT and the onset and development of parkinsonism. Chapter 3 reports on the results obtained from an experiment that subjected young Sprague-Dawley rats, 35 days of age, to a lower and a higher dose of 6-OHDA delivered to the MFB. Control rats received equivalent saline infusions. At 14 days post-surgery, the rats were evaluated for forelimb akinesia. For the higher dose of 6- OHDA the female rats were less impaired than males in making adjustment steps in response to a weight shift and in the vibrissae-evoked forelimb placing test. In addition, Tyrosine hydroxylase (TH) immunoreactivity was significantly higher for the female rats. Early gender differences in cell survival factors and/or other promoters of neuroplasticity may have contributed to the beneficial outcome seen in the females. For example, nerve growth factor (NGF) was found to be higher in the female rats following administration of the DA neurotoxin. It is unclear whether gonadal steroids are involved, and, if so, whether female hormones are protective or whether male hormones are prodegenerative. Determining the mechanisms for the improved outcome seen in the young female rats may lead to potential treatment strategies against PD. 5 Many studies have shown that early life stress may lead to impaired brain development, and may be a risk factor for developing psychiatric diseases, including clinical depression. However, few studies have investigated the impact that early stress may have on the onset and development of neurodegenerative disorders such as PD. The study reported on in chapter 5 conjointly subjected rat pups to a maternal separation (MS) paradigm that is a well characterised model of adverse early life events, and a unilateral, intrastriatal injection of 6- OHDA. The combined effects of these models on motor deficits and brain protein levels were investigated. Specifically, the animals were assessed for behavioral changes at 28 days postlesion with a battery of tests that are sensitive to the degree of DA loss sustained. The results show that animals that had been subjected to MS display poorer performance in the vibrissae and single-limb akinesia test compared to non-MS control animals (that had also been subjected to the toxin exposure). In addition, there was a significant increase in the loss of TH staining in MS rats compared to non-MS ones. The results from this study therefore suggest that exposure to adverse experiences during the early stages of life may contribute towards making dopaminergic neurons more susceptible to subsequent insults to the CNS occurring during mature stages of life. Therefore, taken together, early exposure to stress may predispose an individual towards the onset and development of neurodegenerative disease, which especially becomes a threat during the later stages of adult life. Moreover, within the framework of these characteristics, the capacity of a widely-used pharmacological agent (statins) was tested for possible future therapeutic application in PD (chapter 7). Although the precise cause of sporadic PD remains an enigma, evidence suggests that it may associate with defective activity of complex I of the mitochondrial electron transport chain. Mitochondrial DNA transmit and express this defect in host cells, resulting in increased oxygen free radical production, depressed antioxidant enzyme activities, and greater susceptibility to apoptotic cell death. Simvastatin is a member of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) group of drugs that are widely used for lowering cholesterol levels in patients who display elevated concentrations of low-density lipoprotein cholesterol. The study aimed to investigate the effects that statin-treatment have on motor-function and at the mitochondrial-protein level, using rotenone, a mitochondrial complex I inhibitor, as a rat-model of PD. Adult male Sprague-Dawley rats were treated either with simvastatin (6mg/day for 14 days) or with a placebo. Two different tests to assess motor function were used: The apomorphine-rotation test, and the vibrissae-elicited forelimb placement test. Following the drug administration protocol, the nigrostriatal tract was unilaterally lesioned with either rotenone (3 μg/4 μl) or, for the controls, were sham-operated by infusing the vehicle (DMSO:PEG) only. Five days later the rats were killed and a highly purified concentration of isolated mitochondria was prepared from the substantia nigra (SN) sections. 2- 6 Dimensional electrophoresis (2-DE) with subsequent identification of the spots using electronspray ionization quadruple time-of-flight mass spectrometrical (ESI-Q-TOF MS) was performed and the results BLAST-searched using bio-informatics tools for naming the identified peptides. The motor test results indicate that while unilateral rotenone causes behavioral asymmetries, treatment with simvastatin improved motor function relative to the rotenoneinduced ones. Mass Spectroscopy identified 23 mitochondrial proteins that differ significantly in protein expression (p < 0.05) following simvastatin treatment. The altered proteins were broadly classified according to their cellular function into 6 categories, with the majority involved in energy metabolism. This study effectively illustrated how neuroproteomics, with its sophisticated techniques and non-biased ability to quantify proteins, provides a methodology with which to study the changes in neurons associated with neurodegeneration. As an emerging tool for establishing disease-associated protein profiles, it also generates a greater understanding as to how these proteins interact and undergo post-translational modifications. Furthermore, due to the advances made in bioInformatics, insight is created concerning their functional characteristics. Chapter 4 summarises the most prominent proteomics techniques and discuss major advances made in the fast-growing field of neuroproteomics in PD. Ultimately, it is hoped that the application of this technology will lead towards a presymptomatic diagnosis of PD, and the identification of risk factors and new therapeutic targets at which pharmacological intervention can be aimed. The final chapter (chapter 8) provides a retrospective look at the academic work that had been performed for the purpose of this thesis, recaps on the main findings, and also highlights certain aspects of the project and provides relevant suggestions for future research. Lastly, the appendix provides a detailed overview of the methods followed for the experiments described in this thesis. It provides not only a comprehensive description of the techniques that had been followed, but provides information concerning the care taken with the animals (i.e. post-surgery) in order to control for the potential influence of experimental variables on the results.

A homeostase do colesterol é mediada por proteínas envolvidas na absorção (NPC1L1), regulação (SREBP1, SREBP2, SCAP), síntese (HMGCR) e remoção plasmática (LDLR). Os fármacos inibidores da síntese (vastatinas) e absorção (ezetimiba) do colesterol são potentes agentes hipocolesterolemiantes. Alterações em vários genes têm sido associadas a diferenças na resposta a diversos agentes terapêuticos. Com a finalidade de estudar os efeitos de hipolipemiantes e polimorfismos sobre a expressão dos genes HMGCR, LDLR, SREBF1a, SREBF2, SCAP e NPC1L1, foram selecionados 25 indivíduos com hipercolesterolemia familial (HF), 72 com hipercolesterolemia não familial (HNF) e 125 indivíduos normolipidêmicos e sem doença cardiovascular (NL). Os indivíduos HF foram tratados com sinvastatina (40 mg/dia/4 sem) combinada ou não com ezetimiba (10 mg/dia/4sem) e os HNF foram tratados com atorvastatina (10 mg/dia/4sem). Amostras de sangue foram obtidas antes e após o tratamento para a extração de DNA e RNA e analise do perfil lipídico sérico. A expressão de mRNA dos genes SREBF1a, SREBF2, SCAP, HMGCR, LDLR e NPC1L1 em células mononucleares do sangue periférico (CMSP) foi determinada por RT-PCR em tempo real empregando-se o gene da GAPD como controle endógeno. Os polimorfismos SREBF1a 36delG, SREBF2 G1784C e SCAP A2386G foram determinados por PCR-RFLP. Os indivíduos HF apresentaram maior expressão de mRNA dos genes NPC1L1, HMGCR e LDLR que os grupos HNF e NL (p<0,05). O efeito da atorvastatina sobre a expressão dos genes estudados parece depender da expressão basal nos indivíduos HNF. A variação da expressão após o tratamento com atorvastatina nos pacientes do grupo HNF esteve correlacionada nos genes: SREBF1a e SREBF2; SREBF1a e SCAP; SREBF1a e LDLR; SREBF2 e SCAP; SREBF2 e LDLR; HMGCR e LDLR. O tratamento com sinvastatina e ezetimiba não modificou o padrão de expressão dos genes estudados no grupo HF. Os polimorfismos SREBF2 G1784C e SCAP A2386G parecem estar relacionados com diminuição da expressão de mRNA após o tratamento com atorvastatina. Foi observado que os portadores do genótipo GG do polimorfismo SREBF2 G1784C apresentaram maiores concentrações séricas de colesterol total e LDL-C após o tratamento com atorvastatina. O polimorfismo SCAP A2386G parece estar associado com maiores concentrações de apoB em pacientes do grupo HNF antes do tratamento com atorvastatina. Os resultados são sugestivos que os genes HMGCR, LDLR e NPC1L1 são regulados diferentemente de acordo com o estado metabólico do indivíduo e a taxa de expressão de mRNA é influenciada pelos polimorfismos SREBF2 G1784C e SCAP A2386G após o tratamento com atorvastatina / The regulation of cholesterol is mediated by proteins involved in the absorption (NPC1L1), regulation (SREBP1, SREBP2, SCAP), synthesis (HMGCR) and removal of plasma cholesterol (LDLR). Potent hypocholesterolemic agents inhibit cholesterol synthesis (statins) and its absortion (ezetimibe). Changes in several genes have been associated to different responses to various therapeutic agents. In order to evaluate the association between genes involved in the metabolism of cholesterol and their response to lipid lowering drugs, patients with familial (FH, n = 25) and non familial hypercholesterolemia (NHF, n = 72) were selected. Additionally, 125 normolipidemic individuals and without cardiovascular disease were selected (NL). The HF group were treated with simvastatin (40 mg/day/4 weeks) combined or not with ezetimibe (10 mg/day/4weeks). The NHF group were treated with atorvastatin (10 mg/day/4weeks). Blood samples were obtained prior to and following treatment for extraction of DNA and RNA, and serum lipid profile analysis. The mRNA expression of SREBF1a, SREBF2, SCAP, HMGCR, LDLR, and NPC1L1 genes was determined by real time RT-PCR using the GAPD gene as endogenous control. The polymorphisms SREBF1a-36delG, SREBF2 G1784C, and SCAP A2386G were determined by PCR-RFLP. Individuals with HF showed higher expression of mRNA of genes NPC1L1, HMGCR and LDLR when compared with HNF and NL groups (p <0.05). The effect of atorvastatin on the gene expression seems to depend on the baseline expression in HNF subjects. The change of expression after treatment with atorvastatin in group HNF was correlated as followed: SREBF1a and SREBF2; SREBF1a and SCAP; SREBF1a and LDLR; SREBF2 and SCAP; SREBF2 and LDLR; HMGCR and LDLR. Treatment with simvastatin and ezetimibe did not change the gene-expression profile in HF group. The polymorphisms SREBF2 G1784C, and SCAP A2386G appear to be related to a decreased expression of mRNA after treatment with atorvastatin. HNF group Carriers of GG genotype of SREBF2 G1784C polymorphism had higher serum concentrations of total cholesterol and LDL-C after therapy. The SCAP A2386G polymorphism seems to be associated with higher concentrations of apoB in patients from HNF group prior to treatment with atorvastatin. The results suggest that the HMGCR, LDLR and NPC1L1 genes are regulated according to the metabolic status of the individual, and the expression rate of mRNA is influenced by SREBF2 G1784C and SCAP A2386G polymorphisms after atorvastatin therapy.

As estatinas são os agentes mais eficazes para a redução de colesterol no tratamento de doenças cardiovasculares, e algumas destas moléculas podem ser produzidas através de processos biológicos como o cultivo semi-sólido de fungos filamentosos. O objetivo deste estudo foi determinar a capacidade de produção de estatinas e compostos antimicrobianos por cinco cepas de fungos isolados de Cana de Açúcar. Para isso, extratos obtidos a partir dos cultivos foram analisados por métodos analíticos como CLAE e RMN para determinar a presença de estatinas; adicionalmente, os extratos foram testados contra diferentes modelos biológicos incluindo bactérias, leveduras, fungos filamentosos, células de ovário de hamster chinês, e parasitas. De acordo com os resultados obtidos, os cinco fungos avaliados não produzem estatinas, e em relação ao biomonitoramento dos extratos foi observado um efeito biológico sobre os parasitas e as células de mamífero, no entanto, é possível que o efeito obtido seja uma resposta dos compostos do substrato dos cultivos (Farelo de trigo). / Statins are the most effective cholesterol lowering agents for the treatment of cardiovascular disease, and some of these molecules can be produced through biological process such as the solid state fermentation. The aim of this study was determinate the capability of production of statins and antimicrobials compounds by five strains of fungi isolated from Brazilian sugar cane. For this purpose, extracts were obtained from the cultures and analyzed through analytical methods as HPLC and NMR in order to determinate the presence of statins; in addition, the extracts were tested against different biological models including bacteria, yeast, filamentous fungi, chinese hamster ovary cells, and parasites. According to the results obtained, the five fungal strains tested did not produce statins, and the extracts produced a biological effect against the parasites and mammalian cells, nevertheless it is possible that this effect observed was a response of the compounds from the culture substrate (wheat bran).

No description available.

在2型糖尿病病人身上，常常併發高膽固醇血症，HMG CoA 還原酶的抑制劑常常用作治療這類病症。由於高膽固醇血症與胰島素抵抗和2型糖尿病有著密切關係，我們推測辛伐他汀對於2型糖尿病的發展有著保護和有利的作用。在這項研究中，我們主要測試了辛伐他汀 （10 nM； 24 hr）對於胰島β細胞主要功能的影響，包括其對於葡萄糖的胰島素分泌功能影響。我們假設，在肥胖/糖尿病（db⁺/ db⁺）小鼠分離的胰島，辛伐他汀可以恢復葡萄糖 （5 mM和15 mM）引起的胰島素分泌（加上降低的胰島素含量）。 / 在這個項目中，我們運用24周大的基因糖尿糖C57BL/KSJ +db/+db (db⁺/db⁺)肥鼠和相同年齡的無糖尿病C57BL/KSJ +m/+m (db⁺/m⁺)小鼠作為動物模型。通過應用obese/diabetic (db+/db+)和lean/non-diabetic (db+/m+)中分離的胰腺胰島和胰島β細胞，我們研究了胰腺胰島功能性障礙的潛在機理以及辛伐他汀對於恢復葡萄糖 （5 mM和15 mM）引起的胰島素分泌（加上降低的胰島素含量）的有利作用。資料清晰的顯示，葡萄糖引起的胰島素分泌和胰島素含量在obese/diabetic (db+/db+)的胰腺胰島中明顯低於在lean/non-diabetic (db⁺/m⁺)的胰腺胰島中。在24hr的辛伐他汀處理後，辛伐他汀恢復了葡萄糖 （5 mM和15 mM）引起的胰島素分泌（加上降低的胰島素含量）及葡萄糖 （15 mM）引起的胞內鈣離子變化。 / 在這個項目中，我們證明鈣敏感受體 （CaSR）在obese/diabetic (db⁺/db⁺)中的表達量明顯較低，而辛伐他汀的處理可以顯著性增加鈣敏感受體在obese/diabetic (db⁺/db⁺)胰島中的表達。有人建議說，obese/diabetic (db⁺/db⁺)的胰島中被抑制的鈣敏感受體表達與胰島β細胞的胰島分泌功能障礙有關。這暗示了辛伐他汀可能通過變構啟動鈣敏感受體來恢復obese/diabetic (db⁺/db⁺)胰島中葡萄糖引起的胰島素分泌和胰島含量。實驗也同樣証明辛伐他汀調節的PLA₂信號通路對於辛伐他汀改善obese/diabetic (db⁺/db⁺)胰島β細胞的胰島素分泌功能起著至關重要的作用。除此之外，我們的實驗結果證明高濃度的葡萄糖處理顯著的增加了obese/diabetic (db⁺/db⁺)細胞膜肌動蛋白骨架的密度，而辛伐他汀顯著的減少了這一變化。因此，obese/diabetic (db⁺/db⁺)胰島β細胞的胰島素分泌障礙是由肌動蛋白細胞骨架聚集阻礙胰島素顆粒胞吐引起的。而辛伐他汀通過解聚和重組肌動蛋白細胞骨架來改善obese/diabetic (db⁺/db⁺)胰島β細胞的胰島素分泌功能。 / 在這項研究中，我們的實驗結果證明葡萄糖可以顯著提高obese/diabetic (db⁺/db⁺)胰島β細胞內ROS的含量。而辛伐他汀處理部分降低了胰島β細胞內ROS的含量。除此之外，我們還研究了5 mM和15 mM葡萄糖對於內質網應力（ER-stress）相關的蛋白比如PERK, eIF2α 和IRE1表達的影響。這些內質網跨膜蛋白可以感應ER-stress從而啟動應力感測器來開啟複雜的信號通路。與lean/non-diabetic (db⁺/m⁺)相比，PERK and eIF2α在obese/diabetic (db⁺/db⁺)的胰島中表達量更低，這表明obese/diabetic (db⁺/db⁺)胰島β細胞的功能性障礙可能與ER-stress有關。而辛伐他汀的處理明顯的增加了這些蛋白的表達量，由此證明辛伐他汀還通過對抗ER-stress來保護obese/diabetic (db⁺/db⁺)胰島β細胞。 / 總而言之，我們的資料第一次證明了辛伐他汀通過PLA₂信號通路變構啟動鈣敏感受體來保護obese/diabetic (db⁺/db⁺)胰島β細胞（比如：恢復葡萄糖引發的胰島素分泌和提高減少的胰島素含量），還通過提高obese/diabetic (db⁺/db⁺)胰島β細胞中被抑制的ER-stress相關蛋白的表達量來抵抗ER-stress帶來的損傷。 / Diabetics often have hyperlipidemia as a co-morbidity. Despite the well-documented cholesterol-lowering properties of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) in treating hypercholesterolemia, the beneficial effects of statins consumption in T2DM treatment are confusing. In the current study, we examined the effects of the simvastatin (10 nM; 24 hr) on β-cell function leading to insulin secretory response to glucose. We hypothesized that statins restore the blunted glucose (5 mM and 15 mM)-induced insulin secretion (plus the reduced insulin content) of isolated pancreatic islets of obese/diabetic (db⁺/db⁺) mice. / In the present study, genetically diabetic C57BL/KSJ +db/+db (db⁺/db⁺) mice at 24 week of age and their age-matched non-diabetic littermates C57BL/KSJ +m/+m (db⁺/m⁺) were used. Our results clearly showed that the suppressed glucose (5 mM and 15 mM)-induced insulin release (plus insulin content) and glucose (15 mM)-induced [Ca²⁺]i changes of isolated pancreatic islets of obese/diabetic (db⁺/db⁺) was restored after simvastatin (10 nM; 24 hr) treatment. / The biochemical existence of CaR in pancreatic islets of lean/non-diabetic (db⁺/m⁺) and obese/diabetic (db⁺/db⁺) mice was confirmed. The suppressed/down-regulated expression of CaR was associated to the blunted insulin secretion in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice, and it was markedly up-regulated by simvastatin (10 nM; 24 hr). The involvement of CaR-mediated PLA₂ signaling in simvastatin (10 nM; 24 hr)-induced restoration of glucose (15 mM)-induced insulin secretion in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice was investigated. Our results also showed that the increased density of plasma membrane actin cytoskeleton of obese/diabetic (db⁺/db⁺) mice was significantly decreased by simvastatin (10 nM; 24 hr) treatment. The simvastatin-induced depolymerization and remodeling of actin cytoskeleton may improve insulin secretion capability in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice. / The glucose (15 mM)-induced intracellular ROS level was significantly higher in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice. The elevated ROS level was partially diminished by simvastatin (10 nM; 24 hr) treatment. The protein expressions of PERK and eIF2α (ER stress proteins) were lower in pancreatic islet cells isolated from obese/diabetic (db⁺/db⁺) mice, suggesting that abnormal expresstion/activity of PERK and eIF2α would be coupled to the ER-stress mediated failure of pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice. As simvastatin (10 nM; 24 hr) up-regulated the protein expression of these proteins, this drug exerted protective effect on pancreatic β-cells against ER stress and restored the blunted glucose (15 mM)-induced insulin secretion (plus the reduced insulin content) in obese/diabetic (db⁺/db⁺) mice. / In conclusion, our results demonstrate, for the first time, that simvasatatin (a HMG-CoA reductase inhibitor) (10 nM; 24 hr) provides beneficial effects (i.e. restoration of the blunted glucose-induced insulin release plus the reduced insulin content) in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice via the allosteric modification/up-regulation of extracellular calcium-sensing receptor through the PLA₂ signaling pathway, and provides protective/antioxidant effects against oxidative stress caused by chronic hyperglycemia in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice by up-regulating protein expression of the suppressed ER stress sensors and antioxidant enzyme. / 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. / Au, Lai Shan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 458-532). / Abstracts also in Chinese.

Biocatalysis represents an attractive route to the production of various compounds which are difficult or impossible to synthesise and isolate using traditional chemical synthesis. In particular, the production of chiral molecules is a function ideally suited to biocatalysis, due to the natural stereospecificity of enzymes. The synthesis of such chiral molecules is essential in the production of pharmaceuticals, additives for the food and drinks industry and the creation of specialist polymers. CYP105AS1, isolated from Amycolatopsis orientalis, is a cytochrome P450 enzyme which produces the inactive 6-epi-pravastatin of the blockbuster anti-cholesterol drug pravastatin. Previous directed evolution efforts have engineered this enzyme to produce a five-point mutant, known as P450prava, which partially reversed the stereospecificity of the enzyme to produce a majority pravastatin product mixture. This thesis details work to use structure-led engineering approaches to redesign the active site of P450prava to introduce stringent stereospecificity. A combinatorial approach of manual and computational rational design was pursued, leading to the creation of a novel T95F/V180M double mutant of P450prava. This double mutant was found to have successfully eliminated the unwanted 6-epi-pravastatin enantiomer from the product mix, leaving a pure pravastatin product. P450prava was also shown to bind and hydroxylate other statin substrate molecules, demonstrating its versatility in the production of drug metabolites and other high-value oxyfunctionalised molecules. This property, along with its proven tolerance of significant active site engineering efforts, demonstrates the viability of the P450prava as a platform for the creation of novel biocatalysts for the production of various hydroxylated products from diverse substrate molecules.