Avaliou-se presença de espaços vazios, presença de fendas na interface cimento/dentina, o perímetro de penetração do cimento obturador nos canais e nos istmos.e a ação antimicrobiana intra-dentinária do cimento endodôntico (AH Plus) quando submetido ou não a agitação ultrassônica no momento da obturação. Para a avaliação do preenchimento de istmos foram utilizadas 30 raízes mesiais de primeiros molares inferiores, as quais foram divididas em 2 grupos (n=15). A instrumentação foi realizada com o sistema reciprocante Reciproc (R25) e finalizado com instrumento rotatório Mtwo (35.04). Em seguida, o cimento AH Plus foi espatulado com Rodamina B e foi inserido nos canais com espiral de Lentulo no35; o cimento do grupo 1 foi submetido a agitação ultrassônica enquanto o do grupo 2 não foi agitado, sendo que todos os canais foram obturados por meio da técnica do cone único e armazenados em estufa a 37oC durante 7 dias. As raízes foram seccionadas transversalmente a 2, 4 e 6 mm do ápice radicular para análise em esteromicroscópio e microscopia confocal a laser. Para a avaliação da ação antimicrobiana foram utilizados 30 incisivos de bovinos, os quais foram seccionados em cilindros de 6 mm de espessura. Os canais tiveram seu diâmetro padronizado com uma lima tipo K no 80 e então contaminados com Enterococcus faecalis (ATCC 29212) por 4 dias. Após esse período os espécimes foram divididas em 3 grupos (n=30). No grupo 1 o cimento foi agitado com o ultrassom e obturados com a técnica de cone único; no grupo 2 o cimento não foi agitado e o grupo 3 foi o grupo controle. As raízes foram seccionadas longitudinalmente no sentido vestíbulopalatino e coradas com corante LIVE/DEAD para avaliar a viabilidade bacteriana em microscopia confocal a laser. Para análise da agitação ultrassônica do cimento obturador no preenchimento de canais e istmos, foi utilizado o teste não paramétrico de Mann-Whitney (p <0.05) e para a análise da ação antimicrobiana intradentinária, foi utilizado o teste não paramétrico Kruskal-Waliis e o Teste Dunn (p <0.05). Os resultados indicaram que a agitação ultrassônica do cimento AH Plus favoreceu o preenchimento de istmos e a penetração intratubular. A agitação ultrassônica do cimento AH Plus reduziu significantemente a viabilidade bacteriana. Conclui-se que a agitação ultrassônica do cimento AH Plus favoreceu maior penetração intratubular, menor presença de fendas, menos espaços vazios e maior ação antimicrobiana. / In this study it was assessed the presence voids, gaps in the sealer/dentin interface, the intratubular sealer penetration in canals and isthmuses and intratubular antimicrobial effect of the AH Plus sealer when ultrasonic activation was performed during obturation. 30 mesial roots of mandibular first molars were selected and divided into 2 groups (n=15). Root canal instrumentation was performed with the reciprocating system Reciproc (R25) and finished with the Mtwo (35.04) rotary instrument. Then the AH Plus sealer was mixed with Rhodamine B and was inserted into the root canals with a Lentulo spiral #35. In group 1 the ultrasonic activation of the sealer was performed while in group 2 was not activated. Then, the root canal filling was performed using the single cone technique. After 7 days of storage the roots were sectioned at 2, 4 and 6 mm from the apex and analyzed in the stereomicroscope and by confocal laser scanner microscopy. To the antimicrobial test 30 bovine incisors cut into cylinders with 6 mm in thickness were used. The root canals diameter was standardized with a K file # 80 and then contaminated with Enterococcus faecalis (ATCC 29212) for 4 days. After this period the samples were divided into 3 groups (n = 30). In group 1 the ultrasonic activation of the sealer was perfomed and filled with single cone technique; in group 2 the sealer was not ultrasonic activated and in group 3 the canals were not obturated (control group). The roots were sectioned in the buccal-palatal direction and stained with LIVE / DEAD stain to assess the bacterial viability by confocal laser microscopy. For analysis of ultrasonic activation of the sealer on the filling quality of the canals and isthmuses, a non-parametric Mann-Whitney test (p <0.05) was used. For the analysis of the intradentin antimicrobial action, a Kruskal-Waliis nonparametric and Dunn\'s test was used (p <0.05). The results showed that the ultrasonic agitation of the AH Plus sealer favored the isthmus filling and the intratubular penetration. Also, the ultrasonic activation of AH Plus sealer reduced bacterial viability. It was concluded that the ultrasonic agitation of the sealer favored higher intratubular penetration, lower presence of gaps and voids and higher antimicrobial activity.

A rodamina B e outros marcadores fluorescentes vêm sendo empregados na avaliação morfológica da interface de adesão com o auxílio de microscopia confocal de varredura a laser. Acrescentando-se rodamina B a sistemas adesivos, é possível observar as características de espessura da camada de adesivo, micromorfologia da camada híbrida, extensão e quantidade de tags, bem como diagnosticar defeitos ou alterações na interface de adesão. A literatura revela, entretanto, uma falta de precedentes sobre as quantidades de marcadores nos componentes resinosos. Além disso, ainda não se avaliou o efeito da concentração de rodamina B sobre a qualidade da análise da interface de adesão, bem como sobre o comportamento fotofísico dos marcadores nos sistemas adesivos. Este estudo foi realizado com o propósito de sistematizar um método de adição de rodamina B a sistemas adesivos, incluindo-se estratégias para a determinação de concentrações mínimas de rodamina nos componentes resinosos, porém, ainda viáveis para a realização da análise morfológica da interface de adesão através de microscopia confocal de varredura a laser. Para tal, os sistemas adesivos não simplificados e considerados padrão-ouro em suas categorias Adper&#x2122; Scotchbond&#x2122; Multi-Purpose (convencional de três passos) e Clearfil&#x2122; SE Bond (auto-condicionante de dois passos) foram modificados com rodamina B em cinco diferentes concentrações: C1 (0,5 mg/mL), C2 (0,10 mg/mL), C3 (0,02 mg/mL), C4 (0,004 mg/mL) e C5 (0,0008mg/mL) e o comportamento fluorescente da rodamina nos adesivos foi avaliado tanto no âmbito espectroscópico (espectroscopia de fotoluminescência), como no âmbito microscópico (microscopia confocal de varredura a laser). Para a modificação dos adesivos, utilizou-se uma técnica precisa de proporcionamento na qual o marcador fluorescente foi incorporado às resinas fluidas a partir de soluções de rodamina B em etanol. Os espectros de fluorescência mostraram diferenças nos máximos das bandas de emissão e de excitação, de acordo com as concentrações de rodamina B em cada material dispersante. A análise microscópica dos espécimes em dentina confirmou que é possível utilizar sistemas adesivos contendo rodamina B em concentrações muitas vezes menores do que as mencionadas na literatura. Ambos os sistemas avaliados puderam ser visualizados, microscopicamente, quando marcados com rodamina B nas três concentrações pré-selecionadas para a avaliação da interface de adesão: C2 (0,10 mg/mL), C3 (0,02 mg/mL) e C4 (0,004 mg/mL), sendo que C3 forneceu os melhores resultados em relação ao contraste e à saturação nas fotomicrografias. Concluiu-se que o comportamento fotofísico da rodamina B é influenciado tanto pela concentração, como pelo sistema adesivo no qual o marcador está dissolvido. A concentração de rodamina B também interfere na qualidade da análise morfológica da interface de adesão, sendo que o excesso do marcador pode dificultar a distinção de detalhes micromorfológicos. / Rhodamine B and other fluorescent markers have been used for the assessment of the bonding interface via confocal laser scanning microscopy. By adding rhodamine into adhesive systems, it is possible to study morphological characteristics of the hybrid layer, the extent and amount of resin tags and the adhesive layer thickness, as well as detecting potential defects or alterations at bonding interface. Meantime the literature reveals lack of standards among the quantities of fluorescent markers in resin-based polymers. The effects of rhodamine B concentration on the quality of the microscopic analysis, as well as on the photophysics of labeled adhesive systems were not assessed up to now. This study aimed to systematize methodologies for adding rhodamine B into adhesive systems, including suitable strategies for determining the minimum serviceable rhodamine concentrations to perform the morphological analysis of the bonding interface. Two non-simplified adhesive system considered gold standard categories Adper&#x2122; Scotchbond&#x2122; Multi-Purpose (3 steps conventional) and Clearfil&#x2122; SE Bond (2 steps self-etching) were modified with rhodamine B at five concentrations: C1 (0.5 mg/ml), C2 (0.10 mg/ml), C3 (0.02 mg/ml), C4 (0.004 mg/ml) and C5 (0.0008 mg/ml ) and the fluorescent behavior of the labeled resins was assessed both by photoluminescence spectroscopy and by confocal laser microscopy The preparation of the labeled resins was proceed by means of a precise proportioning technique in which the fluorescent marker was incorporated into the bonding agents from solutions of rhodamine B in ethanol. The fluorescence spectra showed differences in the intensity and wavelength fluorescence according to the rhodamine B concentration. Microscopic analysis of the dentin specimens confirmed adhesive systems can be modified with rhodamine B at far lower concentrations than those mentioned in the literature. Both evaluated systems were microscopically visualized while labeled with Rhodamine B at three pre-selected concentrations: C2 (0.10 mg/mL), C3 (0.02 mg/ml) and C4 (0.004 mg/ml), and C3 offered the best outcome with respect to contrast and saturation in photomicrographs. It was concluded that the photophysical behavior of rhodamine B depends on both the concentration and labeled adhesive system. The rhodamine B concentration also interferes with the quality of morphological analysis of the bonding interface, and the excess of the fluorescent marker may jeopardize the discrimination of morphological details.

Orientador: Giselle Maria Marchi Baron / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-26T05:50:55Z (GMT). No. of bitstreams: 1 Gaglianone_LiviaAguilera_D.pdf: 3416784 bytes, checksum: 47ff2260453d6852e725b4b6356954ae (MD5) Previous issue date: 2014 / Resumo: O objetivo do presente estudo foi avaliar o efeito do pré-aquecimento e da composição de infiltrantes no grau de conversão (GC), nas propriedades físicas e profundidade de penetração em lesões cariosas de mancha branca em esmalte. Os grupos foram assim divididos, de acordo com a composição do material: [Produto comercial Icon®]; [INF 1 - 25% BisEMA, 75% TEGDMA]; [INF 2 - 25% BisEMA, 65% TEGDMA, 10% etanol]; [INF 3 - 25% BisEMA, 65% TEGDMA, 10% HEMA]; [INF 4 - 100 % TEGDMA]; [INF 5 - 90% TEGDMA, 10% etanol]; [INF 6 - 90% TEGDMA, 10% HEMA]; e cada uma dessas resinas foi testada em diferentes condições de temperatura (25°C e 55°C). Para avaliação do GC (n=3), mensurações antes e após fotoativação das amostras (LED Bluephase 5i ¿ Ivoclar Vivadent, 1000 mW/cm2, por 60 s) foram realizadas em espectrômetro de raios infravermelhos e o cálculo foi feito considerando a razão entre espectros não-polimerizados e polimerizados do material. As mesmas amostras de GC (n=3) foram utilizadas no teste de sorção (So) e solubilidade (SL), sendo que as amostras polimerizadas foram pesadas após dessecação inicial (m1), armazenadas em água destilada por 7 dias e pesadas para obtenção de m2, e após nova secagem, foram novamente pesadas até obtenção da massa final (m3). O módulo de elasticidade (ME) e resistência à flexão (RF) dos espécimes (n=10), em forma de barra (7 mm X 2 mm X 1 mm), foram obtidos com teste de 3 pontos em Máquina de Ensaio Universal (0,5 mm/min). O ângulo de contato entre os materiais não polimerizados e a superfície polida de um sólido foi obtido utilizando um goniômetro com uma câmera acoplada. A análise da profundidade de penetração dos materiais testados em lesões artificiais de mancha branca sub-superficiais foi realizada por meio de imagens obtidas por Microscopia Confocal de Varredura a Laser (MCVL), com impregnação de marcadores fluorescentes (rodamina e fluorosceína de sódio) aos materiais avaliados e fragmentos dentais cariados, respectivamente. Os dados foram submetidos à ANOVA 2-critérios e teste de Tukey (?=5%). O aumento da temperatura não influenciou ME e RF. Maior ME foi atribuído ao INF 3 enquanto que os menores valores foram obtidos por INF 5. A 25oC, nenhuma resina testada alcançou 50% de conversão, mas, todos os valores de GC foram superiores a 60% quando pré-aquecidos. Houve redução significante de So a 55oC. Avaliando SL, INF 5 (TEGDMA + etanol) mostrou altos valores, enquanto Icon aquecido foi associado a baixa solubilidade. A maioria dos grupos apresentou aumento do ângulo de contato, a 55oC. Em contrapartida, grupos contendo HEMA mostraram diminuição do ângulo após aquecimento do material. Não houve diferença entre as temperaturas para profundidade de penetração. O material à base de TEGDMA (INF 4) apresentou profundidade de penetração mais homogênea, enquanto que o Icon foi associado a infiltração mais superficial. Pode-se concluir que o aumento prévio da temperatura do infiltrante foi capaz de afetar algumas das propriedades avaliadas (principalmente, GC e So/SL), e que essa abordagem foi mais vantajosa para materiais de composição mais hidrófoba / Abstract: The aim of the present study was to evaluate the composition of infiltrant blends and the pre-heat effect on degree of conversion (DC), physical properties and penetration depth into enamel white spot caries lesions. Groups were set up as follows: [commercially available product Icon®]; [INF 1 - 25% BisEMA, 75% TEGDMA]; [INF 2 - 25% BisEMA, 65% TEGDMA, 10% etanol]; [INF 3 - 25% BisEMA, 65% TEGDMA, 10% HEMA]; [INF 4 - 100 % TEGDMA; INF 5 - 90% TEGDMA, 10% etanol]; [INF 6 - 90% TEGDMA, 10% HEMA]; and each one of these resins was tested under different temperature conditions (25°C and 55°C). DC (n=3) was measured using infrared spectroscopy and calculated by comparing the spectra from samples before and after light activation (LED Bluephase 5i ¿ Ivoclar Vivadent, 1000 mW/cm2, for 60 s). For water sorption (WS) and solubility (SL) test, the same DC samples (n=3) were subjected to water degradation method. After desiccation, polymerized samples were initially weighed (m1), stored in distilled water for 7 days (m2), and, after further drying, a constant mass (m3) were obtained. For the mechanical test (elastic modulus - E / flexural strength - FS), ten bar shaped specimens (7 mm X 2 mm X 1 mm) were prepared to be tested using the three-point flexural test, at a crosshead speed of 0.5 mm/min. Unpolymerized infiltrants contact angle in relation to a polished solid surface were obtained using a goniometer. Penetration depth into artificial enamel white spot caries lesions was evaluated using dual fluorescence confocal microscopy (rodhamine and sodium fluoroscein). All data were subjected to 2-way ANOVA and Tukey¿s test, and the significance level was set at a = 0.05. Pre-heated condition presented no influence in E and FS. Higher E values were attributed to INF3 while low ones were obtained by INF 5. At 25oC, DC did not reach 50% of conversion for all resins tested, but, with the pre-heating, all DC increased to more than 60%. There was significant WS reduction at 55oC. Analysing SL, INF 5 (TEGDMA + etanol) presented high values, and low means were found for heated Icon. Most of the groups were associated to increased contact angle, at 55oC. In contrast, the presence of HEMA led to a decrease of this parameter after material pre-heating. Finally, there was no statistical difference between temperatures for penetration depth. TEGDMA-based material (INF 4) presented more homogeneous penetration whereas Icon was associated to shallow infiltration. It can be concluded that the increase of infiltrant¿s temperature, prior polymerization step, was able to affect some of the evaluated properties (mainly DC and WS/SL). Overall, this approach was mostly beneficial for blends containing hydrophobic monomers / Doutorado / Dentística / Doutora em Clínica Odontológica

Orientador: Wander José da Silva / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-23T13:26:50Z (GMT). No. of bitstreams: 1 Goncalves_LeticiaMachado_D.pdf: 3139804 bytes, checksum: 4f3ad00e23ae5ec0336267a151dbe2e0 (MD5) Previous issue date: 2012 / Resumo: O fluconazol (FLZ) é um antifúngico amplamente utilizado no tratamento da candidose associada ao uso de prótese dental. No entanto, o sucesso desta terapia pode ser dificultado pela presença da matriz extracelular dos biofilmes de Candida albicans. Estudos já foram conduzidos avaliando o efeito do FLZ em biofilmes de C. albicans, no entanto, estudos que simulem uma condição na qual os biofilmes são desenvolvidos na presença do FLZ são escassos. Ainda, na tentativa de avaliar a organização tridimensional destes biofilmes, várias técnicas de microscopia já foram descritas, apesar de pouca atenção ser dada na análise da matriz extracelular. Frente ao exposto, o objetivo deste trabalho foi avaliar a matriz extracelular de biofilmes de C. albicans na presença de concentração salivar de FLZ. Dois estudos foram conduzidos e, em ambos, discos (10 mm x 2 mm) de resina acrílica à base de poli (metil metacrilato) foram confeccionados e cobertos com uma película de saliva sobre a qual biofilmes de C. albicans foram desenvolvidos. O primeiro estudo foi conduzido com o objetivo de definir uma metodologia para análise da matriz extracelular. Biofilmes de C. albicans ATCC 90028 foram desenvolvidos em meio de cultura sem suplementação (controle) ou suplementado com glicose ou sacarose, por 72 horas. Durante o desenvolvimento dos biofilmes foi adicionado o corante Concanavalina A (ConA) para a marcação da matriz extracelular. Após o desenvolvimento, os biofilmes foram corados com SYTO 9 para a marcação das células. As imagens obtidas por microscopia confocal foram analisadas pelo software COMSTAT. Para confirmação dos resultados, os biofilmes foram coletados e analisados bioquimicamente para determinação da composição da matriz de polissacarídeos pelo método fenol-sulfúrico. Os dados foram analisados por ANOVA seguido do teste de Tukey com nível de significância de 5%. Para o segundo estudo, biofilmes de C. albicans (ATCC 90028) e de dois isolados clínicos (P01 e P34) foram desenvolvidos em meio de cultura contendo FLZ na concentração biodisponível na saliva (2,56 ?g/mL), durante 48h. O grupo controle foi desenvolvido sem a presença de fluconazol. O número de células viáveis foi quantificado por diluição seriada, e a matriz extracelular analisada bioquimicamente, além da avaliação por microscopia confocal. Os dados foram analisados pelo teste t de Student com nível de significância de 5%. No primeiro estudo, observou-se que o uso de ConA permitiu uma marcação eficaz da matriz extracelular, confirmada pela análise bioquímica (p < 0,05). No segundo estudo, houve uma redução de 80% no número de células viáveis dos biofilmes desenvolvidos na presença de FLZ (p < 0,001). Considerando-se a proporção de polissacarídeos produzidos pelo número de células viáveis, observou-se que nos grupos experimentais, C. albicans foi capaz de produzir mais matriz extracelular do que os grupos controle (p < 0,05). Este resultado foi confirmado pelas imagens obtidas por microscopia confocal. Conclui-se que a microscopia confocal é uma ferramenta efetiva na análise da matriz extracelular de biofilmes de C. albicans, e que os biofilmes de C. albicans desenvolvidos na presença de FLZ apresentaram maior produção de matriz extracelular / Abstract: Fluconazole (FLZ) is a commonly used antifungal agent to treat patients with Candida-associated denture stomatitis. However, the success of this therapy may be compromised by the presence of extracellular matrix of C. albicans biofilms. Although studies have been conducting evaluating the effect of FLZ on C. albicans biofilms, studies that simulate the condition in which biofilms were allowed to grow in the presence of FLZ are limited. Additionally, in order to evaluate the three-dimensional organization of these biofilms, several microscopic techniques have been described, although little attempt has been paid to assess the extracellular matrix. The objective of this study was to investigate the extracellular matrix of C. albicans biofilms in the presence of salivary concentration of FLZ. For this, two studies were conducted. For both studies, discs (10 mm x 2 mm) were fabricated using poly (methyl methacrylate). Salivary pellicle was formed on disc surface and C. albicans biofilms were developed. In the first study a methodology for analyzing the extracellular matrix was defined. C. albicans ATCC 90028 biofilms were developed in culture media without (control) or with supplementation by glucose or sucrose for 72 hours. During development, biofilms were stained with Concanavalin A (ConA) in order to label the extracellular matrix. After, cells were also labeled with SYTO-9. Images obtained by confocal microscopy were analyzed by COMSTAT software. In order to confirm the results, biofilms were subjected by the biochemical phenol-sulfuric method. Data were analyzed by ANOVA followed by Tukey's test at a significance level set at 5%. For the second study, biofilms of an ATCC 90028 and two clinical isolates (, P01 and P34) were developed for 48 hours. FLZ at 2.56 ?g/mL, concentration bioavailable in saliva, was added to culture media of experimental groups. Biofilms were investigated for the number of viable cells by serial dilution and extracellular matrix production was analyzed by phenol-sulfuric method and confocal microscopy. Data were analyzed by Student's t-test, with significance level set at 5%. In the first study, the use of ConA provided an effective labeling of extracellular matrix, which was confirmed by phenol-sulfuric method (p < 0.05). In the second study, a reduction of 80% was observed in the number of viable cells for C. albicans biofilms developed in the presence of FLZ (p < 0.001). Considering the proportion of polysaccharides produced by the number of viable cells, it was observed that in the experimental groups, C. albicans was able to produce more extracellular matrix than the control groups (p < 0.05). This result was confirmed by confocal images. It was concluded that confocal microscopy is an effective tool to investigate the extracellular matrix of C. albicans biofilms; and C. albicans biofilms developed in the presence of FLZ present increased extracellular matrix production / Doutorado / Protese Dental / Doutora em Clínica Odontológica

Previous research into microbial colonies and biofilms shows a significant gap in our current understanding of how bacterial structures develop. Despite the huge body of research undertaken into the formation, genetic makeup, composition, and optimal growth conditions of colonies, no study has been successful in identifying all individual bacteria in a colony in three-dimensions as a function of time. This lack of bacterial cell lineage in such a simple class of organisms is conspicuous in the light of what is known about other organisms, such as Caenorhabditis elegans [1]. In this thesis I show that using laser scanning confocal microscopy in conjunction with developments in sample preparation and post acquisition image analysis, it is possible to fully reconstruct all individual bacteria within an Escherichia coli (E. coli ) microcolony grown in viscoelastic media. Additionally, I show that by further pushing the resolution of confocal microscopes, commercial systems are capable of extracting three-dimensional information on protein structures inside bacteria at early stages of growth. This thesis is in three parts. The first part shows that by pushing the resolution of a commercial laser scanning confocal microscope system it is possible to achieve single cell resolution of a bacterial colony growing in three dimensions in a viscoelastic medium (agarose) from a seed bacterium. The growth of individual bacteria is examined as the concentration of agarose in the media is altered. Results show there is a nonlinear dependence between the rate of growth of a bacterium and the concentration of the agarose in the media with a peak in growth rate at 3% (weight) concentrations of agarose in M9 media. The second part of this work presents a study of how an initially two-dimensional colony growing between a glass slide and agarose gel suddenly invades the third spatial dimension by buckling. The results show that the cells within the centre of the colony flex and buckle, due to confinement by their neighbours, creating additional layers. Indeed, flexing is not limited to the buckling event but occurs throughout the early growth cycle of a colony. The final part of this thesis shows that by further pushing the resolution of confocal microscopes, commercial systems are capable of extracting three-dimensional information about the temporal evolution of the spatial distribution of the FtsZ septation ring within the cell. As the bacterial colony grows from a seed bacterium to a microcolony, the error in placing the division accurately at the cell centre is seen to increase as the number of bacteria within the colony increases and spatial confinement occurs.

Whey is a by-product of cheese manufacture, containing a mixture of proteins of commercial value, each having unique attributes for nutritional, biological and food ingredient applications. A tremendous amount of whey, normally treated as a waste product, is produced worldwide each year. This work describes the cation-exchange adsorption of the two major whey proteins, alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) with the purpose of optimising a process for isolating them from whey. Adsorption of pure BLG and ALA was studied onto SP Sepharose FF using 0.1M acetate buffer. Batch experiments were carried out at various pH values for ALA and BLG, and the relevant Langmuir isotherm parameters, dissociation constant, Kd, and maximum binding capacity, qm, were determined. The optimum pH for separation was chosen to be pH 3.7. At pH 3.7, both Kd and qm pertaining to ALA were found to have higher numerical values than those of BLG, implying different characteristics of adsorption of the two proteins on this adsorbent. The Kd for the former protein was almost four times larger than the latter, while qm was 1.3 times higher. Packed-bed column adsorption was performed using a 1-ml column at pH 3.7, flow rate 1 ml/min and initial concentration of 3 mg/ml for BLG and 1.5 mg/ml for ALA both in 0.1M sodium acetate buffer. The t1/2 for the resulting ALA breakthrough was 75% longer than its BLG counterpart. The above results suggest the possibility of the occurrence of competitive adsorption between the proteins when adsorbed simultaneously. In traditional batch uptake experiments, the kinetic rate constants of ALA and BLG in both the single- and two-component systems were determined using the simple kinetic model. The values so obtained implied that BLG was adsorbed faster than ALA. In the confocal laser scanning microscopy experiments, the different behaviour of ALA and BLG in the single-component system with regard to their penetration within the adsorbent beads suggested that the two proteins have different transport mechanisms governing their adsorption. The two-component system results showed that ALA was able to displace BLG in spite of the lower affinity of the former protein to the adsorbent. The packed-bed adsorption and elution of a mixture of ALA and BLG were then investigated under the above conditions but using a 5-ml column. BLG breakthrough occurred first, and its concentration in the outlet exceeded its feed value by 1.6 fold before declining to the feed value, followed by the breakthrough of ALA. ALA displaced and eluted all the BLG from the column in a pure form. Pure ALA could then be eluted with good recovery. The single- and two-component breakthrough curves for ALA and BLG were simulated by the simple kinetic model using the isotherm parameters, but the overshoot phenomenon could only be predicted after correcting these parameters. The evidence of the competitive nature of adsorption observed in binary mixtures was used to develop a facile separation procedure for the two proteins from aqueous solutions of whey concentrate powders. A novel consecutive two-stage separation process was developed to separate ALA and BLG from whey concentrate mixtures. Almost all the BLG in the feed was recovered, with 78% being recovered at 95% purity and a further 20% at 86% purity. In addition, 67% of ALA was recovered, 48% at 54% purity and 19% at 60% purity. The correction factors employed for the pure binary mixture were used to simulate the breakthrough curves of the two proteins in experiments conducted with whey concentrate in each of the two stages of the novel separation process, and there was agreement between the experimental and theoretical results.

Physical forces in the cellular microenvironment play an important role in governing cell function. Forces transmitted through the cell cause distinct deformation of the nucleus, and possibly play a role in force-mediated gene expression. The work presented in this thesis drew upon innovative strategies employing simultaneous atomic force and laser-scanning confocal microscopy, as well as parallel optical stretching experiments, to gain unique insights into the response of eukaryotic cell nuclei to external force. Non-destructive approaches confirmed the existence of a clear anisotropy in nuclear mechanical properties, and showed that the nucleus' mechanical response to extracellular forces is differentially governed by both nuclear and cytoskeletal prestress: nuclear prestress regulates shape and anisotropic deformation, whereas cytoskeletal prestress modulates the magnitude and degree of deformation. Importantly, the anisotropic mechanical response was conserved among diverse differentiated cell types from multiple species, suggesting that nuclear mechanical anisotropy plays an important role in cell function.

Serotonin (5-HT)-containing neuroepithelial cells (NECs) of the gill filament are believed to be the primary O2 chemosensors in fish. In the mammalian carotid body (CB), 5-HT is one of many neurotransmitters believed to play a role in transduction of hypoxic stimuli, with acetylcholine (ACh) being the primary fast-acting excitatory neurotransmitter. Immunohistochemistry and confocal microscopy was used to observe the presence of the vesicular acetylcholine transporter (VAChT), a marker for the presence of ACh, and its associated innervation in the gills of zebrafish. VAChT-positive cells were observed primarily along the afferent side of the filament, with some cells receiving extrabranchial innervation. No VAChT-positive cells were observed in the gills of goldfish; however, certain key morphological differences in the innervation of goldfish gills was observed, as compared to zebrafish. In addition, in zebrafish NECs, whole-cell current is dominated by an O2-sensitive background K+ current; however, this is just one of several currents observed in the mammalian CB. In zebrafish NECs and the CB, membrane depolarization in response to hypoxia, mediated by inhibition of the background K+ (KB) channels, is believed to lead to activation of voltage-gated Ca2+ (CaV) channels and Ca2+-dependent neurosecretion. Using patch-clamp electrophysiology, I discovered several ion channel types not previously observed in the gill chemosensors, including Ca2+-activated K+ (KCa), voltage-dependent K+ (KV), and voltage-activated Ca2+ (CaV) channels. Under whole-cell patch-clamp conditions, the goldfish NECs did not respond to hypoxia (PO2 ~ 11 mmHg). Employing ratiometric calcium imaging and an activity-dependent fluorescent vital dye, I observed that intact goldfish NECs respond to hypoxia (PO2 ~ 11 mmHg) with an increase in intracellular Ca2+ ([Ca2+]i) and increased synaptic vesicle activity. The results of these experiments demonstrate that (1) ACh appears to play a role in the zebrafish, but not goldfish gill, (2) goldfish NECs likely signal hypoxic stimuli primarily via the central nervous system (CNS), (3) goldfish NECs express a broad range of ion channels as compared to the NECs of zebrafish, and (4) goldfish NECs rely on some cytosolic factor(s) when responding to hypoxia (PO2 ~ 11 mmHg). This thesis represents a further step in the study of neurochemical and physiological adaptations to tolerance of extreme hypoxia.

Neste estudo avaliou-se o efeito in vitro de 4 compostos isolados de espécies vegetais, as amidas piplartina e piperina, a lignana grandisina e o alcaloide epiisopiloturina; 1 composto isolado da pele de anfíbio, o peptídeo antimicrobiano dermaseptina 01; e de 6 extratos etanólicos obtidos de vegetais, Piper tuberculatum, P. crassinervium, P. diospyrifolium, P. fuligineum, P. gaudichaudianum e Pothomorphe umbellata em adultos (machos e fêmeas com 49 dias) e esquistossômulos (recém-transformados, 1, 3, 5 e 7 dias) de Schistosoma mansoni linhagem BH. O estudo avaliou: 1) a viabilidade de vermes adultos; 2) a capacidade reprodutiva, avaliada pelo acasalamento e oviposição; 3) o efeito no tegumento em parasitas adultos; 4) a viabilidade e o efeito no tegumento em esquistossômulos; 5) a citotoxicidade de compostos e extratos em células de mamífero (célula Vero). Além disso, com microscopia confocal, neste estudo é proposto um novo modelo experimental que avalia, quantitativamente, o efeito de esquistossomicidas no tegumento dos esquistossomos. / In this study, the in vitro effect of 4 compounds isolated from plant species, amides piplartine and piperine, the lignin grandisin and alkaloid epiisopiloturine; 1 compound isolated from amphibian skin, the antimicrobial peptide dermaseptin 01; and 6 ethanolic extracts of plants, Piper tuberculatum, P. crassinervium, P. diospyrifolium, P. fuligineum, P. gaudichaudianum and Pothomorphe umbellata was evaluated in adults worm pairs (49-day-old) and schistosomula (newly-transformed, 1-, 3-, 5-, and 7-day-old) of Schistosoma mansoni BH strain. The effect of compounds and extracts against schistosomes was examined regarding: 1) adult worms survival, 2) the reproductive fitness as assessed by mating and oviposition; 3) alterations on adult worms tegumental surface; 4) viability and morphological alterations on schistosomula; 5) the cytotoxicity of compounds and extracts in mammalian cells (Vero cells) In addition, this study shows a new experimental model to evaluate quantitatively the effect of schistosomicidal drugs on the tegument of the schistosomes.

The house fly, Musca domestica can transmit human pathogens including Escherichia coli O157:H7 through regurgitation of ingested bacteria from the crop which is a foregut organ of house fly and stores the excess ingested nutrients. Interactions between the ingested bacteria and the crop have a direct influence on bacteria persistence, survival and ultimately fly vector competence. In this research, in situ crop vessel assay was developed to investigate bacterial growth within fly crops up to 48 hours post-ingestion. Flies were fasted for 12 h prior to feeding E. coli O157:H7 pEGFP and then fed bacteria with red food color which was added to confirm that flies had consumed the bacteria. After feeding, flies with red abdomens were aseptically dissected and crops were removed and maintained in sterile phosphate buffered saline in microtiter plates held at 32˚C. For each time point (0, 24 and 48 hours post-ingestion), five crops were homogenized individually using a tissue grinder and bacterial levels (CFU/crop) were monitored using plate counts. Confocal microscopy of intact crops was used to monitor biofilm development. There was no statistical difference in cell numbers (CFU/crop) over the 48 h incubation period. Microscopy showed that upon prolonged incubation, GFP-expressing E. coli within the crop produced biofilms. This method showed greater reproducibility in studying crop bacteria level than using a live fly feeding study. But this system was not recommended to study the interaction between bacteria and the crop of housefly.