Problem investigated: Chronic wounds are not only a major burden to the patient arising from general pain and discomfort but also generate economic costs to both these individuals and the health care system. Various electrical stimulation regimes have been employed to study the effects of electrical stimulation on wound healing both in vivo and in vitro. In was hypothesised that electrical stimulation using various waveforms can modulate cell function, particularly cell migration. The aim of this thesis was to study the effects of electrical stimulation on cellular migration, in particular endothelial cells and fibroblasts, key cell types involved in wound healing. The impact of collagen matrix on cell migration was also assessed. Methods: Cells were seeded on either glass or collagen I substrate and stimulated with various electrical regimes via platinum electrodes connected to a constant current source. Cell migration was accessed by manual tracking of cell nuclei over a period of 3 hours from digital time-lapse images acquired during stimulation. Data from cell tracking were analysed for directional migration, migration rates and mean square displacement. Results: No directional cell migration for both endothelial cells and fibroblasts were observed when stimulated with either alternating or biphasic currents. However, surface substrate had impacted on cell motility with opposite effects being observed for the two cell types. Endothelial cells tended to migrate at a faster rate on collagen I substrate than on glass, compared with fibroblasts, which displayed a slower rate of migration on collagen I substrate. Significant changes in mean square displacement of biphasic current stimulated cells on collagen I substrate compared to unstimulated cells were also observed. Conclusion: This thesis has illustrated cell migration can be modulated by electrical stimulation, in particular asymmetric biphasic current. It has also been demonstrated surface substrate can impact cell migration.

<p>Deep brain stimulation (DBS) is a surgical treatment technique, which involves application of electrical pulses via electrodes inserted into the brain. Neurons, typically located in the basal ganglia network, are stimulated by the electrical field. DBS is currently widely used for symptomatically treating Parkinson’s disease patients and could potentially be used for a number of neurological diseases. In this study, computational modeling was used to simulate the electrical activity of neurons being affected by the electrical field, to gain better understanding of the mechanisms of DBS. The spatial and temporal distribution of the electrical field was coupled to a cable model representing a human myelinated axon. A passing fiber with ends infinitely far away was simulated. Results show that excitation threshold is highly dependent on the diameter of the fiber and the influence (threshold-distance and threshold-diameter relations) can be controlled to some extent, using charge-balanced biphasic pulses.</p>

Deep brain stimulation (DBS) is a surgical treatment technique, which involves application of electrical pulses via electrodes inserted into the brain. Neurons, typically located in the basal ganglia network, are stimulated by the electrical field. DBS is currently widely used for symptomatically treating Parkinson’s disease patients and could potentially be used for a number of neurological diseases. In this study, computational modeling was used to simulate the electrical activity of neurons being affected by the electrical field, to gain better understanding of the mechanisms of DBS. The spatial and temporal distribution of the electrical field was coupled to a cable model representing a human myelinated axon. A passing fiber with ends infinitely far away was simulated. Results show that excitation threshold is highly dependent on the diameter of the fiber and the influence (threshold-distance and threshold-diameter relations) can be controlled to some extent, using charge-balanced biphasic pulses.

Prieširdžių virpėjimas (PV) dažniausiai klinikinėje praktikoje pasitaikanti širdies aritmija. PV dažnis populiacijoje – 0,4–1 proc. ir su amžiumi didėja, o tarp vyresnių nei 80 metų žmonių išauga iki 8 proc. Per pastaruosius 20 metų hospitalizavimo dėl PV atvejų padaugėjo 66 proc. PV metu išnyksta normali prieširdžių sistolė, vyksta prieširdžių remodeliavimasis, širdies ertmėse susidaro trombai, trinka hemodinamika. Tai susiję su sunkiomis komplikacijomis: širdies funkcijos nepakankamumu, insultu ir padidėjusiu mirštamumu. Veiksmingiausias PV nutraukimo būdas – elektrinė kardioversija, kuri klinikinėje praktikoje taikoma nuo 1962 m. Darbo tikslas – įvertinti prieširdžių virpėjimo gydymą elektrine kardioversija ir veiksnius, sąlygojančius jos efektyvumą. Darbo uždaviniai: 1. Nustatyti veiksnius, turinčius įtakos elektrinės kardioversijos efektyvumui. 2. Nustatyti ir įvertinti optimalią pradinio elektros impulso energiją, reikalingą prieširdžių virpėjimui nutraukti. 3. Nustatyti veiksnius, turinčius įtakos ankstyvajam prieširdžių virpėjimo atkryčiui po elektrinės kardioversijos. 4. Įvertinti veiksnius, nulemiančius elektrinės kardioversijos komplikacijas. Kauno medicinos universiteto Kardiologijos klinikos Kardiologijos intensyviosios terapijos skyriuje 2005–2007 m. atliktas prospektyvusis atsitiktinių imčių klinikinis tyrimas. Ištirta 224 prieširdžių virpėjimu sergančių pacientų, kuriems nuspręsta taikyti elektrinę kardioversiją sinusinio ritmo atkūrimui. Atsitiktinai... [toliau žr. visą tekstą] / Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. AF occurs in 0.4 – 1 % of population and its frequency increases with age and reaches 8 % above 80 years age. During the last 20 years hospitalization for AF rate increased by 66 %. In AF normal atrial systole does not occur, atrial remodeling starts, intracardiac thrombi start forming, haemodynamics becomes compromised. That leads serious complications – heart failure, stroke and increased mortality rate. The most effective AF termination method is direct current cardioversion, which has been used in clinical practice since 1962. The aim of the study was to evaluate the treatment of atrial fibrillation by direct current cardioversion and to identify the factors, contributing to its effectiveness. The objectives of the study: 1. To identify the factors, contributing to the effectiveness of direct current cardioversion. 2. To estimate and to evaluate the optimal energy of the initial electrical impulse required to terminate atrial fibrillation. 3. To identify the factors, contributing to immediate recurrence of atrial fibrillation after successful direct current cardioversion. 4. To evaluate the factors, influencing complications after direct current cardioversion. A prospective randomized clinical trial has been held in Intensive Care Unit of the Department of Cardiology of Kaunas Medical University Hospital between 2005 and 2007. We examined 224 patients with AF, selected for electrical... [to full text]

Problem investigated: Chronic wounds are not only a major burden to the patient arising from general pain and discomfort but also generate economic costs to both these individuals and the health care system. Various electrical stimulation regimes have been employed to study the effects of electrical stimulation on wound healing both in vivo and in vitro. In was hypothesised that electrical stimulation using various waveforms can modulate cell function, particularly cell migration. The aim of this thesis was to study the effects of electrical stimulation on cellular migration, in particular endothelial cells and fibroblasts, key cell types involved in wound healing. The impact of collagen matrix on cell migration was also assessed. Methods: Cells were seeded on either glass or collagen I substrate and stimulated with various electrical regimes via platinum electrodes connected to a constant current source. Cell migration was accessed by manual tracking of cell nuclei over a period of 3 hours from digital time-lapse images acquired during stimulation. Data from cell tracking were analysed for directional migration, migration rates and mean square displacement. Results: No directional cell migration for both endothelial cells and fibroblasts were observed when stimulated with either alternating or biphasic currents. However, surface substrate had impacted on cell motility with opposite effects being observed for the two cell types. Endothelial cells tended to migrate at a faster rate on collagen I substrate than on glass, compared with fibroblasts, which displayed a slower rate of migration on collagen I substrate. Significant changes in mean square displacement of biphasic current stimulated cells on collagen I substrate compared to unstimulated cells were also observed. Conclusion: This thesis has illustrated cell migration can be modulated by electrical stimulation, in particular asymmetric biphasic current. It has also been demonstrated surface substrate can impact cell migration.

The replacement of missing teeth by osseointegrated dental implants is a commonly utilised treatment option in dentistry. However, successful treatment outcomes are dependent on sufficient bone quantity in the proposed surgical site for implant placement (Buser et al., 2004). Surgical augmentation of bone defects is commonly performed prior to or during implant placement. Bone augmentation procedures of the maxillary sinus or guided bone regeneration (GBR) procedures of alveolar ridge defects have utilised a variety of bone graft materials in block or particulate form, either alone or in combination with resorbable or non-resorbable barrier membranes. Objective: The aim of this study was to determine whether Hydroxyapatite β-Tricalcium Phosphate (HA-TCP) either alone or combined with Enamel Matrix Derivative (EMD) or recombinant human Platelet Derived Growth Factor-BB (rhPDGF-BB) is osteoinductive when implanted into a nonosseous site. Methods: Twenty CD-1 adult male mice underwent intramuscular implantation into both hindlimbs of an empty gelatine capsule or a gelatine capsule containing one of the following: 10 mg of uncoated particulate HA-TCP, (Straumann Bone Ceramic®, HA-TCP), EMD coated HA-TCP, (Emdogain®, HATCP + EMD) or rhPDGF-BB coated HA-TCP (HA-TCP + PDGF). Ten animals were sacrificed at four and eight weeks with five specimens from each group retrieved at each time point. The area of graft placement was radiographed and after graft retrieval, a semi-quantitative histological examination was performed with the aim of assessing the inflammatory changes, reparative processes and osteoinduction within the graft site. Results: At both 4 and 8 weeks, histological analysis failed to demonstrate any osteoinductive activity in any of the specimens from the three experimental groups. A minimal chronic inflammatory response and foreign body reaction was seen in the experimental groups which reduced over time. The particles were embedded within fibrous connective tissue and were encapsulated by a dense cellular layer consisting of active fibroblasts and occasional macrophages with the thickness of this layer decreasing over time. At 4 weeks, a greater density of the fibrous connective tissue was demonstrated in the HA-TCP + EMD group (P<0.001) while a greater thickness in the capsule thickness was seen in the HA-TCP group (P=0.022) although no differences were seen after 8 weeks. Greater neovascularisation was seen in the HA-TCP + PDGF group after 8 weeks (P=0.043) while greater amounts of adipose tissue surrounding the particles were detected in the HA-TCP + PDGF group at 4 weeks (P=0.002) and in the HA-TCP + EMD group at eight weeks (P=0.002). Conclusions: The results of this study suggest that the use of commercially available HA-TCP alone or in combination with EMD or rhPDGF-BB is biocompatible but not osteoinductive in the murine model. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1522641 / Thesis (D.Clin.Dent.) -- University of Adelaide, School of Dentistry, 2010

La séparation de deux énantiomères est un procédé d’intérêt pour l’industrie pharmaceutique. En effet, souvent un seul des énantiomères exerce l'activité biologique requise. Cette problématique est abordée ici par l'utilisation d'enzymes énantiosélectives qui est une alternative intéressante aux méthodes conventionnelles (chromatographie chirale, synthèse asymétrique ou cristallisation). Cette approche a été mise en oeuvre pour les molécules de la famille des profènes (l’Ibuprofène, le Kétoprofène ou le Naproxène par exemple) qui sont des acides 2- arylpropioniques et constituent une classe importante de médicaments anti-inflammatoires non stéroïdiens. Tout d’abord, un travail de recherche dans le domaine de la catalyse enzymatique et de l’ingénierie d’enzymes a été effectué. Des enzymes aux potentialités prometteuses, issues de la levure Yarrowia lipolytica, pour la résolution de ces trois anti-inflammatoires ont été identifiées. S’appuyant sur la modélisation moléculaire, l’ingénierie moléculaire a été utilisée pour accéder à des enzymes performantes tant du point de vue de la sélectivité que de l’activité. Les réactions ont été réalisées conventionnellement dans un système diphasique phase aqueuse/décane car les profènes et les esters associés sont des substrats hydrophobes et très faiblement solubles dans l'eau, la réaction doit donc être effectuée dans un solvant apolaire en contact avec une phase aqueuse où l'enzyme libre est dissoute. Ce mode opératoire permet d'éviter une immobilisation de l'enzyme. Une enzyme pour chaque substrat avec une énantiosélectivité suffisante a pu être développée, à savoir Lip2p V232A pour l’ibuprofène, V235S pour le naproxène et V232F pour le kétoprofène. Les lipases de Candida rugosa se sont également avérées intéressantes pour la résolution des profènes mais moins que les lipases précédemment évoquées. Le deuxième aspect de cette thèse s’est intéressé à la mise oeuvre de cette technique de résolution enzymatique dans un procédé innovant de Chimie Verte où le dioxyde de carbone supercritique (CO2SC) remplace le décane. En effet, les solvants organiques tels que le décane peuvent être toxiques mais aussi difficiles à éliminer, à la fin du processus, ce qui conduit à des étapes fastidieuses et coûteuses de purification. Dans un premier temps l’étude a porté sur la résolution de l’acide 2-bromo phényl acétique par l’hydrolyse de son ester octylique (ester + eau <=> acide (profène) + alcool). L'acidification de la phase aqueuse en contact avec CO2SC (formation d'acide carbonique) s’est montrée préjudiciable pour obtenir des conversions élevées. Cet inconvénient a été atténué en utilisant des concentrations assez élevées de sels (Na2HPO4 et KH2PO4) pour tamponner la phase aqueuse. Une étude spécifique en cellule haute pression utilisant des sondes solvatochromiques a permis d’établir que l’utilisation de concentrations élevées de sels (de l’ordre de 1000 mmol/L) permettait de maintenir un pH de l‘ordre de 6. Dans ces conditions, des conversions élevées ont pu être obtenues pour des temps de réactions de l’ordre de 100 h. Cependant la cinétique s’est avérée plus lente par rapport à celle observée avec le décane. L’explication de cette différence n’est pas encore totalement élucidée mais deux pistes ont été privilégiées : la formation de carbamates dues aux interactions entre le CO2SC et les acides aminés composant l’enzyme ou une mauvaise ouverture du volet moléculaire qui recouvre le site actif de l’enzyme liée à la moindre hydrophobicité du CO2SC hydraté. Cette résolution énantiomérique a également été mise en oeuvre dans un système sans phase aqueuse en utilisant l‘enzyme sous sa forme immobilisée sur support solide en utilisant la réaction réverse, l’estérification. Utilisant les mêmes approches, la résolution énantiomérique de l’ibuprofène a également été réalisée. Les meilleurs résultats obtenus en système diphasique phase aqueuse/CO2SC permettent en 75 heures une résolution quasi-totale. / The separation of the two enantiomers (or enantiomeric resolution) is a process of interest to the pharmaceutical industry. Indeed, very often, only one of the enantiomers has the required biological activity. This issue is addressed here by the use of enantioselective enzymes which is an interesting alternative to conventional methods (chiral chromatography, asymmetric synthesis, crystallization). This approach has been implemented for separation of molecules of the profens family (Ibuprofen, Naproxen or Ketoprofen for example) that are 2-arylpropionic acids and are an important class of anti-inflammatory drugs (NSAIDs). One first aspect of this work was to use molecular modeling and molecular engineering to identify enzymes, from the yeast Yarrovia lipolytica, with promising potential from the point of view of selectivity and activity. Reactions were carried out conventionally in a biphasic aqueous phase/decane system since the profens and related esters are hydrophobic substrates and very sparingly soluble in water so that the reaction must be carried out in an apolar solvent in contact with an aqueous phase where the enzyme is dissolved. This procedure avoids immobilization of the enzyme. An enzyme with good enantioselectivity for each substrate was developed, namely Lip2p V232A for ibuprofen, V235S for naproxen and V232F for ketoprofen. Lipases of Candida rugosa also proved useful for the resolution of ibuprofen, naproxen and ketoprofen but were less efficient than the lipases mentioned above. The second aspect of this thesis focused to the implementation of this enzymatic resolution technology in an innovative process for Green Chemistry where decane was replaced by supercritical carbon dioxide (scCO2).Indeed, organic solvents such as decane may be toxic but also difficult to remove at the end of the process, which leads to cumbersome and costly purification steps. Initially the study focused on enzymatic resolution of 2- bromo phenyl acetic acid by hydrolysis of its octyl ester (ester + water <=> acid (profen) + alcohol). Acidification of the aqueous phase in contact with scCO2 (formation of carbonic acid) proved to be detrimental to obtain high conversions. This disadvantage was mitigated by using relatively high concentrations of salts (1000 mmol/L) to buffer the aqueous phase. A specific study in a high pressure cell using solvatochromic probes showed that the use of high concentrations of salts allowed maintaining a pH of about 6. In these conditions, high conversions could be obtained for reaction times of 100 hours. However kinetics proved to be slower compared to those observed using decane as the organic phase. The explanation for this difference is not yet fully understood but two hypotheses can be evoked: formation of carbamates due to interactions between the scCO2 and amino acids or a bad opening of the "lid" that covers the active site related to a lower hydrophobicity of hydrated scCO2. This enantiomeric resolution was also implemented in a non-aqueous phase system using the enzyme in its immobilized form on a solid support, using the reverse reaction: esterification of profen. Using the same approach, the enantiomeric resolution of ibuprofen was also carried out. Best results using aqueous phase/scCO2 system allowed total resolution within 75 hours.

Made available in DSpace on 2016-12-08T17:19:39Z (GMT). No. of bitstreams: 1 Gisele Dalmonico.pdf: 8707795 bytes, checksum: 3f7704c26d00226d450da2c37f79fcb0 (MD5) Previous issue date: 2011-07-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The caicium phosphate (Ca / P) are the family of biocerarnics best known for biological applications. There are several polytypes of structures in the ratio Ca / P, which typically form the group of cornpounds caiied apatite. Currentiy there are many studies targeting the use of nanostructured powders of caicium phosphate for the reconstniction of bone tissue, implant fixation, weli as coating for metal substrates and as an element in the drug delivery matrix. This paper discusses the process of synthesis and characterization of nanostructured powders of hydroxyapatite, caicium &#946;-phosphate compositions and preparation of biphasic HA/&#946;-TCP. Method was used to obtain the wet compositions of hydroxyapatite and calcium &#946;-phosphate. Later biphasic cornpositions were prepared by mechanical fragmentation process atritor mill. The characterization studies were perfonued on the compositions obtained from the rotary eYaporator, the specimens calcined in two-phase compositions and the compositions sintered. Tests were performed to characterize morphoiogical, microstructural, nanostructurai, mineraiogical and thermai behavior for the different compositions of nanostructured powders. lhe mechanical and physical properties were determined for the different compositions ofbioniaterials obtained from sintering. / Os Fosfatos de Cálcio (Ca/P) são a família de biocerâmicas mais conhecidas pelas suas aplicações biológicas. Existem vários polítipos de estruturas na razão Ca/P que tipicamente formam o grupo de compdstos chamados de apatita. Na atualidade existem muitos estudos direcionando a utilização de pós nanoestruturados de fosfatos de cálcio para a reconstrução de tecido ósseo, fixação de implantes, também corno material de revestimento de substratos metálicos e como elemento màtricial na liberação de medicamentos. Este trabalho aborda o processo de síntese e caracterização de pós nanoestruturados de hidroxiapatita, de fosfato de cálcio-&#946;; e elaboração de composições bifásicas HAITCP-&#946;. Foi utilizado o método via úmida para obtenção das composições de hidroxiapatita e de fosfato de cálcio-&#946;. Posteriormente foram elaboradas as composições bifásicas através do processo de fragmentação mecânica em moinho atritor. Os estudos de caracterização foram realizados sobre as composições obtidas do evaporador rotativo, nos corpos de prova calcinados, nas composições bifásicas e nas composições sinterizadas. Foram realizados os ensaios de caracterização morfológica, microestrutural, nanoestrutural, mineralógica e do comportamento térmico, para as diferentes composições de pós nanoestruturados. As propriedades mecânicas e físicas foram determinadas para as diferentes composições de biornateriais obtidos da sinterização.

Orientadoesr: Elias Basile Tambourgi, Ranulfo Monte Alegre / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-16T02:07:01Z (GMT). No. of bitstreams: 1 Padilha_GiovanadaSilva_D.pdf: 2518753 bytes, checksum: 6b6f99c293a070aff342e419f243fe69 (MD5) Previous issue date: 2010 / Resumo : O presente trabalho foi dividido em três etapas e teve como objetivo a caracterização, purificação e encapsulamento da lípase a partir da cepa de Burkholderia cepacia. Para produção da enzima, o microorganismo foi cultivado em meio contendo sais, extrato de levedura, peptona bacteriológica e 6% de óleo de soja, por fermentação líquida em biorreator do tipo Bioflo III. As fermentações foram conduzidas a 150 rpm em 30ºC. Após 96 horas de cultivo, o sobrenadante foi separado das células por centrifugação e foi utilizado como extrato enzimático, uma vez que a Burkholderia cepacia produz lípase extracelular. O extrato bruto apresentou atividade usando azeite de oliva como substrato. Na primeira etapa do trabalho, analisou-se a condições ótimas de trabalho e estabilidade sob condições diversas e na presença de diferentes íons. A temperatura ótima foi a 37ºC, no entanto a 50ºC a enzima perdeu 10% da atividade em relação à temperatura em 37ºC. O cálculo de energia de ativação, seguindo a equação de ARRHENIUS, foi de 10,1 kJ/mol. O efeito do pH sobre a hidrólise do azeite de oliva pela lípase foi investigado com diferentes tampões na faixa de pH entre 3 e 11. Foram mantidas aproximadamente 80% da atividade entre os pHs 5 e 7, com atividade máxima no pH 8. Atividades razoáveis foram obtidas mesmo nos valores mais ácidos de pH e menores atividades nos valores entre 9 e 11. No entanto, no pH 11 observou-se queda de 80% de atividade em relação à atividade ótima da lípase. A estabilidade da atividade enzimática em diferentes tampões e valores de pH 5, 8 e 11 também foi investigada, onde nas 4 horas de incubação a diferentes valores de pH, a enzima mostrou-se bastante estável. Um estudo de estabilidade com diferentes íons por incubação da lípase durante 30 dias também foi realizado. Íons como Mn2+, Co2+, I- e Ca2+ mantiveram ou aumentaram a atividade enzimática, mas a enzima foi inibida na presença de Fe2+, Hg2+ e Al3+. À temperatura de 37 ºC e pH 8 foi feita a determinação dos parâmetros cinéticos. Os dados obtidos experimentalmente permitiram a obtenção da curva cinética, que mostrou a influência da emulsão óleo e água contendo diferentes proporções de azeite de oliva (0,1 a 50% v/v) na velocidade de hidrólise pela lípase. De acordo com a metodologia de LINEWEAVER-BURK, calculou-se os valores de Km e Vmáx de 43,90 mg/mL e 0,0258 U/mg, respectivamente. A segunda etapa foi purificar a lípase de Burkholderia cepacia em sistema bifásico aquoso polietileno glicol (PEG)/fosfato. Foram preparadas soluções estoques de PEG com massas molares de 1500, 4000 e 6000 Da (50% m/m) e tampão fosfato nos pHs 6, 7 e 8 (20% m/m de KH2PO4/K2HPO4). Um planejamento fatorial 23 foi feito para avaliar a massa molecular do PEG, pH e comprimento da linha de amarração (tie-line) no coeficiente de partição da atividade específica da lípase. Os resultados mostraram que maiores valores de atividade específica foram obtidos ao usar a menor massa molecular de PEG (1500 Da) em pH 6 e 8 e maior comprimento da linha de amarração. Após a purificação, determinou-se 33 kDa a massa molecular e 6,0 o ponto isoelétrico da lípase de Burkholderia cepacia. Na terceira etapa do trabalho, a enzima foi encapsulada por gelificação iônica usando alginato de sódio e cloreto de cálcio. Para a encapsulação foi feito um planejamento fatorial 22 variando o tamanho do bico atomizador (2 e 0,5 mm) e a concentração de CaCl2 (2 e 4% m/v). A enzima imobilizada foi caracterizada quanto à eficiência de encapsulamento, estabilidade, tamanho médio e sua distribuição e morfologia. No estudo de estabilidade verificou-se, em todas as condições de processo, que a enzima imobilizada manteve maior estabilidade em relação ao extrato bruto durante os 28 dias analisados. Nas análises de microscopia óptica, as microcápsulas formadas apresentaram formas esféricas, com média de tamanho de 400 e 100 ?m para os bicos de 2 e 0,5 mm, respectivamente. Nas análises por microscopia eletrônica de varredura (MEV), as microcápsulas foram secas em estufa e mantiveram as formas esféricas, com redução de tamanho, mesmo após esse processo de secagem. Usando o bico atomizador de 2 mm nas concentrações de 2 e 4% (m/v) de CaCl2, a microcápsula seca apresentou um tamanho de 139 e 150 µm, respectivamente, contra a média de 400 µm das hidratadas. Para o bico atomizador de 0,5 mm nas concentrações de 2 e 4% (m/v) de CaCl2, as microcápsulas apresentaram tamanhos de 15 e 6 µm, respectivamente / Abstract: This work was divided into three stages and aimed the characterization, purification and encapsulation of the lipase from a strain of Burkholderia cepacia. For enzyme production, the microorganism was grown in medium containing salts, yeast extract, bacteriological peptone and 6% soybean oil, liquid fermentation in bioreactor type Bioflo III. The fermentations were performed at 150 rpm and 30ºC. After 96 hours, the supernatant was separated from the cells by centrifugation and it was used as enzyme extract, since the Burkholderia cepacia produces extracellular lipase. In the first stage of this work, the enzymatic activity and stability were analyzed under different conditions and in the presence of different ions. The crude extract showed activity using olive oil as substrate. The optimum temperature was 37ºC, however at 50ºC the enzyme remained activity, with loss of activity of 10% compared to activity at 37°C. The calculation of activation energy, ARRHENIUS equation, was 10.1 kJ/mol. The effect of pH on hydrolysis of olive oil by lipase was investigated in the pH range between 3 and 11. About 80% of the activity was kept in the pH range between 5 and 7, with maximum activity at pH 8. Reasonable activities were obtained even in the more acidic pH values and lower activities in the values between 9 and 11. At pH 11 we observed a decrease of 80% of activity relative to the optimal activity of the enzyme. The stability of the enzyme activity of the crude extract in different buffers and pH values of 5, 8 and 11 was also investigated, where the 4 hour incubation at different pH values, the enzyme was stable. A stability study with different ions for incubation the lipase for 30 days was also realized. Ions such as Mn2+, Co2+, I- and Ca2+ remained stable or increased enzyme activity but the enzyme was inhibited in the presence of Fe2+, Hg2+ and Al3+. The determination of kinetic parameters was performed at 37°C and pH 8. The data obtained experimentally allowed obtaining the kinetic curve, which showed the influence of oil-water emulsion containing different proportions of olive oil (0.1 to 50% v/v) at the rate of hydrolysis by the lipase. According to the method of LINEWEAVER-BURK, it was calculated Km and Vmax values of 43.90 mg/mL and 0.0258 U/mg, respectively. The second step was to purify the lipase from Burkholderia cepacia in a polyethylene glycol (PEG)/phosphate aqueous two-phase system. Stock solutions were prepared with PEG molecular mass of 1500, 4000 and 6000 Da (50% w/w) and phosphate buffer in pHs 6, 7 and 8 (20% w/w KH2PO4/K2HPO4). A factorial design 23 was made to evaluate the molecular mass of PEG, pH and length of the tie-line in the partition coefficient of the enzyme specific activity. The results showed that higher values of specific activity of the enzyme were obtained when using the lower molecular mass PEG (1500 Da) at pH 6 and 8 and greater tie-line length. After purification, 33 kDa was determined for the molecular mass and 6.0 for the isoelectric point of the lipase from Burkholderia cepacia. In the third stage, the enzyme was encapsuled by ionic gelation using sodium alginate and calcium chloride. For the encapsulation, a factorial design 22 was made with 2 and 0.5 mm atomizer sizes and CaCl2 (2 and 4% w/v) concentration. The immobilized enzyme was characterized as to its encapsulation efficiency, stability, size and distribution size, and morphology. In the 28 day stability study, it was found in all process conditions that the immobilized enzyme remained more stable compared to the crude extract. In optical microscopy, the formed microspheres were spherical with average size of 400 and 100 µm for the 2 and 0.5 mm nozzles respectively. In the scanning electron microscope (SEM) analyses, the microspheres were dried in oven and maintained their spherical shapes with size reduction, even after the drying process. Using a 2 mm atomizer in the 2 and 4% (w/v) CaCl2 concentrations, the dry microcapsule presented a size of 139 and 150 µm respectively, against the average of hydrated 400 ?m. For the 0.5 mm atomizer in 2 and 4% (w/v) concentrations the dry microcapsules were 15 and 6 ?m, respectively / Doutorado / Sistemas de Processos Quimicos e Informatica / Doutor em Engenharia Química

O amido de milho é uma importante fonte de energia para os seres humanos, além de ser um polímero natural utilizado nas indústrias químicas, farmacêuticas e de alimentos. O amido possui grande capacidade de retenção de água e gelatinizam na presença de água e em elevadas temperaturas. Nestas condições, suas propriedades reológicas e mecânicas se modificam e fazem com que seja interessante o uso na fabricação de diversos materiais. Os processos de aglomeração e recobrimento de partículas, em combinação com outros métodos, podem proporcionar o aprimoramento de ingredientes alimentícios em pó, por meio da alteração do tamanho e forma das partículas. Além disso, o processo de aglomeração também permite alterar o índice de compactação, incorporar aditivos às partículas e aplicar películas de revestimento. Assim, este trabalho teve como objetivo realizar o estudo do tamanho e forma de partículas de amido de milho aglomeradas com alginato de sódio, produzidas por meio de formação de gotas a partir de suspensão de amido-alginato de sódio, e posterior gelificação iônica em cloreto de cálcio. Os processos de aglomeração e recobrimento foram estudados por meio dos métodos de gotejamento e método de dispersão em óleo. O método de gotejamento, ou dripping, consistiu no gotejamento da suspensão de amido-alginato em solução de cloreto de cálcio. O método de agitação em líquidos imiscíveis consistiu na gelificação iônica da suspensão sob agitação continua, em que o óleo de soja é a fase contínua e a suspensão de amido-alginato é a fase dispersa. Após a secagem em estufa a 60 °C, as partículas foram caracterizadas pelas análises de tamanho e forma. As alterações na microestrutura dos grânulos e investigação da resistência foram observadas por ensaios de calorimetria diferencial de varredura. As partículas também foram caracterizadas por análises de imagem de tamanho e forma. Os grânulos de amido foram produzidos utilizando-se a partir de suspensões de amido-alginato, e resultaram em frações de amido iguais a (50, 60, 70, 80 e 90)%. O aumento da concentração de amido de milho na suspensão de alginato resultou no aumento da viscosidade e também grânulos secos com maior tamanho. Para o método de dispersão em óleo, o aumento da frequência de agitação de 4 Hz para 11 Hz produziu menores tamanho de gota e, consequentemente, em grânulos secos com menores tamanhos. A aplicação destas técnicas de aglomeração por formação de gotas pode ser uma ferramenta útil para a produção de microcápsulas ou agregação de sistemas particulados contento dois ou mais ingredientes com distribuições de tamanho distintas. / Cornstarch is an important source of energy for humans, as well as being a natural polymer seen in the chemical, pharmaceutical and food industries. Starch has a high retention capacity of water and gelatin in the presence of water and in heat. Under these conditions, its rheological and mechanical properties change and make it interesting to use as a material medium. Agglomeration and particle coating processes, in combination with other methods, can provide the enhancement of powdered food ingredients by altering the size and shape of the particles; agglomeration process can also replace, adopt applications and apply coating films. The aim of this work is to study the size and shape of particles of agglomerated particles with sodium alginate produced by the formation of droplets from the suspension of sodium starch alginate and subsequent ionic gelation in calcium chloride. The agglomeration and recoating processes were studied by means of the drip methods and oil dispersion method. The dripping method consisted of the drip of the starch-alginate suspension in to calcium chloride solution. The method of stirring in immiscible liquids consisted of ionic gelation of the suspension under continuous stirring, wherein the soybean oil is the continuous phase and the starch-alginate suspension is the dispersed phase. After oven drying at 60 ° C, the particles were characterized by size and shape analyzes. Changes in microstructure and resistance of the beads investigation were observed by differential scanning calorimetry assays. Particles were also characterized by size and shape image analyzes. The starch granules were produced using starch-alginate suspensions, and resulted in starch fractions equal to (50, 60, 70, 80 and 90%). Increasing the concentration of corn starch in the alginate suspension resulted in increased viscosity and also larger dried granules. For the oil dispersion method, increasing the stirring frequency from 4 Hz to 11 Hz produced smaller droplet size and consequently smaller dried granules. The application of these agglomeration techniques by droplet formation can be a useful tool for the production of microcapsules or aggregation of particulate systems containing two or more ingredients of different size distribution.