Cette thèse a pour objectif de proposer de nouvelles méthodes permettant l'étude de l'évolution du métabolisme. Pour cela, nous avons choisi de nous pencher sur le problème de comparaison du métabolisme de centaines de micro-organismes.Afin de comparer le métabolisme de différentes espèces, il faut dans un premier temps connaître le métabolisme de chacune de ces espèces.Les protéomes des micro-organismes avec lesquels nous souhaitons travailler proviennent de différentes bases de données et ont été séquencés et annotés par différentes équipes, via différentes méthodes. L'annotation fonctionnelle peut donc être de qualité hétérogène. C'est pourquoi il est nécessaire d'effectuer une ré-annotation fonctionnelle standardisée des protéomes des organismes que nous souhaitons comparer.L'annotation de séquences protéiques peut être réalisée par le transfert d'annotations entre séquences orthologues. Il existe plus de 39 bases de données répertoriant des orthologues prédits par différentes méthodes. Il est connu que ces méthodes mènent à des prédictions en partie différentes. Afin de tenir compte des prédictions actuelles tout en ajoutant de l'information pertinente, nous avons développé la méta-approche MARIO. Celle-ci combine les intersections des résultats de plusieurs méthodes de détections de groupes d'orthologues et les enrichit grâce à l'utilisation de profils HMM. Nous montrons que notre méta-approche permet de prédire un plus grand nombre d'orthologues tout en améliorant la similarité de fonction des paires d'orthologues prédites. Cela nous a permis de prédire le répertoire enzymatique de 178 protéomes de micro-organismes (dont 174 champignons).Dans un second temps, nous analysons ces répertoires enzymatiques afin d'en apprendre plus sur l'évolution du métabolisme. Dans ce but, nous cherchons des combinaisons de présence/absence d'activités enzymatiques permettant de caractériser un groupe taxonomique donné. Ainsi, il devient possible de déduire si la création d'un groupe taxonomique particulier peut s'expliquer par (ou a induit) l'apparition de certaines spécificités au niveau de son métabolisme.Pour cela, nous avons appliqué des méthodes d'apprentissage supervisé interprétables (règles et arbres de décision) sur les profils enzymatiques. Nous utilisons comme attributs les activités enzymatiques, comme classe les groupes taxonomiques et comme exemples les champignons. Les résultats obtenus, cohérents avec nos connaissances actuelles sur ces organismes, montrent que l'application de méthodes d'apprentissage supervisé est efficace pour extraire de l'information des profils phylogénétiques. Le métabolisme conserve donc des traces de l'évolution des espèces.De plus, cette approche, dans le cas de prédiction de classifieurs présentant un faible nombre d'erreurs, peut permettre de mettre en évidence l'existence de probables transferts horizontaux. C'est le cas par exemple du transfert du gène codant pour l'EC:3.1.6.6 d'un ancêtre des pezizomycotina vers un ancêtre d'Ustilago maydis. / This thesis has for objective to propose new methods allowing the study of the evolution of the metabolism. For that purpose, we chose to deal with the problem of comparison of the metabolism of hundred microorganisms.To compare the metabolism of various species, it is necessary to know at first the metabolism of each of these species.We work with proteomes of the microorganisms coming from various databases and sequenced and annotated by various teams, via various methods. The functional annotation can thus be of heterogeneous quality. That is why it is necessary to make a standardized functional annotation of this proteomes.The annotation of protein sequences can be realized by the transfer of annotations between orthologs sequences. There are more than 39 databases listing orthologues predicted by various methods. It is known that these methods lead to partially different predictions. To take into account current predictions and also adding relevant information, we developed the meta approach MARIO. This one combines the intersections of the results of several methods of detection of groups of orthologs and add sequences to this groups by using HMM profiles. We show that our meta approach allows to predict a largest number of orthologs while improving the similarity of function of the pairs of predicted orthologs. It allowed us to predict the enzymatic directory of 178 proteomes of microorganisms (among which 174 fungi).Secondly, we analyze these enzymatic directories in order to analyse the evolution of the metabolism. In this purpose, we look for combinations of presence / absence of enzymatic activities allowing to characterize a taxonomic group. So, it becomes possible to deduct if the creation of a particular taxonomic group can give some explanation by (or led to) the appearance of specificities at the level of its metabolism.For that purpose, we applied interpretable machine learning methods (rulers and decision trees) to the enzymatic profiles. We use as attributes the enzymatic activities, as classes the taxonomic groups and as examples the fungi. The results, coherent with our current knowledge on these species, show that the application of methods of machine learning is effective to extract informations of the phylogenetic profiles. The metabolism thus keeps tracks of the evolution of the species.Furthermore, this approach, in the case of prediction of classifiers presenting a low number of errors, can allow to highlight the existence of likely horizontal transfers. It is the case for example of the transfer of the gene coding for the EC:3.1.6.6 of an ancestor of pezizomycotina towards an ancestor of Ustilago maydis.

Made available in DSpace on 2017-05-12T14:36:23Z (GMT). No. of bitstreams: 1 DISSERTACAO AMANDA ALVES MESTRADO EM CIENCIAS FARMACEUTICAS _UNIOESTE 2015.pdf: 10598736 bytes, checksum: 51f0f3eb83858fee62392b7892930766 (MD5) Previous issue date: 2015-12-07 / Lignocellulosic biomass are the raw material most abundant and promising as a natural and renewable resource. These plant materials are complex carbohydrate polymer composed mainly of cellulose, hemicellulose and lignin, which are linked by covalent bonds and can be transformed into value-added products, such as biofuels. The degradation of lignocellulosic material is made mainly from enzymes produced by microorganisms such as filamentous fungi, yeast and bacteria. Ethanol production from agricultural residues, based on the enzymatic hydrolysis, it takes basically four stages: production of enzymes, pretreatment, enzymatic hydrolysis and fermentation. Pretreatment is a work that will break the lignin cellulose complex, reducing the degree of crystallinity of the cellulose and increase the porosity of the material, by increasing the surface area of the biomass. However, pre-treatment products can generate inhibitors which include phenolic and other aromatic, aliphatic acids, aldehydes, furans, inorganic ions. The fermentation and simultaneous saccharification is an important approach for producing cellulosic or ethanol of second generation, where the enzymatic hydrolysis of cellulose and fermentation are simultaneously carried out in the same reactor, in order to obtain ethanol at a high rate and decrease formation of inhibitor compounds. Enzymatic hydrolysis requires, first, that the lignocellulosic biomass is pretreated to increase access to enzymatic attack, so that later the cellulose is broken down by cellulase action. Xylanases include the group of enzymes responsible for the hydrolysis of xylan, the major constituent of hemicellulose. The key enzymes involved in this process are β-1,4-endoxylanase and β-D-xylosidase. Endoxylanase cleave glycosidic linkages of the main chain of xylan releasing xylo-oligosaccharides, which are used by β-xylosidase to produce monomers of xylose. The alfaproteobacteria Caulobacter crescentus is non pathogenic, Gram negative, mainly found in aquatic environments and on many types of soils. This bacterium has about seven genes directly associated with xylan degradation and five of them encoding β-xylosidases. To date, there are only three studies on the β-xylosidase II from C. crescentus. The first characterization of this enzyme showed that it is capable of hydrolyzing substrates such as xylobiose, xylotriose and xilopentose whose optimum pH is 6 and optimum temperature is 55°C, although it is stable at 50°C, which shows a thermotolerance, indicating strong enough to be used in different biotechnological applications. The stability and reusability of enzymes are of fundamental importance, since they reflect significantly on the cost of the final product, and one way to achieve this is with the immobilization of enzymes, consisting of confinement thereof in a matrix or support, which can be inert polymers or inorganic materials, so that its catalytic activity is retained and the enzyme can be used repeatedly and continuously. In the present report, it was found that the β-xylosidase II (CcXynB2) of Caulobacter crescentus increased by 62% of its activity in 5 mM KCl probably as a consequence of a positive role of K+ ions. CCxynB2 was measured against various compounds described as inhibitors of hydrolysis and fermentation of lignocellulosic biomass and showed 61% more tolerant incubation with ethanol (200 mM) at 37 °C for 48 h in the absence of alcohol. The specific activities of CcXynB2 were evaluated in the presence of 10mM phenol or galacturonic acid, 100 mM hydroxymethylfurfural or ferulic acid, 1 mM acetic acid, 200 mM arabinose, glucose or xylose and it was found that were equal (100%) or much higher than the values obtained in the total absence of these compounds after 48 h. When the inhibitors were used in combination, the CcXynB2 retained 67% of its initial activity after testing at 37°C during 48 h. The enzymatic hydrolysis of hemicellulose from corncob was conducted with CcXynB2 alone or in synergism with xylanase and commercial β-glycosidase, which were more efficient in performed the saccharification of hemicellulose from 37-50 °C. The immobilized CcXynB2 in mobile phase resin led to a protective effect of specific activity, which was proportionally parallel to decreased temperatures (60 to -20°C). The data presented here indicate that CcXynB2 is promising and has potential to work in simultaneous saccharification and fermentation processes for cellulosic ethanol production. To our knowledge, is the first time that similar results are reported in the literature to bacterial β-xylosidases. Thus, this work contribute positively by providing essential information to improve the use of β-xylosidase II of Caulobacter crescentus. / Biomassas lignocelulósicas constituem a matéria-prima mais abundante e promissora como recurso natural e renovável. Esses materiais vegetais são polímeros de carboidratos complexos compostos basicamente por celulose, hemicelulose e lignina, que estão unidos entre si por ligações covalentes e podem ser convertidos em produtos de valor agregado, como os biocombustíveis. A degradação dos materiais lignocelulósicos é feita a partir de enzimas produzidas principalmente por micro-organismos como fungos filamentosos, leveduras e bactérias. Para obter etanol a partir de resíduos agroindustriais, baseando-se na hidrólise enzimática, são necessárias, basicamente, quatro etapas: produção de enzimas, pré-tratamento, hidrólise enzimática e fermentação. O pré-tratamento é o processo que irá dissociar o complexo lignina-celulose, reduzir o grau de cristalinidade da celulose e aumentar a porosidade dos materiais, através do aumento da área superficial da biomassa. No entanto, o pré-tratamento pode gerar produtos inibidores, que incluem compostos fenólicos e outros aromáticos, ácidos alifáticos, aldeídos, furanos, íons inorgânicos. A fermentação e sacarificação simultânea é uma estratégia importante para a produção de etanol celulósico ou de segunda geração, onde a hidrólise enzimática da celulose e a fermentação são desenvolvidas simultaneamente no mesmo reator, com o intuito de obter etanol em altas taxas e diminuir a formação de compostos inibidores. A hidrólise enzimática necessita, primeiramente, que a biomassa lignocelulósica seja pré-tratada para aumentar o acesso ao ataque enzimático, para que posteriormente a celulose seja quebrada pela ação de celulases. As xilanases compreendem o grupo de enzimas responsáveis pela hidrólise do xilano, principal constituinte da hemicelulose. As principais enzimas envolvidas nesse processo são β-1,4-endoxilanase e a β-D-xilosidase. Endoxilanases clivam as ligações glicosídicas da cadeia principal do xilano liberando xilo-oligossacarídeos, que são utilizados pelas β-xilosidases para liberar xilose. A alfaproteobactéria Caulobacter crescentus é não patogênica, Gram negativa, encontrada principalmente em ambientes aquáticos e em muitos tipos de solos. Essa bactéria apresenta cerca de sete genes envolvidos diretamente na degradação do xilano, sendo que cinco deles codificam para β-xilosidases. Até o momento, existem apenas três trabalhos sobre a β-xilosidase II de C. crescentus. A primeira caracterização da enzima mostrou que esta é capaz de hidrolisar substratos como xilobiose, xilotriose e xilopentose, cujo pH ótimo é 6 e temperatura ótima é 55ºC, embora seja mais estável em 50ºC, o que demonstra uma modesta termotolerância, indicando ser suficientemente resistente para diferentes aplicações biotecnológicas. A estabilidade e a possibilidade de reutilização de enzimas são de fundamental importância, pois refletem significativamente no custo do produto final, e uma forma de conseguir isso é com a imobilização de enzimas, que consiste no confinamento da mesma em uma matriz ou suporte, que podem ser polímeros inertes ou materiais inorgânicos, de modo que sua atividade catalítica fique retida e a enzima possa ser usada repetidamente e continuamente. No presente trabalho, verificou-se que a β-xilosidase II (CcXynB2) de Caulobacter crescentus aumentou 62% da sua atividade em 5 mM de KCl provavelmente em consequência de um papel positivo dos íons K+. CcXynB2 foi avaliada frente a diferentes compostos descritos como inibidores do processo de hidrólise e fermentação da biomassa lignocelulósica e mostrou-se 61% mais tolerante a incubação com etanol (200 mM) a atividades específicas da CcXynB2 foram avaliadas na presença de 10 mM fenol ou ácido galacturônico, 100 mM de hidroximetilfurfural ou ácido ferúlico, 1 mM de ácido acético, 200 mM de arabinose, glicose ou xilose, e verificou-se que foram iguais (100%) ou muito superiores aos valores obtidos na ausência total destes compostos após 48 h. Quando os inibidores foram usados em associação, a CcXynB2 reteve 67% da sua atividade inicial após 48 h de ensaio a 37ºC. A hidrólise enzimática da hemicelulose de sabugo de milho foi conduzida com CcXynB2 isoladamente ou em sinergismo com xilanase e β-glicosidase comerciais, as quais foram mais eficientes em sacarificar a hemicelulose entre 37-50ºC. A imobilização da CcXynB2 em resina de fase móvel levou a um efeito protetor da atividade específica, que ocorreu de forma paralela à diminuição de temperatura (60 a -20ºC). Os dados apresentados aqui indicam que a CcXynB2 é promissora e possui potencial para atuar em processos de sacarificação e fermentação simultânea para produção de etanol celulósico. Segundo nosso conhecimento, é a primeira vez que resultados similares são relatados na literatura para β-xilosidases bacterianas. Dessa forma, este trabalho pode contribuir positivamente, fornecendo informações fundamentais para aprimorar o uso da β-xilosidase II de Caulobacter crescentus

Made available in DSpace on 2017-07-10T13:59:26Z (GMT). No. of bitstreams: 1 DISSERTACAO AMANDA ALVES MESTRADO EM CIENCIAS FARMACEUTICAS _UNIOESTE 2015.pdf: 10598736 bytes, checksum: 51f0f3eb83858fee62392b7892930766 (MD5) Previous issue date: 2015-12-07 / SIM(não especificado) / Lignocellulosic biomass are the raw material most abundant and promising as a natural and renewable resource. These plant materials are complex carbohydrate polymer composed mainly of cellulose, hemicellulose and lignin, which are linked by covalent bonds and can be transformed into value-added products, such as biofuels. The degradation of lignocellulosic material is made mainly from enzymes produced by microorganisms such as filamentous fungi, yeast and bacteria. Ethanol production from agricultural residues, based on the enzymatic hydrolysis, it takes basically four stages: production of enzymes, pretreatment, enzymatic hydrolysis and fermentation. Pretreatment is a work that will break the lignin cellulose complex, reducing the degree of crystallinity of the cellulose and increase the porosity of the material, by increasing the surface area of the biomass. However, pre-treatment products can generate inhibitors which include phenolic and other aromatic, aliphatic acids, aldehydes, furans, inorganic ions. The fermentation and simultaneous saccharification is an important approach for producing cellulosic or ethanol of second generation, where the enzymatic hydrolysis of cellulose and fermentation are simultaneously carried out in the same reactor, in order to obtain ethanol at a high rate and decrease formation of inhibitor compounds. Enzymatic hydrolysis requires, first, that the lignocellulosic biomass is pretreated to increase access to enzymatic attack, so that later the cellulose is broken down by cellulase action. Xylanases include the group of enzymes responsible for the hydrolysis of xylan, the major constituent of hemicellulose. The key enzymes involved in this process are β-1,4-endoxylanase and β-D-xylosidase. Endoxylanase cleave glycosidic linkages of the main chain of xylan releasing xylo-oligosaccharides, which are used by β-xylosidase to produce monomers of xylose. The alfaproteobacteria Caulobacter crescentus is non pathogenic, Gram negative, mainly found in aquatic environments and on many types of soils. This bacterium has about seven genes directly associated with xylan degradation and five of them encoding β-xylosidases. To date, there are only three studies on the β-xylosidase II from C. crescentus. The first characterization of this enzyme showed that it is capable of hydrolyzing substrates such as xylobiose, xylotriose and xilopentose whose optimum pH is 6 and optimum temperature is 55°C, although it is stable at 50°C, which shows a thermotolerance, indicating strong enough to be used in different biotechnological applications. The stability and reusability of enzymes are of fundamental importance, since they reflect significantly on the cost of the final product, and one way to achieve this is with the immobilization of enzymes, consisting of confinement thereof in a matrix or support, which can be inert polymers or inorganic materials, so that its catalytic activity is retained and the enzyme can be used repeatedly and continuously. In the present report, it was found that the β-xylosidase II (CcXynB2) of Caulobacter crescentus increased by 62% of its activity in 5 mM KCl probably as a consequence of a positive role of K+ ions. CCxynB2 was measured against various compounds described as inhibitors of hydrolysis and fermentation of lignocellulosic biomass and showed 61% more tolerant incubation with ethanol (200 mM) at 37 °C for 48 h in the absence of alcohol. The specific activities of CcXynB2 were evaluated in the presence of 10mM phenol or galacturonic acid, 100 mM hydroxymethylfurfural or ferulic acid, 1 mM acetic acid, 200 mM arabinose, glucose or xylose and it was found that were equal (100%) or much higher than the values obtained in the total absence of these compounds after 48 h. When the inhibitors were used in combination, the CcXynB2 retained 67% of its initial activity after testing at 37°C during 48 h. The enzymatic hydrolysis of hemicellulose from corncob was conducted with CcXynB2 alone or in synergism with xylanase and commercial β-glycosidase, which were more efficient in performed the saccharification of hemicellulose from 37-50 °C. The immobilized CcXynB2 in mobile phase resin led to a protective effect of specific activity, which was proportionally parallel to decreased temperatures (60 to -20°C). The data presented here indicate that CcXynB2 is promising and has potential to work in simultaneous saccharification and fermentation processes for cellulosic ethanol production. To our knowledge, is the first time that similar results are reported in the literature to bacterial β-xylosidases. Thus, this work contribute positively by providing essential information to improve the use of β-xylosidase II of Caulobacter crescentus. / Biomassas lignocelulósicas constituem a matéria-prima mais abundante e promissora como recurso natural e renovável. Esses materiais vegetais são polímeros de carboidratos complexos compostos basicamente por celulose, hemicelulose e lignina, que estão unidos entre si por ligações covalentes e podem ser convertidos em produtos de valor agregado, como os biocombustíveis. A degradação dos materiais lignocelulósicos é feita a partir de enzimas produzidas principalmente por micro-organismos como fungos filamentosos, leveduras e bactérias. Para obter etanol a partir de resíduos agroindustriais, baseando-se na hidrólise enzimática, são necessárias, basicamente, quatro etapas: produção de enzimas, pré-tratamento, hidrólise enzimática e fermentação. O pré-tratamento é o processo que irá dissociar o complexo lignina-celulose, reduzir o grau de cristalinidade da celulose e aumentar a porosidade dos materiais, através do aumento da área superficial da biomassa. No entanto, o pré-tratamento pode gerar produtos inibidores, que incluem compostos fenólicos e outros aromáticos, ácidos alifáticos, aldeídos, furanos, íons inorgânicos. A fermentação e sacarificação simultânea é uma estratégia importante para a produção de etanol celulósico ou de segunda geração, onde a hidrólise enzimática da celulose e a fermentação são desenvolvidas simultaneamente no mesmo reator, com o intuito de obter etanol em altas taxas e diminuir a formação de compostos inibidores. A hidrólise enzimática necessita, primeiramente, que a biomassa lignocelulósica seja pré-tratada para aumentar o acesso ao ataque enzimático, para que posteriormente a celulose seja quebrada pela ação de celulases. As xilanases compreendem o grupo de enzimas responsáveis pela hidrólise do xilano, principal constituinte da hemicelulose. As principais enzimas envolvidas nesse processo são β-1,4-endoxilanase e a β-D-xilosidase. Endoxilanases clivam as ligações glicosídicas da cadeia principal do xilano liberando xilo-oligossacarídeos, que são utilizados pelas β-xilosidases para liberar xilose. A alfaproteobactéria Caulobacter crescentus é não patogênica, Gram negativa, encontrada principalmente em ambientes aquáticos e em muitos tipos de solos. Essa bactéria apresenta cerca de sete genes envolvidos diretamente na degradação do xilano, sendo que cinco deles codificam para β-xilosidases. Até o momento, existem apenas três trabalhos sobre a β-xilosidase II de C. crescentus. A primeira caracterização da enzima mostrou que esta é capaz de hidrolisar substratos como xilobiose, xilotriose e xilopentose, cujo pH ótimo é 6 e temperatura ótima é 55ºC, embora seja mais estável em 50ºC, o que demonstra uma modesta termotolerância, indicando ser suficientemente resistente para diferentes aplicações biotecnológicas. A estabilidade e a possibilidade de reutilização de enzimas são de fundamental importância, pois refletem significativamente no custo do produto final, e uma forma de conseguir isso é com a imobilização de enzimas, que consiste no confinamento da mesma em uma matriz ou suporte, que podem ser polímeros inertes ou materiais inorgânicos, de modo que sua atividade catalítica fique retida e a enzima possa ser usada repetidamente e continuamente. No presente trabalho, verificou-se que a β-xilosidase II (CcXynB2) de Caulobacter crescentus aumentou 62% da sua atividade em 5 mM de KCl provavelmente em consequência de um papel positivo dos íons K+. CcXynB2 foi avaliada frente a diferentes compostos descritos como inibidores do processo de hidrólise e fermentação da biomassa lignocelulósica e mostrou-se 61% mais tolerante a incubação com etanol (200 mM) a atividades específicas da CcXynB2 foram avaliadas na presença de 10 mM fenol ou ácido galacturônico, 100 mM de hidroximetilfurfural ou ácido ferúlico, 1 mM de ácido acético, 200 mM de arabinose, glicose ou xilose, e verificou-se que foram iguais (100%) ou muito superiores aos valores obtidos na ausência total destes compostos após 48 h. Quando os inibidores foram usados em associação, a CcXynB2 reteve 67% da sua atividade inicial após 48 h de ensaio a 37ºC. A hidrólise enzimática da hemicelulose de sabugo de milho foi conduzida com CcXynB2 isoladamente ou em sinergismo com xilanase e β-glicosidase comerciais, as quais foram mais eficientes em sacarificar a hemicelulose entre 37-50ºC. A imobilização da CcXynB2 em resina de fase móvel levou a um efeito protetor da atividade específica, que ocorreu de forma paralela à diminuição de temperatura (60 a -20ºC). Os dados apresentados aqui indicam que a CcXynB2 é promissora e possui potencial para atuar em processos de sacarificação e fermentação simultânea para produção de etanol celulósico. Segundo nosso conhecimento, é a primeira vez que resultados similares são relatados na literatura para β-xilosidases bacterianas. Dessa forma, este trabalho pode contribuir positivamente, fornecendo informações fundamentais para aprimorar o uso da β-xilosidase II de Caulobacter crescentus

Esta dissertação apresenta um estudo de diferentes metodologias de imobilização (fisissorção, quimissorção com carboxibenzaldeído e quimissorção com glutaraldeído) da lipase de Burkholderia cepacia em nanopartículas magnéticas e sua aplicação na resolução cinética de alcoóis secundários racêmicos. O método de imobilização por fisissorção resultou na imobilização de 0,21 mg de proteína em 20 mg de nanopartículas magnéticas. Para a mesma quantidade de nanopartículas magnéticas, o método de quimissorção com carboxibenzaldeído imobilizou 0,26 mg de proteína contra 0,28 mg de proteína pelo método de quimissorção com glutaraldeído, a melhor relação encontrada neste trabalho. A atividade enzimática foi avaliada na resolução cinética de alcoóis secundários racêmicos [(RS)-2-bromo-1-(fenil)etanol, (RS)-2-bromo-1-(4-nitrofenil)etanol, (RS)-1-(4-nitrofenil)etanol e (RS)-1-(fenil)-1,2-etanodiol] via reação de transesterificação enantiosseletiva. O efeito de diferentes parâmetros reacionais para a resolução cinética foi estudado, como agente acilante, quantidade de substrato, solvente, quantidade de nanopartículas magnéticas (suporte), velocidade de agitação, tempo e temperatura reacionais. Os melhores parâmetros encontrados foram acetato de vinila como agente acilante, tolueno como solvente e sob agitação de 800 rpm. Observou-se que após 30 dias de estocagem da lipase imobilizada por fisissorção sua atividade foi mantida. Além disso, estudou-se a reciclagem da enzima imobilizada, durante a resolução cinética. A melhor temperatura e tempo reacional foram determinados para cada método de imobilização. A quimissorção com glutaraldeído foi o melhor método de imobilização para a reciclagem da enzima, pois durante 8 ciclos de resolução cinética a conversão (50 %) e a enantiosseletividade (>99 %) foram mantidas. Com base nesses resultados, pode-se concluir que o processo de imobilização permite um aumento da estabilidade da enzima quando comparada com a enzima livre, permitindo sua reutilização por vários ciclos reacionais. / This dissertation describes studies about different immobilization methodologies (physisorption, chemisorption with carboxibenzaldehyde and chemisorption with glutaraldehyde) of the Burkholderia cepacia lipase on magnetic nanoparticles and its application in the enzymatic kinetic resolution of chiral secondary alcohols. The physisorption method immobilized 0.21 mg of protein per 20 mg of magnetic nanoparticles. Using the same amount of magnetic nanoparticles, the chemisorption method with carboxibenzaldehyde immobilized 0.26 mg of protein against 0.28 mg for the chemisorption with glutaraldehyde, the best result found in this work. The enzymatic activity was determined in the enzymatic kinetic resolution of chiral secondary alcohols [(RS)-2-bromo-1-(phenyl)ethanol, (RS)-2-bromo-1- (4-nitrophenyl)ethanol, (RS)-1-(4-nitrophenyl)ethanol and (RS</I<)-1-(phenyl)- 1,2-ethanodiol] via enantioselective transesterification reaction. The effect of several reaction parameters for the kinetic resolution was studied, such as acetyl donor, substrate concentration, solvent, amount of magnetic nanoparticles (support), agitation speed, reaction time and temperature. The best results were obtained using vinyl acetate as acetyl donor, toluene as solvent, and 800 rpm as agitation speed. Regarding the physisorption method, after 30 days as storing time the enzymatic activity remained the same. Besides, the reusability of immobilized lipase was evaluated. The best temperature and reaction time in the kinetic resolution were determined for each immobilization method. The chemisorption with glutaraldehyde was the best immobilization method for the enzyme reusability, because even after 8 cycles of the kinetic reaction, the conversion (50 %) and enantioselectivity (>99 %) remained the same. Based on these results, it is possible to conclude that the immobilization process increased the enzyme stability when compared to the free enzyme, allowing its reusability for many reaction cycles.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Overt hypothyroidism is characterized by decreased of thyroid hormones synthesis, with elevation of thyroid-stimulating hormone (TSH). It s a common disorder in population with highest prevalence in women and with aging. Frequently is associated with lipid metabolism alterations, represented by lipid parameters elevation and consequently with atherosclerosis development. The association between hypercholesterolemia and oxidative stress already well established, however oxidative stress presence in hypothyroidism is controversy. The oxidative stress is characterized by an increase in oxygen reactive species (ROS) generation or antioxidant system deficiency. In the present study were determined biochemical and oxidative stress biomarkers in overt hypothyroidism patients. The biochemical markers, total cholesterol (TC), LDL-cholesterol(C-LDL), cholesterol HDL (C-HDL) and trycerides (TG) were measured in serum samples in these patients. Lipid peroxidation was measured by thiobarbituric acid reactive substances (TBARS) levels. Antioxidant system evaluation was performed by superoxide dismutase (SOD) and catalase (CAT) actvities and non-enzymatic antioxidants levels were evaluated by reduced glutathione (GSH) and vitamin E (VIT E) levels.The results demonstrated an increase in biochemical markers (TC, C-LDL and TG) in hypothyroid group, when compared to control group. In relation to lipid peroxidation, was observed an increased in TBARS levels in patients with hypothyroidism when compared to control group. The same was observed for antioxidants defenses SOD and CAT. Nonenzymatic antioxidants, such vitamin E, were higher in hypothyroid group in relation to controls, while GSH levels remained unchanged with hypothyroidism. These results suggest the association between overt hypothyroidism and hypercholesterolemia and oxidative stress. The high levels of cholesterol presented by hypothyroidism patients, has a stronger influence under oxygen reactive species (ROS) generation and in consequence under the oxidative stress. The increase in SOD and CAT activities, suggest antioxidant system induction as a mechanism to combat the ROS generation and high lipid peroxidation. In conclusion, overt hypothyroidism has association with hypercholesterolemia and oxidative stress biomarkers increase. / O hipotireoidismo clínico é caracterizado pela diminuição na síntese dos hormônios tireoideanos, com elevação dos níveis do hormônio tireoestimulante (TSH). É uma desordem comum na população com maior incidência no sexo feminino e com a progressão da idade. Freqüentemente está associado a alterações no metabolismo lipídico, representadas pela elevação nos parâmetros lipídicos e consequentemente com o desenvolvimento de aterosclerose. A associação entre hipercolesterolemia e estresse oxidativo já é bem estabelecida, entretanto a presença de estresse oxidativo no hipotireoidismo é controversa. O estresse oxidativo é caracterizado por um aumento na produção de espécies reativas de oxigênio (ERO) ou deficiência do sistema antioxidante. Neste trabalho determinaram-se marcadores bioquímicos e de estresse oxidativo em pacientes com hipotireoidismo clínico. Os marcadores bioquímicos, colesterol total (CT), colesterol-LDL (LDL-C), colesterol HDL (HDL-C) e triglicerídeos (TG) foram medidos em soro dos pacientes. A peroxidação lipídica foi medida através dos níveis de substâncias reativas ao ácido tiobarbitúrico (TBARS). A avaliação do sistema antioxidante enzimático foi realizada através da medida da atividade das enzimas superóxido dismutase (SOD) e catalase (CAT) e os níveis de antioxidantes não-enzimáticos através dos níveis de tióis totais (SH) e vitamina E (VIT E). Os resultados demonstraram um aumento dos marcadores bioquímicos (CT, LDL-C e TG) no grupo hipotireóideo, com relação ao grupo controle. Em relação a peroxidação lipídica, observou-se um aumento dos níveis séricos de TBARS de pacientes com hipotireoidismo quando comparados com o grupo controle. Esse aumento também foi observado para as defesas antioxidantes enzimáticas, SOD e CAT. Com relação aos antioxidantes não-enzimáticos, ocorreu um aumento nos níveis séricos de VIT E no grupo hipotireóideo com relação ao grupo controle, enquanto que para SH não foi observada diferença entre os grupos estudados. Estes resultados sugerem a associação do hipotireoidismo clínico com hipercolesterolemia e estresse oxidativo. Os altos níveis de colesterol apresentados pelos pacientes com hipotireoidismo, exercem forte influência sobre a geração de espécies reativas de oxigênio (ERO) e por conseqüência sobre o estresse oxidativo. Os aumentos das enzimas SOD e CAT, sugerem a indução do sistema antioxidante enzimático, na tentativa de combater a formação de ERO e a elevada peroxidação lipídica. Concluí-se então, que o hipotireoidismo clínico, está associado à hipercolesterolemia e ao aumento dos biomarcadores de estresse oxidativo.

Diabetes mellitus (DM) is a chronic disease characterized by hyperglycemia, which is related to oxidative stress and plays an important role in the development of other diseases and tissue damage, such as liver and kidney damage. Thus, it is important for studies with potential antioxidant that may reduce the deleterious effects of oxidative stress due to diabetes. In this sense, N-acetylcysteine (NAC) is used as a hepatoprotective drug in the treatment of acute poisoning by paracetamol to reduce oxidative damage. Along these lines, the aims of this study were to evaluate biomarkers of oxidative stress such as reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), malondialdehyde (MDA) and delta aminolevulinate acid dehydratase (ALA-D) in liver and kidneys of controls and animals with diabetes induced after treatment with NAC. Treatments consisted of intraperitoneal administration of 25 mg / kg and 75 mg / kg N-acetylcysteine. In the liver, MDA levels were significantly increased in the diabetic group compared to controls, treatment with 75 mg / kg reduced the levels of MDA, being similar to the control group. GSH levels of the enzyme was found to be highest in kidney and liver of diabetic animals than controls, and treatment with NAC led to these reduced levels in the liver of diabetic animals, but in the kidney, no changes. The levels of SOD and GPx decreased in the liver of diabetic animals compared to control, and administration of NAC did not alter these indices. Diabetes also reduced the activity of ALA-D in the liver, and treatment with 25 mg/ kg NAC did with this activity increased significantly. In the kidney, both doses of NAC increased the levels of ALA-D in diabetic animals. The results suggest that NAC may be more effective in the liver, the organ that suffers most oxidative changes, and especially in groups of diabetic animals. / O diabetes mellitus (DM) é uma doença crônica caracterizada pela hiperglicemia, que está relacionada Com o estresse oxidativo, o qual possui papel importante no desenvolvimento de outras patologias e danos teciduais, tais como dano hepático e renal. Dessa forma, faz-se importante à realização de estudos com possíveis antioxidantes, que possam diminuir os efeitos deletérios do estresse oxidativo decorrentes do diabetes. Neste sentido, a N-acetilcisteína (NAC) é um medicamento utilizado como hepatoprotetor por estimular a síntese de Glutationa Reduzida, diminuindo o dano oxidativo. Nesta linha, o objetivo deste trabalho foi avaliar o efeito antioxidante da NAC nos tecidos renal e hepático de ratos diabéticos através dos biomarcadores do estresse oxidativo como: glutationa reduzida (GSH), glutationa peroxidase (GPx), superóxido dismutase (SOD), malondialdeído (MDA) e ácido delta aminolevulinato desidratase (ALA-D) no fígado e rins de animais controles e com diabetes induzida, tratados e não tratados com NAC. Os tratamentos consistiram em administrações intraperitoneais de 25 mg/Kg e 75 mg/Kg de N-acetilcisteína. No fígado, os níveis de MDA foram significativamente aumentados no grupo diabético comparados ao grupo controle. O tratamento com 75 mg/Kg foi capaz de reduzir os níveis de MDA, ficando semelhantes ao grupo controle. Os níveis da GSH mostrou-se mais elevada no rim e no fígado dos animais diabéticos do que dos controles, e o tratamento com a NAC fez com que esses níveis fossem reduzidos no fígado dos animais diabéticos, entretanto no rim, não houve alterações. Os níveis de SOD e GPx diminuíram no fígado dos animais diabéticos quando comparados ao controle, e a administração de NAC não alterou esses índices. O diabetes também diminuiu a atividade da ALA-D no fígado, e o tratamento com a 25 mg/Kg NAC fez com essa atividade aumentasse significativamente. No tecido renal, ambas as doses de NAC elevaram os níveis de ALA-D nos animais diabéticos. Diante dos resultados encontrados, comparando-se os tecidos renal e hepático dos ratos controles com os diabéticos tratados com NAC, sugere-se que a NAC demonstrou diminuir o dano oxidativo mais no fígado do que no rim.

L’usage excessif d’antibiotiques a provoqué l’émergence de résistance, constituant un problème sanitaire mondial. L’antibiotique triméthoprime (TMP) inhibe l’enzyme dihydrofolate réductase (FolA) des bactéries, interrompant la production d’un précurseur essentiel dans la synthèse des purines et empêchant ainsi la croissance bactérienne. Cependant, certaines bactéries produisent une seconde dihydrofolate réductase : une DfrB, appartenant à une famille d’enzymes hautement résistantes au TMP. Actuellement, dix membres de la famille DfrB ont été identifiés, qui partagent une identité de séquence élevée (74 – 98 %). Les enzymes DfrB sont constituées de domaines identiques de 78 acides aminés, de type ‘SH3-like’, qui s’homotétramérisent afin de former l’enzyme active. Les DfrB ne partagent aucune homologie de séquence ou de structure avec les FolA et aucun antibiotique n’a encore été développé pour contourner la résistance au TMP causée par les DfrB. Afin de mieux comprendre le domaine SH3-like, des homologues (DfrB-H) partageant 10 à 80 % d’identité avec la DfrB1 ont été identifiés et caractérisés. Ils possèdent une activité dihydrofolate réductase (Dfr) et confèrent de la résistance au TMP. De plus, afin de vérifier si les gènes dfrB se retrouvent dans divers environnements, une recherche dans une base de données métagénomiques a été entreprise, permettant de caractériser 10 nouvelles séquences homologues aux DfrB connues. En 2012, le groupe Pelletier a rapporté le premier inhibiteur spécifique d’une DfrB, et plusieurs autres depuis. Seule la DfrB1 a été caractérisée concernant son profil d’inhibition ainsi que sa thermostabilité inhabituelle. Ici, une méthode semi-automatisée sera développée pour caractériser les profils d’inhibition, de thermostabilité, de résistance au TMP et d’activité enzymatique de toutes les DfrB et des homologues identifiés, afin de les comparer à ceux de la DfrB1. Pour atteindre ces objectifs, des nouvelles méthodes à haut débit de détermination d’activité ainsi que des tests de concentration minimale inhibitrice (CMI) furent développés. Ces méthodes ont permis de déterminer que les profils de thermostabilité et d’inhibition de plusieurs DfrB et DfrB-H sont comparables aux profils de la DfrB1. De plus, le criblage de dizaines de composés potentiellement inhibiteurs a été effectué afin de poursuivre la recherche d’inhibiteurs spécifiques aux DfrB. En outre, nous signalons 10 nouvelles séquences homologues de DfrB qui confèrent une résistance élevée au TMP et possèdent une activité Dfr. La caractérisation de tous les membres DfrB et les homologues nous permettra d’acquérir une meilleure connaissance de leur mécanisme de résistance, de leur prévalence dans divers environnements et de soutenir notre développement de nouveaux inhibiteurs des DfrB. / The intensive usage of antibiotics has provoked the emergence of antibiotic resistance, causing a worldwide health issue. The antibiotic trimethoprim (TMP) targets the microbial dihydrofolate reductase enzyme (FolA), abrogating the production of an essential precursor in the synthesis of purines and thus preventing bacterial proliferation. However, some bacteria produce an additional dihydrofolate reductase: the highly TMP-resistant DfrB. Currently, ten DfrB family members have been identified, that share high sequence identity (74 – 98 %). DfrB enzymes consist of identical, 78 amino acid-long SH3-like domains, that homotetramerize to form the active enzyme. DfrB share no sequence or structural homology with FolA and no antibiotic has yet been developed to circumvent the TMP resistance caused by DfrB. In order to gain insight into the SH3-like domain of DfrB, homologues (DfrB-H) sharing 10 to 80 % identity with DfrB1 were identified and characterized, which displayed dihydrofolate reductase (Dfr) activity and conferred high TMP resistance. Also, to investigate if dfrB genes are identified in various environments, a metagenomic database search was undertaken to characterize ten new DfrB1 homologue sequences. In 2012, the Pelletier group reported the first specific inhibitor of a DfrB, and several others since. Only DfrB1 has been characterized regarding its inhibition profile as well as its unusual thermostability. Here, semi-automated methods will be developed to compare the inhibition, thermostability, TMP-resistance and enzymatic activity profiles of all DfrB and DfrB homologues to those of DfrB1. To address this objective, new high-throughput activity assays as well as Minimal Inhibitory Concentration (MIC) assays were developed. Using those methods, we determined that thermostability and inhibition profiles of several DfrB and DfrB-H were comparable to those of DfrB1. Also, a screen of several dozen potential inhibitory compounds was performed, to attempt to identify further specific DfrB inhibitors. In addition, we report 10 new DfrB homologues that confer high TMP resistance and possess Dfr activity. The characterization of all DfrB members and DfrB homologues will allow us to acquire greater knowledge on their antimicrobial resistance mechanism, their prevalence in different environments and support our development of new DfrB-specific inhibitors.

The diversity of microorganisms and the influence of their enzymatic activities in soil are critical to the maintenance of good soil health. Changes in these parameters may be the earliest predictors of soil quality changes, potentially indicating anthropogenic influences. The goal of this study was to investigate the soil microbial diversity and function of grasslands along an urbanization gradient. Soil samples were collected in the Potchefstroom municipal area, South Africa, at specific sites. Sampling sites were described as urban, suburban and rural - according to the V-I-S (Vegetation-Impervious surface-Soil) model of Ridd (1995). Soil samples were collected over a warmer, wet season (May) and a colder, dry season (August) over two years (2007 and 2008). Collected soil samples were characterised using certain physical and chemical parameters. Plant species composition and abundance were determined at each site, along with basic site data (soil compaction, percentage ground cover, percentage bare ground, percentage organic material present). The Shannon-Weaver diversity index was used to calculate biodiversity values for all the investigated sites regarding collected plant species composition. The microbial component of the soil was quantified and characterized using culture-dependent and culture-independent techniques. Culture-dependent techniques included the investigation of the aerobic heterotrophic bacteria and fungi. Organisms were plated out on different media, and the bacterial component was broadly grouped using morphology. Dominant organisms were identified by sequencing of PCR amplified 16S ribosomal DNA fragments. Shannon-Weaver index for bacterial diversity was determined for each of the sites. Denaturing gradient gel electrophoresis (DGGE) profiling of selected bacterial communities were also conducted. Microbial community function was determined using enzyme assays of five major groups of enzymes, namely (i) dehydrogenase; (ii) β-glucosidase; (iii) acid phosphatase, (iv) alkaline phosphatase and (v) urease. Plant species results were then brought into context with microbiological diversity and functionality results using multivariate statistics. Physical and chemical parameters of the collected soil samples revealed patterns present along the urbanization gradient. The pH values were mostly higher in the sub-urban and urban sites than in the rural sites. Electrical conductivity values were generally highest in the sub-urban sites. Plant species composition revealed trends along the urbanization gradient. Ordinations clearly grouped the plant species into rural, sub-urban and urban groups regarding plant species composition. Rural sites had the highest number of plant species. Shannon-Weaver values regarding the plant diversity supported the plant species composition data indicating higher plant diversity in the rural areas, followed by the sub-urban and the urban areas. Plant structural data indicated that forbs were most numerous in the rural sites, and less so in the urban sites. Higher average aerobic heterotrophic bacterial levels were present in the urban soil samples. The bacterial levels were lower in the sub-urban and rural soil samples. Subsequent identification of the dominant bacteria in the soil samples revealed organisms of the genus Bacillus dominated the aerobic heterotrophic bacterial communities in the soil samples. Bacillus species dominated the soil samples along the urbanization gradient. Shannon-Weaver indices based on culture-dependent methods indicated that urban sites had the highest biodiversity. These results could have been exaggerated, because of an overestimation of the number of bacterial morphotypes present in samples. Fungal levels were higher in the soil from samples collected at the rural samples sites. The culture-independent method (DGGE) was not optimized and inconclusive results were obtained. Enzyme assays revealed that potential dehydrogenase, β-glucosidase and urease activity followed a trend along the urbanization gradient, with urban samples registering the highest values and rural sites the lowest. Enzymes involved in carbohydrate catabolism (β-glucosidase and dehydrogenase) registered significantly higher potential activity in urban sites than the sub-urban and rural sites. The results could indicate that urban sites have the potential to lose carbon at higher rates than the rural sites. This aspect may need further investigation. Higher potential urease activity could indicate higher N-cycling in the urban soil environment. Ordination results for soil-, plant- and microbial diversity as well as microbial functionality indicated certain trends along the urbanization gradient. Plant species composition and structure data indicated that urbanization has a definite effect on the plant communities in the urban ecosystem. Results regarding aerobic heterotrophic bacteria populations and potential enzyme activity of the dehydrogenase, β-glucosidase (both active in the carbon cycle) and urease (active in the nitrogen cycle) illustrated clear trends along the urbanization gradient. In conclusion, results indicated that urbanization has an effect on plant species composition, and the population and function of aerobic heterotrophic bacteria and the fungal population. Furthermore, this study demonstrated the potential of using microbial diversity and activity as tools to investigate carbon utilization and storage along an urban-rural gradient. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2011

The diversity of microorganisms and the influence of their enzymatic activities in soil are critical to the maintenance of good soil health. Changes in these parameters may be the earliest predictors of soil quality changes, potentially indicating anthropogenic influences. The goal of this study was to investigate the soil microbial diversity and function of grasslands along an urbanization gradient. Soil samples were collected in the Potchefstroom municipal area, South Africa, at specific sites. Sampling sites were described as urban, suburban and rural - according to the V-I-S (Vegetation-Impervious surface-Soil) model of Ridd (1995). Soil samples were collected over a warmer, wet season (May) and a colder, dry season (August) over two years (2007 and 2008). Collected soil samples were characterised using certain physical and chemical parameters. Plant species composition and abundance were determined at each site, along with basic site data (soil compaction, percentage ground cover, percentage bare ground, percentage organic material present). The Shannon-Weaver diversity index was used to calculate biodiversity values for all the investigated sites regarding collected plant species composition. The microbial component of the soil was quantified and characterized using culture-dependent and culture-independent techniques. Culture-dependent techniques included the investigation of the aerobic heterotrophic bacteria and fungi. Organisms were plated out on different media, and the bacterial component was broadly grouped using morphology. Dominant organisms were identified by sequencing of PCR amplified 16S ribosomal DNA fragments. Shannon-Weaver index for bacterial diversity was determined for each of the sites. Denaturing gradient gel electrophoresis (DGGE) profiling of selected bacterial communities were also conducted. Microbial community function was determined using enzyme assays of five major groups of enzymes, namely (i) dehydrogenase; (ii) β-glucosidase; (iii) acid phosphatase, (iv) alkaline phosphatase and (v) urease. Plant species results were then brought into context with microbiological diversity and functionality results using multivariate statistics. Physical and chemical parameters of the collected soil samples revealed patterns present along the urbanization gradient. The pH values were mostly higher in the sub-urban and urban sites than in the rural sites. Electrical conductivity values were generally highest in the sub-urban sites. Plant species composition revealed trends along the urbanization gradient. Ordinations clearly grouped the plant species into rural, sub-urban and urban groups regarding plant species composition. Rural sites had the highest number of plant species. Shannon-Weaver values regarding the plant diversity supported the plant species composition data indicating higher plant diversity in the rural areas, followed by the sub-urban and the urban areas. Plant structural data indicated that forbs were most numerous in the rural sites, and less so in the urban sites. Higher average aerobic heterotrophic bacterial levels were present in the urban soil samples. The bacterial levels were lower in the sub-urban and rural soil samples. Subsequent identification of the dominant bacteria in the soil samples revealed organisms of the genus Bacillus dominated the aerobic heterotrophic bacterial communities in the soil samples. Bacillus species dominated the soil samples along the urbanization gradient. Shannon-Weaver indices based on culture-dependent methods indicated that urban sites had the highest biodiversity. These results could have been exaggerated, because of an overestimation of the number of bacterial morphotypes present in samples. Fungal levels were higher in the soil from samples collected at the rural samples sites. The culture-independent method (DGGE) was not optimized and inconclusive results were obtained. Enzyme assays revealed that potential dehydrogenase, β-glucosidase and urease activity followed a trend along the urbanization gradient, with urban samples registering the highest values and rural sites the lowest. Enzymes involved in carbohydrate catabolism (β-glucosidase and dehydrogenase) registered significantly higher potential activity in urban sites than the sub-urban and rural sites. The results could indicate that urban sites have the potential to lose carbon at higher rates than the rural sites. This aspect may need further investigation. Higher potential urease activity could indicate higher N-cycling in the urban soil environment. Ordination results for soil-, plant- and microbial diversity as well as microbial functionality indicated certain trends along the urbanization gradient. Plant species composition and structure data indicated that urbanization has a definite effect on the plant communities in the urban ecosystem. Results regarding aerobic heterotrophic bacteria populations and potential enzyme activity of the dehydrogenase, β-glucosidase (both active in the carbon cycle) and urease (active in the nitrogen cycle) illustrated clear trends along the urbanization gradient. In conclusion, results indicated that urbanization has an effect on plant species composition, and the population and function of aerobic heterotrophic bacteria and the fungal population. Furthermore, this study demonstrated the potential of using microbial diversity and activity as tools to investigate carbon utilization and storage along an urban-rural gradient. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2011

Existing commercially available microcarriers are very efficient at encouraging cell attachment and proliferation. However, recovery of the cells is problematic as it requires the use of proteolytic enzymes which are damaging to critical cell adhesion proteins. From this perspective, temperature responsive polymers appear to be a valid option. The current innovative study is to produce and engineer microcarriers in terms of particle size, surface coating and properties, as well as thermo-responsiveness for cell release. All these benefits are based on particle production by membrane emulsification to provide a highly controlled particle size. The polymer of choice is poly N-isopropylacrylamide (pNIPAM) because of the sharpness of its phase transition, biocompatibility and transition temperature close to the physiological value. These characteristics make pNIPAM a very attractive material for Tissue Engineering applications. Cells are cultured on the hydrophobic surface at 37??C and can be readily detached without using proteolytic enzymes from the surface by lowering the temperature to room temperature. The Dispersion Cell (MicroPore Technologies Ltd, UK) was successfully employed for the production of W/O emulsions. The generated monomer droplets were additionally solidified by applying a free radical polymerisation to manufacture solid pNIPAM microspheres. Additionally, calcium alginate particles were also generated and further functionalised with amine terminated pNIPAM to form temperature responsive core-shell particles by simply taking advantage of the electrostatic interactions between the carboxyl groups of the alginate and amino groups of the modified pNIPAM. Controlled particle production was achieved by varying process parameters and changing the recipe formulation (e.g. monomer concentration, surfactant concentration, pore size and inter-pore spacing, injection rate, shear stress applied at the membrane s surface). The manufactured particles were then analysed in terms of particle size and size distribution, chemical composition, surface analysis, shrinkage ratio and thermo-responsiveness and further sterilised and used for cell culture and release experiments. Swiss Albino 3T3 fibroblastic cells (ATCC, USA) were utilised to show proof-of-concept for this technology. Cell attachment and proliferation were assessed and successfully demonstrated qualitatively and quantitatively. pNIPAM solid particles, uncoated and with different protein coatings were shown to allow a limited degree of cell attachment and proliferation compared to a commercially available microcarrier. On a different approach, uncoated core-shell structures demonstrated improved capabilities for cell attachment and proliferation, similar to commercially available microcarriers. Having in mind the potential of temperature responsive polymers and the aim of this innovative study, cell detachment from the generated microcarriers was evaluated and compared to a commercially available temperature responsive surface. Necessary time for detachment was recorded and detached cells were recovered and reseeded onto tissue culture plastic surfaces in order to evaluate the replating and reattachment capabilities of the recovered cells. Successful cell detachment was achieved when using the core-shell structures as cell microcarriers, but the necessary time of detachment was of an order higher than that for the commercial temperature responsive surface.