Thesis (Ph. D.)--University of Chicago, 1921. / "Private edition, distributed by the University of Chicago libraries, Chicago, Illinois." "Reprinted from the American journal of physiology, vol. LVII, no. 2, September, 1921." Bibliography: p. 385-386. Also available on the Internet.

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The plant cuticle is located on most surfaces of the plant from seeds to leaves from stem to petal, this is to allow a direct interface between the plant and its environment. These cuticles act as a barrier to prevent waters loss from the plant to the environment and penetration of compounds through the cuticle, like agrochemicals and formulations. The leaf cuticle provides an ideal surface to try to penetrate agrochemicals through. The leaf surface has a larger surface area then most surfaces of the plant allowing ease of application of formulation via spraying. This makes the study of the leaf and its cuticle important, with the interaction of the formulation and the cuticle an area of interest. The main purpose of this thesis is to investigate the applications of a relatively new imaging technique called scanning ion conductance microscopy (SICM). This new technique is utilised to image live cells with the intention to characterise the living processors on the surface. SICM has not been used to image leaf surface before, but its non-contact nature and large z axis range makes it ideal for surface analysis. The first part of this thesis is to describe the comparison of SICM with other conventional techniques used to image leaf surfaces. For example, atomic force microscopy (AFM) and scanning electron microscopy (SEM) and asses the strengths and weaknesses of the technique for leaf imaging. This was achieved by imaging various leaf surfaces and surface features like epicuticular wax (EW) crystals and stomata. Also the possible research routes for the SICM were identified and experiments conducted to ascertain the abilities to perform them. This resulted in wetting being imaged and imaging the drying of a formulation. The other purpose of this thesis is to investigate the possibility of live leaf imaging and characterisation, and the implications of adjuvants on live leaves. This was achieved by thermal characterisation of different leaf surfaces in two states, them being live and intact (but dried). This allowed the understanding of the impact water has on the cuticle and the importance of studying live leaves. This shows that water has a plasticizing effect on the cuticle waxes, and also effects the structure of the cuticle. AFM with scanning thermal microscope (SThM) with local thermal analysis (LTA) were also utilised to investigate the impact of two adjuvants on the surface of live leaf cuticle. These were Brij 98 and Tris (2-ethylhexyl) phosphate (TEHP), Brij 98 in a non-ionic ethoxylated surfactant, while TEHP is a phosphoric acid ester known for its properties has a plasticizer. Both AFM and LTA showed that both resulted in the plasticizing of the cuticle with the area affected showing depression in melting transition compared with that of the native leaf surface. The thesis also shows that it is possible to characterise the impact of adjuvants on live leaf cuticles. This thesis has shown the importance of new techniques being used to image and characterise the leaf surface, showing that image wetting as a possible research route for SICM. The new techniques have resulted in new experiments being performed that provide insight into the interactions of the cuticle with formulations and components of formulations. Also the importance of water in understanding the structure of the cuticle.

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As a high-resolution wide-field near-surface microscopy, total internal reflection fluorescence microscopy (TIRFM) has been widely applied for the study of biomolecules. Unlike those costly, sample consuming and time consuming traditional detection assays, the application of TIRFM enable the direct quantification of biomolecules in a sample pretreatment and enrichment free fashion. Taking advantages of the TIRFM imaging system, in this thesis we have applied the TIRFM imaging system to directly quantify the content of different cancer associated biomarkers. Four different detection approaches for direct cancer biomarkers quantification with the aid of TIRFM were herein presented respectively. In Chapter 2, a direct quantification of nasopharyngeal carcinoma associated miRNAs was described. In the assay, five different miRNAs were chosen as the target analytes, which hybridized with the synthetic complementary LNA, probe in solution. The duplex was labeled with intercalating fluorescence dye YOYO-1 and the signal was then detected by the TIRFM-EMCCD imaging system. The LNA probe exhibited a high binding affinity towards the complementary target miRNAs and a limit of detection of 8 pM was achieved. Since the LOD is far below the reported concentration of miRNAs found in body fluids, this developed assay is of high potential to serve as a tool for non-invasive detection of miRNAs for early disease diagnosis. In Chapter 3, an advanced single-molecule based assay for direct circulating miRNAs detection was developed. The assay was demonstrated to be capable of differentiating the expression of a nasopharyngeal carcinoma (NPC) up-regulator hsa-mir-205 (mir-205) in serum collected from patients of different stages of NPC. To overcome the background matrix interference in serum, locked nucleic acid modified molecular beacon (LNA/MB) was applied as the detection probe to hybridize, capture and detect target mir-205 in serum matrix with enhanced sensitivity and specificity. A detection limit of 500 fM was achieved. The as-developed method was capable of differentiating NPC stages by the level of mir-205 quantified in serum with only 10 μL of serum and the whole assay can be completed in an hour. The experimental results agreed well with reported and while the quantity of mir-205 determined by our assay was found comparable to that of quantitative reverse transcription polymerase chain reaction (qRT-PCR), supporting that this assay can be served as a promising non-invasive detection tool for early NPC diagnosis, monitoring and staging. In chapter 4, a self-assembled protein nanofibril based online pre-concentrating sensor was developed. This solution-based hybridization assay was applied to quantified the amount of target miRNAs, mir-196a. Biotinylated locked nucleic acid (LNA) of complimentary sequence was served as the probe to capture the target miRNA analyte. The target hybridization duplex was immobilized on the backbone of the nanofibril through the biotin-streptavidin interaction. The quantification was achieved by the fluorescence intensity measured with total internal reflection fluorescence microscopy. A detection limit of 1 pM was achieved with trace amount of sample consumption. This assay showed efficient single-base mismatch discrimination. The applicability of quantifying circulating mir-196a in both normal and cancer patient’s serums was also demonstrated. In chapter 5, a magnetic nanoparticles based sandwich immunosensor with carbazole-based cyanine as the fluorescence labeling dye for the direct quantification of prostate cancer related antigen, PSA, was developed. Taking benefit of the magnetic property of the nanoparticles, the target sandwich immunocomposites can be easily online separated from the sample matrix. The as-developed assay can efficiently discriminate the target PSA from other disease related antigens and achieve a LOD of 400 fM (13 pg/mL) and a LOQ of 2 pM (0.66 ng/mL). As the whole detection assay can be completed in 1 h with only 10 μL of sample, this assay is fast and cost effective and of high potential for early disease and cancer diagnosis, staging and monitoring

Cette thèse porte sur l'utilisation et le développement de techniques de microscopie multiphotonique pour l'imagerie d'échantillons biologiques humains. Une plateforme d'imagerie multiphotonique utilisant les contrastes non linéaires sans marquage tels que la fluorescence à deux photons, la génération de seconde harmonique, et les mécanismes Raman cohérent (CARS et SRS) a été conçue et développée au cours de cette thèse, et les travaux expérimentaux suivant deux axes de recherche principaux sont présentés.Dans une première partie , l'imagerie tridimensionnelle et sans marquage des muqueuses du système digestif humain est comparée aux images histologiques classiques avec marquages colorimétriques. Nous montrons que les techniques multiphotoniques utilisées permettent de reconstituer la structure et de discerner les différents éléments moléculaires présents dans les tissus dans le but d'obtenir une caractérisation des zones touchées par le développement de tumeurs cancéreuses. / This thesis focuses on multiphotonic microscopy techniques development and use in order to image human biological samples. A multiphotonic imaging setup using label-free nonlinear contrasts mechanisms such as two-photons fluorescence, second harmonic generation, or stimulated Raman effect (CARS or SRS) has been designed and developped during this PhD, and I present the experimental work in two main research topics.In a first part, we compare label-free 3D imaging with classic histological imaging using colorimetric labels in human digestive system. We show that multiphotonic technics allow to reconstruct the organization and discern the molecular compounds inside the tissues, in order to get a caratérization of the cancerous tumors developpement.The second part is related to the application of our multimodal setup to the quantitative study of real active molecular compounds real time penetration into in vivo human skin. We show that multiphotonic microscopy make possible to mesure active molecules in depth 3D concentration in the skin in order to understand transcutaneous diffusion mechanisms in cosmetic and pharmacological applications.

Este trabalho tem como objetivo principal, o estudo de técnicas de microscopia para a caracterização estrutural de semicondutores, visando o desenvolvimento das técnicas de preparação de amostras, visto que a caracterização estrutural é de suma importância para a obtenção de melhores resultados no processo de produção de filmes de semicondutores do grupo III-V. Dentre as técnicas mais utilizadas na caracterização estrutural, destacam-se as técnicas de microscopia eletrônica de varredura e de transmissão, juntamente com a microscopia de força atômica. Foram utilizadas amostras semicondutoras de InGaAs/GaAs e InAs/GaAs, crescidas pela técnica de MBE (epitaxia por feixe molecular), contendo pontos quânticos, estruturas estas ricas em detalhes. Tais amostras foram preparadas e caracterizadas em cada uma das técnicas em estudo. A microscopia de varredura e de força atômica apresentam fácil preparação. Os resultados obtidos, porém mostram que a técnica de microscopia eletrônica de varredura não oferece resolução suficiente para visualização das heteroestruturas; já a técnica de microscopia de força atômica mostra resultados excelentes da topografia dos pontos quânticos. Para a microscopia de transmissão a preparação de amostras mostra-se muito difícil e demorada, entretanto, o resultado obtido foi muito satisfatório. O processo de preparação passa por etapas de clivagem, \"dimpling\" e \"ion milling\". As imagens obtidas revelam com clareza a estrutura de pontos quântico. Com o estudo realizado, foi possível determinar as principais características de cada técnica, assim como determinar uma metodologia que pode vir a ser aplicada a outros tipos de heteroestruturas semicondutoras / This work has as main objective, the study of microscopy techniques for structural characterization of semiconductors and the development of the techniques of sample preparation, because the structural characterization is of highest importance for the obtaining of better results in the process of production of semiconductors thin films. The techniques more used in the structural characterization, are the techniques of electronic microscopy (Scanning and Transmission), together with the Atomic Force Microscopy. Samples of InGaAs/GaAs and InAs/GaAs were used, grown by the technique of MBE (Molecular Beam Epitaxy), with quantum dots, structures these rich ones in details. Such samples were prepared and characterized in each one of the techniques in study. The Scanning Microscopy and Atomic Force present easy preparation. The obtained results even so they show that the technique of Scanning Microscopy doesn\'t offer enough resolution for visualization of the heteroestructures; already the technique of Atomic Force shows excellent results of the topography of the quantum dots. For the Transmission Microscopy the preparation of samples is shown very difficult and delayed, however, the obtained result was very satisfactory. The preparation process goes by cutting stages, dimpling and ion milling. The obtained images reveal with clarity the quantum structure of points. With the accomplished study, it was possible to determine the main characteristics of each technique, as well as determining a methodology that can come to be applied to the other types of semiconductors heterostructures

Orientador: Sergio Roberto Peres Line / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-10T17:02:28Z (GMT). No. of bitstreams: 1 Frozoni_MarcosRobertodosSantos_M.pdf: 11548696 bytes, checksum: f731de9d3b6f95185faf5cab3e19d566 (MD5) Previous issue date: 2008 / Resumo: A biossíntese do esmalte dentário inicia-se pela secreção, processamento proteolítico e auto-agregação de uma complexa mistura de proteínas, sintetizadas pelos ameloblastos, conhecida como matriz orgânica do esmalte. A formação desta matriz ocorre em três estágios: secreção (inicial), transição e maturação e parece ser fundamental para o controle da orientação e morfologia dos cristais de hidroxiapatita, que constituem a fase mineral do esmalte em desenvolvimento. No estágio de secreção da amelogênese, a matriz orgânica do esmalte apresenta uma organização supramolecular birrefringente, dessa forma, a referida matriz pode ser observada e quantificada por meio de microscopia de luz polarizada. Alterações genéticas e ambientais podem induzir a distúrbios na organização molecular da matriz orgânica extracellular do esmalte (MOECE) dentário no estágio secretório, gerando modificações em sua birrefringência, tais distúrbios podem contribuir para alterações na estrutura do esmalte maduro. Altos níveis de ingestão de flúor causam mudanças na estrutura e concentração das proteínas da matriz orgânica do esmalte, induzindo a falhas na mineralização e formação desorganizada dos cristais do esmalte. Estas alterações caracterizam a fluorose de esmalte e incluem aumento da porosidade, redução do conteúdo mineral e diminuição da microdureza do esmalte maduro. O objetivo deste estudo foi analisar os efeitos do flúor sobre a birrefringência da MOECE no estágio secretório. Quinze camundongos da linhagem A/J foram divididos em 3 grupos e submetidos a um tratamento de 30 dias com dieta exclusiva de ração e água deionizada ad libitum. A água ingerida continha 0, 25, e 50 ppm de flúor (NaF) nos grupos A/J-Controle, A/J-Flúor 25 ppm e A/J-Flúor 50 ppm, respectivamente. Os mesmos procedimentos foram aplicados a quinze camundongos da linhagem NOD (Non Obese Diabetic), caracterizando os grupos NOD-Controle, NOD-Flúor 25 ppm e NOD-Flúor 50 ppm. Após o período acima mencionado, todos os animais foram perfundidos com uma mistura de paraformaldeído 2% com glutaral deído 0,5% em tampão fosfato 0,2 M e suas hemimaxilas foram extraídas e mantidas na mesma solução fixadora por 16h, as amostras foram então descalcificadas em mistura de ácido nítrico 5% com formaldeído 4 % por 6 h sob agitação. Após desidratação e inclusão em parafina, obtive-se cortes longitudinais de 5µm de espessura que foram desparafinizados, hidratados, montados em solução aquosa de glicerina 80% e analisados em microscopia de luz polarizada. Realizou-se a análise da matriz orgânica dos incisivos superiores de modo a se determinar o retardo óptico em nanômetros (nm) na área de maior birrefringência no estágio de secreção da amelogênese. Os valores de retardo ótico foram submetidos à análise estatística (Kruskal-Wallis) e os grupos A/J e NOD foram comparados separadamente. Observou-se um aumento, estatisticamente significante, dos valores de retardo ótico nos grupos A/J-Flúor 25 ppm e A/J-Flúor 50 ppm, quando comparados ao grupo A/J-Controle (p<0,01). O mesmo aconteceu com os grupos NOD-Flúor 25 ppm e NOD-Flúor 50 ppm que mostraram aumento, estatisticamente significante, dos valores de retardo ótico quando comparados ao grupo NOD-Controle (p<0,01). Os grupos A/J-Flúor apresentaram valores semelhantes (p>0,05) o que também ocorreu com os grupos NOD-Flúor. Os resultados do presente estudo mostram que o flúor induz a um aumento da birrefringência da MOECE no estágio de secreção, podendo estar associado ao mecanismo de desenvolvimento da fluorose de esmalte / Abstract: Dental enamel biosynthesis begins with secretion, proteolytic processing and self-assembly of a highly complex mixture of proteins, synthesised by ameloblast, which is known as the enamel organic matrix. This matrix formation occurs at three stages: secretion (initial), transition and maturation and seems to be essential for controlling orientation and morphology of the hydroxyapatite crystals that comprise mineral phase of developing enamel. In the secretory stage of amelogenesis, the enamel organic matrix presents a birefringent supramolecular organization. Therefore, it can be observed and quantified by polarizing microscopy. Genetic and environmental alterations may induce disturbances in the molecular organization of the secretory-stage enamel organic extracellular matrix (EOECM), producing birefringence changes, these disturbances may contribute to mature enamel alterations. High levels of ingested fluoride cause modifications in the structure and concentration of proteins of the enamel organic matrix inducing failures in the mineralization and disorganized enamel crystals formation. These alterations characterize enamel fluorosis, include increased porosity, mineral content reduction and diminished mature enamel micro hardness. The aim of the present study was to analyse the effects of fluoride on the birefringence of secretory stage EOECM. Fifteen A/J inbred mice strain were divided into 3 groups and submitted to a treatment during 30 days with exclusive diet of food and deionized water ad libitum. The ingested water contained 0, 25 and 50 ppm fluoride (NaF) in the groups A/J Control, A/J 25 ppm fluoride and A/J 50 ppm fluoride, respectively. The same procedures were applied to fifteen NOD (Non Obese Diabetic) mice, which formed the groups NOD Control, NOD 25 ppm fluoride and NOD 50 ppm fluoride. After the abovementioned period, all the animals were perfused with 2% paraformaldehyde, 0.5% glutaraldehyde in 0.2 M phosphate buffered solution. Its hemimaxillae were then extracted and maintained in the same fixative solution for 16 h, the samples were decalcified under stirring in 5% nitric acid, 4% formaldehyde for 6 h. After dehydration and embedded in paraffin, longitudinal 5-µm-thick sections were obtained and deparaffined, hydrated and mounted with aqueous 80% glycerine as imbibing medium and analyzed with polarizing microscopy. Optical retardation (nm) of the area that showed the highest birefringence brightness in the EOECM of upper incisors was determined. Optical retardation values were submitted to statistical analysis (Kruskal-Wallis) and A/J and NOD groups were separately compared. An statistically significant increase in optical retardations values was observed in A/J 25 ppm Fluoride and A/J 50 ppm Fluoride, when compared to A/J Control group (p<0.01). The same happened with NOD 25 ppm Fluoride and NOD 50 ppm Fluoride groups which exhibited statistically significant increase in optical retardations values when compared to NOD Control group (p<0.01). A/J Fluoride groups presented similar optical retardation values (p>0.05) which occurred with NOD fluoride groups. The results presented here show the fluoride induces an increase in the birefringence of secretory stage EOECM, which may be associated with enamel fluorosis development. Key words: Enamel, Amelogenesis, Enamel Organic Matrix, Birefringence, Fluoride / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental

Orientadores: José Roberto Lovadino, Débora Alves Nunes Leite Lima / Texto em português e inglês / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T14:53:35Z (GMT). No. of bitstreams: 1 Pini_NubiaInocencyaPavesi_M.pdf: 2670984 bytes, checksum: b4ce5e2b5f02ccd6636853ceb3a2b8fc (MD5) Previous issue date: 2013 / Resumo: Objetivo: Avaliar, in vitro, a influência dos ácidos utilizados para microabrasão e, in situ, o efeito do tempo de contato com a saliva na microdureza e morfologia do esmalte abrasionado. Metodologia: In vitro: Setenta blocos dentais bovinos foram divididos em 7 grupos (n=10). Os grupos experimentais foram tratados com aplicação ativa/passiva dos ácidos H3PO4 35% (E1/E2) ou HCl 6,6% (E3/E4); e controles, tratados com microabrasão com H3PO4+pedra-pomes (C5), HCl+silica (C6) ou nenhum tratamento (C7). In situ: Nove grupos (n=19) de blocos dentais bovinos foram divididos de acordo com o tratamento e o tempo de exposição salivar, sendo 4 grupos tratados com H3PO4+pedra-pomes, 4 com HCl+sílica e 1 grupo controle. Os grupos tratados foram subdivididos em: sem exposição salivar, 1 hora, 24 horas ou 7 dias de exposição em ambiente intrabucal. A microdureza superficial (SMH) foi avaliada antes e após a microabrasão, e após exposição salivar (in situ). A microdureza subsuperficial (CSMH - 10, 25, 50 e 75 ?m) foi analisada após a microabrasão (in vitro) e após a exposição salivar (in situ). Espécimes representativos foram selecionados para a avaliação da morfologia do esmalte por meio da microscopia confocal de varredura a laser (MCVL - in vitro) e por microscopia eletrônica de varredura (MEV - in situ). Para a análise estatística foi realizada análise de variância para medidas repetidas (Proc Mixed), e os testes de Tukey-Kramer e Dunnet (SMH) e ANOVA (parcelas subdivididas) e Tukey-Kramer (CSMH - in situ) (p<0.05). Resultados: In vitro: Não foram encontradas diferenças entre as análises pré e pós-microabrasão entre os grupos controles para SMH. Entre os grupos experimentais, a aplicação ativa demonstrou os maiores valores de SMH, sem diferença entre os ácidos, com a mesma forma de aplicação. A maioria dos grupos apresentou redução do valor de CSMH conforme aumento da profundidade, com diferenças entre os grupos com microabrasão (C5 e C6) e o C7; e entre todos os grupos experimentais e o C7. Comparando a aplicação dos ácidos, a aplicação ativa do H3PO4 (E1) mostrou maior CSMH com diferença estatística em relação ao HCl (E3). A MCVL demonstrou diferentes padrões de condicionamento para cada grupo. In situ: Para as análises de SMH, todos os grupos tratados apresentaram redução na microdureza, com diferenças em relação ao controle e a leitura inicial. Após exposição salivar, os resultados demonstraram que o tratamento com HCl+sílica foi mais propenso à remineralização, já que, com 1 hora foi verificado aumento na SMH, com diferença significante em relação à análise pós-microabrasão. Apenas o tratamento com HCl+sílica foi eficiente em reestabelecer tal propriedade em relação ao controle. A análise de CSMH confirmou a maior capacidade de remineralização do esmalte tratado com HCl+sílica, uma vez que após 7 dias de exposição salivar, os valores de microdureza foram restabelecidos para as camadas mais superficiais do esmalte (10 e 25 ?m). A MEV demonstrou o efeito remineralizador da saliva para ambos os tratamentos. Conclusões: Os ácidos utilizados para microabrasão apresentaram alto poder erosivo quando aplicados individualmente. O tratamento com HCl+sílica resultou em uma superfície de esmalte mais propensa à remineralização / Abstract: Objective: To evaluate, in vitro, the effect of acids used in microabrasion on enamel microhardness, and, in situ, the effects of remineralizing time on enamel surface after microabrasion. Methods: In vitro: Seven groups (n=10) of enamel blocks from bovine incisors were divided in: Experimental groups treated by active/passive application of 35% H3PO4 (E1/E2) or 6.6% HCl (E3/E4); and control groups treated by microabrasion with H3PO4+pumice (C5), HCl+silica (C6), or no treatment (C7). In situ: Nine groups (n=19) of same specimens were divided in according to microabrasion and salivary exposition being 1 control (no treatment) and 4 groups with microabrasion using 35% H3PO4+pumice and 4 groups using 6.6% +silica. One group of each treatment was submitted to 4 frames of salivary exposition, being without exposition and with 1 hour, 24 hours or 7 days of presence on in situ regimen. Surface microhardness (SMH) was evaluated before and after microabrasion, and after salivary exposition (in situ). Cross-sectional microhardness (CSMH) was analyzed after microabrasion (in vitro) and after salivary exposition (in situ). For confocal laser scanning microscopy (CLSM - in vitro) and scanning electron microscopy (SEM - in situ), representative specimens group were selected. Statistical analysis used Proc Mixed, Tukey-Kramer and Dunnet tests (SMH) e ANOVA (subdivided parcels) and Tukey-Kramer tests (CSMH - in situ) (p<0.05). Results: In vitro: For SMH, it was not found statistically differences between the control groups after treatment. Active application resulted in significantly higher microhardness results than passive application, with no difference between acids. For most groups, the CSMH decreased as the depth increased, with differences between the groups treated with microabrasion (C5 and C6) and C7; and between all of experimental groups and C7. A significantly higher mean CSMH result was obtained with active application of H3PO4 compared to HCl. CLSM revealed the conditioning pattern for each group. In situ: For SMH, the groups treated with microabrasion presented reducing in mineral content, with statistical difference in relation to the control and to the initial analysis. The treatment HCl+silica presented lower reduction and were statistically different from the treatment with H3PO4+pumice. After salivary exposition SMH results revealed that surface treated with HCl+silica was more prone to remineralizing effect of saliva, once it was verified since with 1 hour of presence in in situ regimen, with significant differences between the treatments after 7 days of salivary exposition. Just for SMH, the HCl+silica reached values obtained in control group. CSMH analysis showed that 7 days of salivary exposition were efficient in reestablish de values for the outer layers (10 e 25 ?m) of enamel treated with HCl+silica. SEM analysis presented the remineralizing effect in the course of the time. Conclusions: Acids used for enamel microabrasion presented a higher erosive action when solely applicated. Data suggested that enamel surface treated with HCl+silica presented more susceptibility for remineralizing action of saliva than that treated with phosphoric acid and pumice / Mestrado / Dentística / Mestra em Clínica Odontológica