Liu Ming Ju. / "June 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

A β-galactosidase (E.C 3.2.1.23) é uma das enzimas mais empregadas na indústria de alimentos sendo utilizada na hidrólise da lactose. Neste trabalho foram utilizadas duas metodologias para imobilização desta enzima. Na primeira delas foi empregado como suporte um material híbrido à base de sílica que possui um grupo orgânico catiônico covalentemente ligado. A adsorção da enzima a este material apresentou eficiência que variou de 74 a 53% com o aumento da quantidade de enzima aplicada ao suporte. A baixa estabilidade térmica da enzima imobilizada obtida e as prováveis fracas interações envolvidas na sua adsorção a este suporte podem explicar o decréscimo de atividade observada durante as sucessivas bateladas de hidrólise da lactose. Na primeira batelada o grau de hidrólise foi de 90,9% e no final da última batelada (4ª), a enzima foi capaz de converter apenas 13% do substrato. A segunda metodologia utilizada foi imobilização covalente da enzima em um filme de celulose/líquido iônico modificado com uma poliamina e ativado com glutaraldeído. A presença da poliamina foi confirmada por análises de infravermelho. Após a imobilização, a enzima reteve 60% de sua atividade inicial. Bons resultados de hidrólise da lactose em batelada foram obtidos tanto a 7ºC como a 35ºC e foi possível reutilizar a enzima imobilizada por 16 ciclos consecutivos, a 7ºC, sem mudanças significativas na atividade enzimática. O valor de Km para a enzima imobilizada no material híbrido à base de sílica foi de 9,17 mM e para a enzima imobilizada nos filmes de celulose foi de 11,22 mM, ambos apresentaram um acréscimo quando comparados ao Km enzima livre (1,25 mM), devido à dificuldade de acesso do substrato ao sítio ativo da enzima. Não houve mudança no pH e temperatura ótimos da enzima imobilizada em relação à enzima livre em nenhum dos métodos testados. / β-galactosidase (E.C 3.2.1.23) is the most widely used enzymes in the food industry and its employed in the lactose hydrolysis process. In this study, two methodologies were used to test their immobilization. In the first, the enzyme was immobilized by adsorption in one silica based hybrid material that contains a cationic organic group covalently linked. The efficiency of immobilization showed a decrease of 74 to 53% by increasing the protein load applied to the support. The low thermo stability of the immobilized enzyme and the probable weak interactions involved in their adsorption, could explain the decrease in enzyme activity observed in the successive batch hydrolysis of lactose. In the first run, the degree of lactose hydrolysis was 90.9% and, at the end of the last run (4th), the enzyme was able to convert only 13% of the substrate. The second methodology used was the covalent immobilization of the enzyme on a cellulose/ionic liquid film, modified with a polyamine and activated using glutaraldehyde. The presence of a polyamine was confirmed by infrared analysis. After immobilization, the enzyme retained 60% of its initial activity. Highly efficient lactose conversion was achieved in a batch process at 7ºC and 35ºC and was possible to reuse the immobilized enzyme in 16 repeated cycles, at 7ºC, without any drastic decrease in enzyme activity. Km value for the immobilized enzyme in silica based hybrid material was 9.17 mM and for the enzyme immobilized in the film of cellulose/ionic liquid was 11.22 mM, both showing an increase compared with the Km value for free enzyme (1.25 mM), due to the difficulty of access of the substrate to the active sites of the enzyme. The immobilized enzyme did not show any changes in the optimal pH and temperature when compared to the free enzyme in both methods tested.

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Agradecemos a compreensão. on 2017-03-22T14:32:46Z (GMT) / Submitted by JOAO PEDRO CORREA DE CAMARGO null (joaoquimica1991@hotmail.com) on 2017-03-22T15:13:54Z No. of bitstreams: 1 Autoarquivamento da dissertação corrigido .pdf: 2162415 bytes, checksum: 64ada6268776f57730601f9cd0ffc35b (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-03-24T16:41:36Z (GMT) No. of bitstreams: 1 camargo_jpc_me_bot.pdf: 2162415 bytes, checksum: 64ada6268776f57730601f9cd0ffc35b (MD5) / Made available in DSpace on 2017-03-24T16:41:36Z (GMT). No. of bitstreams: 1 camargo_jpc_me_bot.pdf: 2162415 bytes, checksum: 64ada6268776f57730601f9cd0ffc35b (MD5) Previous issue date: 2017-02-09 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Um novo biossensor foi desenvolvido baseado na simples eletrocodeposição do óxido de grafeno reduzido (rGO), polipirrol (PPy) e da enzima acetilcolinesterase (AChE) na superfície do eletrodo de platina (Pt). No intervalo de potencial -0,2 a +0,5 V vs. Ag/AgCl/KCl (3,0 mol L-1), utilizando voltametria de pulso diferencial (DPV), observou-se um processo em +0,1 V e este corresponde a dimerização dos produtos de oxidação eletroquímica da tiolcolina, formando ditio-bis-colina. O biossensor desenvolvido foi avaliado utilizando DPV na análise do pesticida carbaril, o qual inibe a ação da enzima AChE. Os melhores resultados obtidos foram com as seguintes condições otimizadas: 75 mV amplitude de pulso, incremento de potencial de 4 mV, e uma solução tampão fosfato (PBS) 0,2 mol L-1 pH 6,0. Usando tais parâmetros observou-se uma resposta linear para o carbaril no intervalo de 0,1 a 0,5 mol L-1, com um limite de detecção de 11,6 nmolL-1 (2,3 µg/kg), que é um limite adequado para determinar carbaril nas culturas em que este pesticida é aplicado considerando o limite máximo de resíduo permitido pelas legislações brasileiras. O biossensor proposto, Pt/rGO/PPy/AChE, foi aplicado com sucesso na determinação de carbaril em amostras de tomate e repolho. / A new biosensor was developed by a simple electrocodeposition of reduced graphene oxide (rGO), polypyrrole (PPy) and the enzyme acetylcholinesterase (AChE) on surface of platinum (Pt) electrode. In potential range of -0.2 to +0.5 V vs. Ag/AgCl/KCl (3.0 mol L-1), using differential pulse voltammetry (DPV), it was observed a process in + 0.1 V and this corresponds to the dimerization of electrochemical oxidation products of thiocholine, resulting in ditio-bis-choline. The biosensor developed was evaluated using DPV in the analysis of carbaryl, which inhibits the AChE enzyme action. The best results achieved were with the followings optimized conditions: 75 mV pulse amplitude, step potential of 4 mV, and a phosphate buffer solution (PBS) 0.2 mol L-1 and pH 6.0. Using these parameters was observed a linear response to carbaryl in a range of 0.1 to 0.5 µmol L-1, with a detection limit of 11.6 nmol L-1 (2.3 µg/kg), which is an appropriate limit for determination of carbaryl in the cultures which these pesticide is applied, considering the maximum reside limit allowed by Brazilian legislation. The biosensor proposed, Pt/rGO/PPy/AChE, was applied successfully in the determination of carbaryl in samples of cabbage and tomato. / FAPESP: 2015/02136-2

El presente estudio tuvo como objetivo determinar la capacidad de remoción de nitratos (N-NO3⁻) y fosfatos (PO₄³¯) en Aguas Residuales Municipales (ARM) por microalgas libres e inmovilizadas. Se utilizaron cepas provenientes de los afluentes de la Planta de Recuperación de las Aguas del Río Surco (Lima, Perú) y se evaluó su capacidad de remoción de N-NO3⁻ y PO₄³¯ durante 10 días, a nivel laboratorio, en ARM con tratamiento primario, tanto de forma libre como inmovilizadas en discos de alginato de sodio al 4%. Los cepas obtenidas se identificaron como Chlorella sp y Chlamydomonas sp. Ambas tuvieron un buen crecimiento en ARM, especialmente Chlamydomonas sp, la cual reportó los mayores valores en los parámetros cinéticos de crecimiento. El cultivo de Chlorella sp. libre fue el que registró uno de los valores más altos de porcentaje (71.25%) y tasa de remoción (0.43 mg/l/día) de N-NO3⁻, y los máximos valores de dichos parámetros para PO43- (83.69%; 0.09 mg/l/día), así como para el índice de eficacia (EI) de ambos nutrientes, comparado con los de Chlamydomonas sp. Los cultivos inmovilizados de ambas especies reportaron valores altos de remoción, entre 56% a 67% para N-NO3⁻ y 78% a 81% para PO43-, este último fue removido en 24 horas en la mayoría de los cultivos. La inmovilización fue el principal factor que afectó la capacidad de remoción de nutrientes. Ambas cepas mostraron ser eficientes en la remoción de nutrientes en ARM, especialmente de PO43-, con valores cercanos a los máximos reportados para ambas especies en estudios previos.The aim of this investigation was to determinate the nitrate (N-NO3⁻) and phosphate (PO43-) removal capacity of free and immobilized microalgae in Municipal Wastewater (MW). Strains were isolated from Planta de Recuperación de las Aguas del Río Surco (Lima, Perú) influent. Their N-NO3⁻ and PO43- removal capacity were evaluated during 10 days in MW with primary treatment in batch cultures with free and immobilized cells in 4% sodium alginate discs. Strains were identified as Chlorella sp and Chlamydomonas sp. Both showed good growth in MW, especially Chlamydomonas sp., which reported the highest value of kinetic growth parameters. Free Chlorella sp. culture reported one of the highest values of N-NO3- removal percentage (71.25%) and rate (0.43 mg/l/día), and the maximum values of PO43- removal parameters (83.69%; 0.09 mg/l/día) and efficacy index (EI) for both nutrients compared with Chlamydomonas sp. Immobilized cultures reported high values of nutrient removal, between 56% to 67% for N-NO3⁻ and 78% to 81% for PO43-. In most microalgae cultures, high percentages of PO43- (76-80%) were removed in 24 hours. Method of culture (free or immobilized) was the main factor affecting the nutrient removal capacity. Strains were efficient in nutrient removal in MW, especially for PO43-, with values close to the maximum ones reported for both species in previous studies.

A quitosana é um polisacarídeo obtido pela desacetilação das moléculas de quitina, principal constituinte de alguns fungos e do exoesqueleto de crustáceos e insetos. Os grupos amino presentes na quitosana conferem-lhe importantes propriedades biológicas, como a biodegradação, biocompatibilidade, atividade/efeitos imunológicos e atividade antibacteriana. A desacetilação da quitina é um processo cuja conversão é agressiva, já que exige o ataque da quitina em solução de álcalis em alta concentração e à quente, com duração de 6 a 8 horas. Neste trabalho, carapaças de siri da espécie Charybdis hellerii foi fragmentada e pré-tratada para a obtenção da quitosana e cada etapa, desde o pré-tratamento do material in natura à sua conversão em quitosana, foi investigada detalhadamente. Observou-se que dose e taxa de dose não influenciaram no pré-tratamento ou na etapa de desacetilação da quitina; na dose de 20 kGy (gama ou feixe de elétrons), o processo de conversão teve duração de 60 minutos. A quitosana obtida teve baixa massa molar e grau de desacetilação comparável á quitosana padrão (SA), dependendo das condições de irradiação. Além disso, apresentou inibição da atividade bacteriana tanto livre como imobilizada em substratos poliméricos de polipropileno e de polietileno processados também por radiação ionizante. / Chitosan is a polysaccharide obtained from chitins molecule deacetylation, which is the main composition of certain fungi species and crustaceans and insects exoskeleton. The amino groups present in chitosan give it important biological properties such as biodegradability and biocompatibility, activity/immunological effects and antibacterial healing. The deacetylation of chitin is an aggressive process, which reaction processes in 6 to 8 hours under hot concentrated alkali solution. In this work, Charybdis hellerii crab shells was fragmented and pre-treated for chitosan obtention and each conversion step, from in natura material pre-treatment to final chitosan, were investigated in deteails. It was observed dose and dose rate have not influence neither pre-treatment nor chitin deacetylation steps; at 20 kGy (from gamma or electron beam sources), the conversion process was performed in 60 minutes. The obtained chitosan presented low weight and deacetylation degree compared to standard chitosan, considering specific irradiation conditions. Also, obtained chitosan presented bacterial inactivity as a free compoud as immobilized onto polymeric substrates such polypropylene and polyethylene, also processed by ionizing radiation.

L'un des avantages majeurs des réactions de bioconversion résulte du fait que le milieu réactionnel des biocatalyseurs est l'eau. Ce dit avantage constitue l'une des principales limitations de ces réactions de bioconversion lorsqu'il s'agit de substances lipophiles non solubles dans l'eau connue les stérols. L'efficacité d'un procédé de bioconversion de substances lipophiles solides dépend essentiellement du contact et de 1'interaction entre le biocatalyseur et ce substrat lipophile. Les solutions proposées dans la littérature font appel à des solvants et des produits chimiques de natures souvent toxiques, inflammables et explosives. Ces solutions décrites font perdre à la bioconversion son caractère de biotechnologie blanche. Dans ce travail, nous avons montré qu'en plus de ses avantages connus, l'immobilisation passive de biocatalyseurs au sein d'un support poreux peut favoriser l'interaction cellule-substrat lipophile et augmenter le taux de bioconversion sans utiliser de solvants et de produits chimiques. La réaction modèle étudiée est le clivage de la chaine latérale des stérols par des mycobactéries en vue de l'obtention des androsténones précurseurs naturels des stéroïdes, molécules à forte valeur biologique ajoutée. Le support d'immobilisation le plus performant a été le fruit sec de Luffa cylindrica. Par rapport aux supports organiques classiques tels que les gels de polyacrylamide, les mousses de polyuréthane, la silicone et les plastiques, le fruit sec de Luffa cylindrica présente les avantages suivants : (i) c'est un produit naturel, (ii) biodégradable, (iii) peu onéreux, (iv) non toxique pour les microorganismes, (v) stable du point de vue mécanique et thermique, (vi) et réutilisable. / One of the major advantages of using biocatalysts in organic synthesis is that water constitutes the reaction medium. However, water becomes a serious problem when bioconversion deals with lipophilic compounds, in particular those poorly soluble in water such as sterols. Bioconversion of lipophilic compounds depends on the close contact between the hydrophobic substrate and the biocatalyst. Increasing this contact requires usually the use of huge amounts of chemical which are often toxic, flammable and explosive. In this work, we showed that passive cell immobilization in porous materials can increase the contact between microorganisms and lipophilic substrates without using chemicals. The side chain cleavage of sterols was studied as a model multistep microbial bioconversion of lipophilic compounds. This reaction allows the production of androstenones which are the natural precursors of steroids. Among the studied immobilization carriers, the dried fruit of Luffa cylindrica was the most efficient Compared to other organic support carrier such as alginate beads, polyurethane foams, silicones and plastics, the dried fruit of Luffa cylindrica is advantageous since it is natural, renewable, biodegradable, cheap, mechanically strong, free of toxicity and it doesn't need a chemical pretreatment.

La bio-transformation naturelle des médicaments peut produire des métabolites toxiques; l’identification de ces métabolites est essentielle dans la stratégie de choix de molécules thérapeutiques. En appliquant les technologies de fermentation en bioréacteur des cellules hétérologues (souches d’E. coli recombinantes exprimant une iso-enzyme de cytochrome P450 humain avec la réductase humain), la bioconversion du substrat (principe actif) en ses métabolites de dégradation, a été réalisée à grande échelle (g-g). Notre choix s’est porté sur le complexe hCYP3A4/HR fonctionnel produit par un hôte E. coli. Les cellules intactes ou les membranes cellulaires peuvent être exploitées comme biocatalyseur dans un système bioréacteur. Cependant, la faible solubilité des principes actifs dans des milieux de bioconversion aqueuse limitent le rendement. Un bioréacteur biphasique a été étudié. En solution, plusieurs combinaisons eau/solvants organiques conciliant la viabilité des cellules, la solubilité des principes actifs et produits de réaction et la catalyse des complexes enzymatiques ont conduit à l’établissement d’un mélange approprié. Cependant, ces combinaisons présentent toujours une inhibition importante du pouvoir catalytique des complexes enzymatiques. Pour minimiser un effet dénaturant possible des solvants sur le système enzymatique, ce dernier a été maintenu dans un environnement aqueux en immobilisant les cellules et/ou les membranes cellulaires dans une matrice hydrophile. L’alginate de calcium apparaît être une matrice d’immobilisation idéale pour les membranes assurant la fonctionnalité du complexe CYP/HR et permettant en outre un stockage à long terme des préparations. Par contre, l’immobilisation des cellules dans diverses matrices, si elle permet une viabilité et une conservation à long terme des souches recombinantes, ne permet aucune expression de l’activité enzymatique présente dans les cellules. La combinaison d’une localisation du complexe hCYP/HR fonctionnel dans la membrane interne et d’une perméabilité réduite des cellules d’E. coli (immobilisées) en est une explication possible mais non-démontrée. Entre-temps, cette technologie de bioréacteur homogène biphasique ou par immobilisation des membranes cellulaires a été utilisée plusieurs reprises pour produire des métabolites humains à partir de divers principes actifs. Ces métabolites ont été purifiés avec succès, démontrant que cette approche technologique est compétitive comparée aux procédures conventionnelles. Néanmoins, de nouvelles pistes de recherche seraient extrêmement intéressantes. La localisation des complexes enzymatiques recombinants en surface des cellules permettrait de concilier les propriétés hydrophobes des principes actifs et l’environnement hydrophile nécessaire aux enzymes. D’autre part une investigation de complexes enzymatiques résistant aux solvants pourrait remplacer avantageusement l’immobilisation.

Mobility of the horse to initiate motion from the standing position is examined in this thesis. In particular, the thesis focuses on the study of the mobility of a horse with fixed hooves to the ground, and on how its musculoskeletal system is used to free the legs from restraints. Possible leg patterns to initiate motions are investigated. The breaking forces generated at front and hind hooves during static-pulling and dynamic jerking are evaluated. Design of the restraint system that uses ropes to immobilize certain joints in order to prevent the horse from generating these forces is the main objective of this thesis. Such a system could be applied as an alternative to rather massive mechanical devices, the main purpose of which is to block the breaking forces (which are quite large when fully developed).<p> Analysis of the mobility of the horse is based on the mechanics of a skeletal linkage system driven by muscle forces. Only major muscles involved in fighting the restraints are included in the analysis. The force generation capability of a muscle is determined by physiological cross sectional area (PCSA) of the muscle. Possible leg patterns are predicted with the kinematics analysis considering range of motion at each joint in the legs. Corresponding breaking forces generated in each pattern is evaluated with the kinetics analysis. Relationship between the characteristic parameter of the pattern and the breaking force at hoof are established. <p>The horse's computer model is used to justify the analytical result. Fighting mechanisms of the horse are simulated in the dynamic simulation software package. Patterns and the breaking forces developed by the horse model simulation agree well with the analytical results. To the authors best knowledge, this is the first time a computer model is used in analyzing the method of restraining an animal. <p>The mobility of the animal with hoof restraints and methods to remove mobility were further confirmed with a preliminary animal restraint test conducted on a sheep. The sheep was chosen because the leg patterns to initiate motion on a horse are similar to that of sheep, but the sheep is more convenient to handle. The experiment showed that the mobility of the sheep could be removed completely by restraining its hooves, lower legs, and head with easily attached ropes.

It is well established that bone is capable of adapting to changes in its environment; however, little is known regarding how environmental stimuli, specifically loading, are associated with the internal 3D microarchitecture of cortical bone. The aim of this thesis was to develop a small animal model that can be used to experimentally test hypotheses regarding bone adaptation. High resolution micro-CT was validated and employed as a novel method for the visualization and quantification of rat cortical bone microarchitecture in 3D. The use of this imaging method allowed for the measurement of primary vascular canal orientation in 3D, which had never been achieved before. Using this measure along with an immobilization model for unloading allowed me to test how loading is associated with the orientation of these vascular canals. Normally ambulating rat bones (from 10 female rats) had a canal structure that was 9.9° more longitudinal than their immobilized counterparts. This finding that loading has an effect on primary canal orientation brought to light the need to induce remodeling and therefore, secondary vascular canals, in the rat to increase its novelty as a model for looking at bone adaptation. Remodeling was induced by increasing the calcium demands of female rats, either through a calcium restricted diet (n=2) or pregnancy and lactation coupled with a calcium restricted diet (n=2). Mean cortical thickness for the calcium restricted rats and the pregnant and lactating rats that were on a calcium restricted diet were 622 µm and 419 µm, respectively. The mean BMU count for calcium restricted rats seemed to be higher than that of the pregnant and lactating rats; however, the calcium restricted rats seemed to have a lower BMU density. Once this full-scale study is executed the rat will provide a more representative model for studying human bone adaptation.

It is well established that bone is capable of adapting to changes in its environment; however, little is known regarding how environmental stimuli, specifically loading, are associated with the internal 3D microarchitecture of cortical bone. The aim of this thesis was to develop a small animal model that can be used to experimentally test hypotheses regarding bone adaptation. High resolution micro-CT was validated and employed as a novel method for the visualization and quantification of rat cortical bone microarchitecture in 3D. The use of this imaging method allowed for the measurement of primary vascular canal orientation in 3D, which had never been achieved before. Using this measure along with an immobilization model for unloading allowed me to test how loading is associated with the orientation of these vascular canals. Normally ambulating rat bones (from 10 female rats) had a canal structure that was 9.9° more longitudinal than their immobilized counterparts. This finding that loading has an effect on primary canal orientation brought to light the need to induce remodeling and therefore, secondary vascular canals, in the rat to increase its novelty as a model for looking at bone adaptation. Remodeling was induced by increasing the calcium demands of female rats, either through a calcium restricted diet (n=2) or pregnancy and lactation coupled with a calcium restricted diet (n=2). Mean cortical thickness for the calcium restricted rats and the pregnant and lactating rats that were on a calcium restricted diet were 622 µm and 419 µm, respectively. The mean BMU count for calcium restricted rats seemed to be higher than that of the pregnant and lactating rats; however, the calcium restricted rats seemed to have a lower BMU density. Once this full-scale study is executed the rat will provide a more representative model for studying human bone adaptation.