published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

 Retinopathy of prematurity (ROP) commonly occurs in premature babies, with the first phase of vessel cessation followed by a second phase of vessel proliferation. In addition to vascular changes, neuronal abnormalities have also been observed. However, evidence for morphological changes of retinal neurons at the cellular level is lacking. Oxidative stress has been highly indicated in the pathogenesis of ROP. Increased oxidative stress level was demonstrated in preterm babies expecially in those with ROP. The activity of aldose reductase (AR), the first enzyme in the polyol pathway, has been found to contribute to oxidative stress. Therefore, the role of AR in ROP was examined using a mouse model of oxygen-induced retinopathy (OIR), which was a well-established model to mimic human ROP. Studies in examining the effects of AR on retinal vasculature showed that genetic deletion or pharmacological inhibition of AR reduced vaso-obliteration and neovascularization, possibly through regulating VEGF-induced pathway. In addition, morphological changes of various retinal neurons at different time points in the mouse model of OIR were also demonstrated. The degree of effects from hyperoxic and hypoxic exposure appeared to depend on the different stages of maturation of various retinal neurons. AR deficiency showed protective effects on retinal neurons including horizontal cells, rod bipolar cells and amacrine cells, possibly through attenuating the damage on blood vessels as well as facilitating blood vessel re-growth in the avascular area which provide more nutrients and supply to the retinal neurons. To elucidate the protective role of AR deficiency in ROP, the changes in oxidative stress and oxygen-dependent gene expression including HIF-1α and iNOS were investigated. AR deficiency attenuated oxidative stress induction to protect the neonatal retina. In addition, AR deficiency also showed attenuated HIF-1α expression and enhanced iNOS expression. This served to strictly control the HIF-1α level which in turn can tightly regulate VEGF induction in the mouse retinae after OIR. In order to further elucidate the role of AR in the pathogenesis of ROP, effects of AR deficiency on glial cells and microglia were investigated. AR deficiency reduced retinal astrocytic activation in hyperoxia and induced early M?ller cell gliosis in hypoxia. In addition, AR deficiency enhanced the specific function of microglia in different areas with facilitation of revascularization in avascular area and promotion of tufts regression in neovascular area. Moreover, AR deficiency also reduced the activation of a key inflammatory mediator NF-κB, which was considered to contribute to neovascularization. Therefore, AR deficiency demonstrated regulatory roles in reponses of glial cells, microglia and inflammation, contributing to the protective effects on neonatal retina in the mouse model of OIR. Taken together, AR deficiency reduced the vascular and neuronal changes possibly through attenuating oxidative stress and glial responses as well as modulating inflammatory responses, indicating a beneficial role of AR inhibition in OIR. These findings highly suggest the therapeutic potential of AR inhibition in the treatment of ROP. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 89-111) Also available in print.

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published_or_final_version / Physiology / Master / Master of Philosophy

Abiotic stresses are responsible for limiting crop production worldwide. Among diverse abiotic stresses, drought and salinity are the most challenging. Plants under these conditions have diverse strategies for tolerating stress. Osmotic adjustment and osmoprotection occur in plants during salinity and drought stress through accumulation of compatible solutes to a high level without interfering with cellular metabolism. Polyols (sugar alcohols) including sorbitol and ribitol are one such class of compatible solutes. Using plants of wild-type (WT) and three genetically-modified lines of tomato (Solanum lycopersicum cv. ‘Ailsa Craig’), an empty vector line ‘TR22’, and 2 sdh anti-sense lines ‘TR45’, and ‘TR49’ designed to severely limit sorbitol metabolism, the objective of this work was to characterize the sorbitol cycle in tomato in response to abiotic stresses. Sorbitol and ribitol content, as well as the enzymatic activities, protein accumulation, and gene expression patterns of the key sorbitol cycle enzymes ALDOSE-6-PHOSPHATE REDUCTASE (A6PR), ALDOSE REDUCTASE (AR), and SORBITOL DEHYDROGENASE (SDH), were measured in mature leaves in response to drought stress by withholding water and by using polyethylene glycol as a root incubation solution to mimic drought stress, to salt stress by incubating roots in NaCl solution, and to incubation of roots in 100 mM sorbitol and ribitol. A6PR, not previously reported for tomato, and AR both exhibited increased activity correlated to sorbitol accumulation during the drought osmotic, and salt stresses, with SDH also increasing in WT and TR22 to metabolize sorbitol. The level of sorbitol accumulation was considerably lower than that of the common sugars glucose and fructose so was not enough to have a significant impact on tissue osmotic potential but could provide other important osmoprotective effects. Use of the sdh antisense lines indicated that SDH has the key role in sorbitol metabolism in tomato as well as a likely role in ribitol metabolism. Like sorbitol, ribitol also accumulated significantly more in the antisense lines during the stresses. Expression and/or activity of A6PR, AR, and SDH were also induced by the polyols, although it is not clear if the induction was due to a polyol signal, the osmotic effect of the incubation solution, or both. In addition, a unique post-abiotic stress phenotype was observed in the sdh anti-sense lines. After both drought and salt stresses and during a recovery phase after re-watering, the antisense lines failed to recover. This may have been due to their accumulation of ribitol. The sdh anti-sense lines were uniquely sensitive to ribitol but not sorbitol, with an apparent foliar and seed germination toxicity to ribitol. The determination that sorbitol, and perhaps ribitol as well, plays a role in abiotic responses in tomato provides a cornerstone for future studies examining how they impact tomato tolerance to abiotic stresses, and if their alteration could improve stress tolerance.

In the polyol pathway, Aldose reductase (AR) is an important enzyme in reduction of aldehydes and aldosugars to their suitable alcohols. AR, using NADPH as a coenzyme, has a molecular weight of 37 000 dalton. AR in its activated form, known to increase the sorbitol accumulation in lens, is responsible for the cataract formation in diabetis diseases. Therefore, the inhibition of aldose reductase is important to prevent the incedence of cataract formation in diabetus mellitus. In the treatment of diabetis dependent cataract, chemically synthetized drugs were sometimes less than beneficial due to the severe side effects they cause. Recently a huge amount of study has been intensified on developing new drugs from natural compounds and even by utilizing plant extracts for their easily metabolizing polyphenolic compounds. In this study, BLAR, source of enzyme, was obtained as crude via differential centrifugation and ammonium sulfate precipitation. The enzyme assay conditions were optimized for the protein, substrate, coenzyme, and salt concentrations, also for the effects of pH and temperature. Ocimum basilicum, Lavandula stoechas, Melissa officinalis, Glycyrrhiza glabra L. and Tilia tomentosa were selected as commonly used alternative medicine plants. Plant extracts were prepared in ethanol and ethyl acetate and their inhibitory effects were tested on crude bovin lens aldose reductase enzyme. Fifty percent inhibitory concentrations (IC50) were found between values of 25.53 &micro / g/mL and 54.15 &micro / g/mL for ethanol extracts and between 41.55 &micro / g/mL and 82.96 &micro / g/mL for the ethyl acetate extracts of selected plants. In addition, the plant extracts were also characterized for their antioxidant activities by of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method and test of total phenolic content (TPC) .

Aldose reductase, E.C.1.1.1.21, catalyzes the reduction of different types of aldehydes to their corresponding alcohols, and especially reduces various aldo-sugars using NADPH as the coenzyme. Under hyperglycemic conditions aldose reductase is involved in the development of diabetic complications. As a result, interest has been placed over the years on the development of potent aldose reductase inhibitors for possible use in the therapy of these severe diabetic complications. In this study, aldose reductase was isolated from bovine lens by differential centrifugation and ammonium sulfate precipitation. The conditions for the enzyme assay / such as substrate (DL-Glyceraldehyde) and coenzyme (NADPH) concentration, protein amount, effect of sulfate ions, temperature and pH on the enzyme activity were optimized. The inhibitory effects of Punica granatum, Spinacia olaeracea, Allium cepa Allium porrum, Malus flouribunda, Malus domestica extracts were tested on crude bovine lens aldose reductase. Four different types of organic fractions from each crude plant extract were obtained by solvent fractionation. The inhibitory activity of these organic fractions was calculated considering the aldose reductase activity without extracts as 100 %. All six plants were found to inhibit aldose reductase activity to different extent. Among these fractions obtained as / petroleum ether, diethyl ether, ethyl acetate, and n-butanol. Highest inhibitory activity was found for the ethyl acetate fraction. The IC50 values of ethyl acetate fractions of all these plants was calculated as, 25.46 &micro / g/ml, 20.5 &micro / g/ml, 18.5 &micro / g/ml, 12.32 &micro / g/ml, 6.45 &micro / g/ml, 5.4 &micro / g/ml, for Allium porrum, Malus domestica, Spinacia olaeracea, Malus floribunda Allium cepa, Punica granatum respectively.

Le développement du tissu adipeux blanc est finement régulé par des facteurs pro- et anti- adipogéniques. Au cours de l’obésité, son expansion conduit à de nombreuses complications métaboliques. A ce jour, peu de données sont disponibles sur les facteurs qui contrôlent négativement son développement. Dans ce contexte, le laboratoire a dirigé ses recherches sur le rôle de l’aldose réductase murine Akr1b7 dans ce tissu. Akr1b7 est exprimée dans la fraction stromale vasculaire du tissu adipeux blanc et possède un effet anti-adipogénique sur les préadipocytes en culture. La réalisation et l’analyse de souris invalidées pour le gène Akr1b7 nous a permis de démontrer que la perte de Akr1b7 entraîne une expansion de la masse adipeuse par une hypertrophie et une hyperplasie des adipocytes associées à une insulino-résistance. Les souris Akr1b7-/- ne sont pas hyperphagiques mais présentent un métabolisme basal réduit. Akr1b7 qui possède une activité prostaglandine synthase, régule le développement excessif du tissu adipeux par deux mécanismes dépendant de la PGF2α à savoir, l’inhibition de l’adipogenèse et de la lipogenèse. D’autre part, nous avons développé un modèle de souris transgéniques sur-exprimant l’aldose réductase humaine AKR1B1 dans le tissu adipeux. Contre toute attente et à l’inverse de Akr1b7, ce modèle montre un effet pro-adipogénique deAKR1B1. Ces données in vivo révèlent des activités inédites et opposées entre différentes isoformes d’aldose réductase et ouvrent de nouvelles pistes pour appréhender les mécanismes contrôlant l’homéostasie adipeuse et ses dérèglements. / White adipose tissue development is tightly regulated by pro-and anti-adipogenic factors. In obesity, its increased development leads to many metabolic complications. To date, little is known about the factors that control negatively its growth. In this context, the laboratory has focused researches on the murine aldose reductase Akr1b7 role in white adipose tissue. Akr1b7 is expressed in stromal vascular fraction of white adipose tissue and exhibits an anti-adipogenic action on a preadipocyte cell line. Generation and study of Akr1b7-/- knockout mice allows us to demonstrate that lack of Akr1b7 leads to adipose tissue expansion due to hypertrophy and hyperplasia of adipose cells associated to insulin resistance. Akr1b7-/- mice are not hyperphagic but show reduced basal metabolic rate. This phenotype confirms Akr1b7 involvement in adipose tissue physiology. Akr1b7 regulates development of adipose tissue by a PGF2α-dependent inhibition of both adipogenesis and lipogenesis. On the other hand, we have developed a transgenic murine model over-expressing the human aldose reductase AKR1B1 in adipose tissue. Against all odds and contrary to Akr1b7, this model shows a pro-adipogenic effect of AKR1B1. These in vivo data reveal new and opposed activities of different aldose reductase isoforms and open new avenues to understand the mecanisms regulating fat homeostasis and its disturbances.