Recent investigations into the permeability of the kidney capillaries have produced conflicting reports. This study was an attempt to better describe the permeability of the kidney capillaries by using narrow-range macromolecular dextran fractions in four molecular sizes: MW 61,400, MW 77,000, MW 118,000, and MW 147,000. Permeability was measured by dextran concentration differences in plasma and kidney lymph. Permeability decreased as the dextran molecular weight increased. Molecular weights 61,400 and 77,000 penetrated into the kidney lymph. Molecular weight 118,000 exhibited greater difficulty in penetrating to the lymph. The largest fraction penetrated into the kidney lymph with greatest difficulty. Plasma expansion by saline infusion increased the permeability of all dextran fractions.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

As técnicas analíticas empregadas na determinação da qualidade da canade- açúcar são muito importantes para a indústria de processamento dessa matériaprima, uma vez que é a maneira mais eficiente de determinar a quantidade de sacarose, glicose, frutose e fibras que entra no processo. Aos fornecedores de cana-de-açúcar, é o momento da remuneração pela quantidade e qualidade de cana-de-açúcar que entrega ao processo. O Conselho dos Produtores de Cana-deaçúcar, Açúcar e Álcool do Estado de São Paulo - CONSECANA, determina a metodologia analítica de qualidade dessa matéria-prima e os cálculos para o pagamento da cana-de-açúcar ao fornecedor. Com a proibição da queima desde o ano de 2014 em áreas mecanizáveis, a cana-de-açúcar que entra na indústria tem características, como aumento no teor de amido relacionado a composição do ponteiro, que pode influenciar nos resultados analíticos e causar desvios de leituras pelos atuais equipamentos de polarimetria e refratometria. Assim como o amido, a presença de dextrana no caldo também pode causar desvios de leituras. Com esse cenário, e o interesse do Laboratório Hugot de Tecnologia em Sucroderivados da ESALQ-USP em fornecer estudos de metodologias para a determinação de sacarose, glicose e frutose, como parâmetros analíticos no sistema de avaliação da qualidade de matéria-prima (cana-de-açúcar), por técnicas cromatográficas e espectroscópicas; esse projeto tem o objetivo de trazer contribuições complementares as usinas e destilarias do Estado de São Paulo. / The analytical techniques employee in sugarcane quality determination are very important to industrial process of that raw material, once it is de efficient thing to determinate the quantity of sucrose, glucose, fructose and fibers that come to process. For sugarcane suppliers it is time to receive the payment for the quantity and quality of sugarcane ship to process. The Sugarcane, Sugar and Alcohol Board Producers of São Paulo State - Portuguese initials: CONSECANA, determinate the analytical methods of raw material quality and the payment calculations for sugarcane suppliers. Whit the burn prohibition since 2014 in a mechanical areas the sugarcane in a industrial process has characteristics, how increase of starch associated to top leaves composition, that could to influence analytical results and cause reading deviations for currents equipments of polarimetry and refractometry. Thus starch, the presence of dextran in a sugarcane juice could also cause reading deviations. In this scenario and the interesting of Sucroderivatives Technology Hugot Laboratory at ESALQ-USP to provide studies and methods to determinate sucrose, glucose and fructose how analytical parameters in a evaluation system of raw material (sugarcane) quality to chromatography and spectroscopy techniques; this project aims to bring complementary contribution to sugarcane mills of São Paulo State.

A indústria Sucroenergética tem como uma de suas preocupações atuais a presença de polissacarídeos, como o amido e a dextrana, no caldo de cana-deaçúcar. Além de afetar a qualidade do açúcar, estes polissacarídeos podem alterar resultados de análises tecnológicas do caldo e, possivelmente, causar erros no pagamento da cana. Foram objetivos deste estudo: a) isolar e caracterizar o amido isolado de cana da variedade RB 86-7515 e b) avaliar a interferência de adições crescentes deste amido isolado e de dextranas comerciais com pesos moleculares 40.000 e 5.348.000 Dalton nas análises de polarimetria e refratometria do caldo e de uma solução de sacarose (sistema modelo) a 19% (m/m). As adições de amido e dextrana interferiram nas leituras de Pol e Brix, tanto em solução de sacarose como no caldo. Foi constatada forte correlação positiva entre as variáveis (Pol% x doses de amido ou dextrana e Brix% x doses de amido ou dextrana), tanto para o ensaio em solução de sacarose como para caldo. A dextrana teve maior interferência sobre as análises, embora a interferência do amido também tenha sido evidente. Por tratar-se de um sistema complexo (composição mista de várias substâncias, inclusive amido e dextrana originalmente presentes) o caldo de cana apresentou na maioria dos ensaios, incrementos menores de leituras no polarímetro e refratômetro que o sistema modelo. A adição de amido em solução de sacarose causou incrementos de Pol% a partir de dose compreendida entre 500 e 1000 ppm, enquanto no caldo esses acréscimos foram significativos a partir de doses entre 100 e 500 ppm. A adição de dextrana em solução de sacarose causou variação significativa da Pol% a partir de dose entre 100 e 500 ppm, independente do peso molecular, e no caldo entre 1000 e 5000 ppm para as de peso molecular menor e entre 500 e 1000 ppm para a de maior peso. Na análise de Brix% a adição de amido em solução de sacarose causou variação significativa a partir de dose compreendida entre 0 e 100 ppm, e no caldo a partir de dose entre 500 e 1000 ppm. No caso da dextrana, o ensaio com solução de sacarose apresentou variação significativa do Brix% a partir de dose compreendida entre 0 e 100 para a de menor peso e entre 100 e 500 ppm para a de maior peso. No caldo esses acréscimos foram significativos a partir de doses entre 100 e 500 ppm para ambas as dextranas. O amido isolado de cana foi caracterizado revelando a predominância de grânulos esféricos, com diâmetro médio variando de 2,8 a 3,1 ?m e coloração muito próximo à branca. Seu teor de amilose foi de 17,5%, com padrão de cristalinidade do tipo A e cristalinidade relativa de 44,21% (elevada). A endoterma de gelatinização variou entre 66 e 81°C, com entalpia de 10,57 J.g-1. / The Sucroenergetic industry has as one of their current concerns the presence of polysaccharides, such as starch and dextran, in sugar cane juice. In addition to affecting the quality of sugar, these polysaccharides can alter the results of technological analysis of the juice and possibly cause errors in the payment of sugarcane. The aims of this study were: a) isolate and characterize the isolated starch of sugarcane variety RB 86-7515 b) evaluate the influence of increasing additions of isolated starch and commercial dextrans with molecular weights 40,000 and 5,348,000 Daltons in the analysis of polarimetry and refraction of the juice and a sucrose solution (system model) to 19% (w/w). The additions of starch and dextran affected the Brix and Pol readings, both sucrose solution as in the juice. There was a strong positive correlation between the variables (% Pol x doses of starch or dextran and Brix% x doses of starch or dextran) for the test in sucrose solution and juice. Dextran has been more interference in the analysis, although interference of starch has also been evident. Because it is a complex system the juice showed in most trials, smaller increments on polarimetry and refractometry that the model system, by the presence of other substances. The addition of starch in sucrose solution caused increments of Pol% from the dose between 500 and 1000 ppm, while in the juice these additions were significant at doses of 100 to 500 ppm. The addition of dextran in solution of sucrose caused significant variation of Pol% from the dose between 100 and 500 ppm, regardless of molecular weight, and in juice between 1000 and 5000 ppm for the lower molecular weight and between 500 and 1000 ppm for the highest weight. In the analysis of Brix% the starch addition in sucrose solution caused a significant variation from the dose between 0 and 100 ppm, and in juice from the dose between 500 and 1000 ppm. In the case of dextran, the test with sucrose solution presented a significant variation of Brix% from the dose between 0 and 100 to the lower weight and between 100 and 500 ppm for higher weight. In juice these additions were significant from the doses between 100 to 500 ppm for both dextrans. The starch was isolated from sugar cane and was characterized revealing the predominantly spherical granules with an average diameter ranging from 2.8 to 3.1 ?m and coloration close to white. Its amylose content was 17.5%; with crystallinity pattern of type A and relative crystallinity of 44.21% (high). The gelatinisation endotherm varied between 66 and 81 °C with enthalpy of 10.57 J.g-1.

As técnicas analíticas empregadas na determinação da qualidade da canade- açúcar são muito importantes para a indústria de processamento dessa matériaprima, uma vez que é a maneira mais eficiente de determinar a quantidade de sacarose, glicose, frutose e fibras que entra no processo. Aos fornecedores de cana-de-açúcar, é o momento da remuneração pela quantidade e qualidade de cana-de-açúcar que entrega ao processo. O Conselho dos Produtores de Cana-deaçúcar, Açúcar e Álcool do Estado de São Paulo - CONSECANA, determina a metodologia analítica de qualidade dessa matéria-prima e os cálculos para o pagamento da cana-de-açúcar ao fornecedor. Com a proibição da queima desde o ano de 2014 em áreas mecanizáveis, a cana-de-açúcar que entra na indústria tem características, como aumento no teor de amido relacionado a composição do ponteiro, que pode influenciar nos resultados analíticos e causar desvios de leituras pelos atuais equipamentos de polarimetria e refratometria. Assim como o amido, a presença de dextrana no caldo também pode causar desvios de leituras. Com esse cenário, e o interesse do Laboratório Hugot de Tecnologia em Sucroderivados da ESALQ-USP em fornecer estudos de metodologias para a determinação de sacarose, glicose e frutose, como parâmetros analíticos no sistema de avaliação da qualidade de matéria-prima (cana-de-açúcar), por técnicas cromatográficas e espectroscópicas; esse projeto tem o objetivo de trazer contribuições complementares as usinas e destilarias do Estado de São Paulo. / The analytical techniques employee in sugarcane quality determination are very important to industrial process of that raw material, once it is de efficient thing to determinate the quantity of sucrose, glucose, fructose and fibers that come to process. For sugarcane suppliers it is time to receive the payment for the quantity and quality of sugarcane ship to process. The Sugarcane, Sugar and Alcohol Board Producers of São Paulo State - Portuguese initials: CONSECANA, determinate the analytical methods of raw material quality and the payment calculations for sugarcane suppliers. Whit the burn prohibition since 2014 in a mechanical areas the sugarcane in a industrial process has characteristics, how increase of starch associated to top leaves composition, that could to influence analytical results and cause reading deviations for currents equipments of polarimetry and refractometry. Thus starch, the presence of dextran in a sugarcane juice could also cause reading deviations. In this scenario and the interesting of Sucroderivatives Technology Hugot Laboratory at ESALQ-USP to provide studies and methods to determinate sucrose, glucose and fructose how analytical parameters in a evaluation system of raw material (sugarcane) quality to chromatography and spectroscopy techniques; this project aims to bring complementary contribution to sugarcane mills of São Paulo State.

Submitted by William Justo Figueiro (williamjf) on 2015-07-06T23:37:20Z No. of bitstreams: 1 10a.pdf: 1719495 bytes, checksum: 2ed1add8423e889edf7e44b9eb1498f1 (MD5) / Made available in DSpace on 2015-07-06T23:37:20Z (GMT). No. of bitstreams: 1 10a.pdf: 1719495 bytes, checksum: 2ed1add8423e889edf7e44b9eb1498f1 (MD5) Previous issue date: 2014-07 / Nenhuma / A baleia-franca-austral (Eubalaena australis) foi alvo de grande exploração comercial entre os séculos XVII a XX no Hemisfério Sul. Estimativas recentes sugerem que a população mundial da espécie é de 12.000 indivíduos, o que representaria 17 a 21% do seu tamanho original. Apesar de aparente recuperação, o declínio drástico no tamanho da população, resultante dos 400 anos de atividade da caça, pode ter gerado altos níveis de endogamia, baixa capacidade reprodutiva e perda de variabilidade genética da espécie, podendo ter seu potencial adaptativo comprometido, aumentando assim a probabilidade de extinção. Frente ao exposto é de extrema importância a avaliação comparativa da variabilidade genética entre as populações atuais de E. australis e amostras representativas do período de caça, em especial no Brasil. Sendo assim, os objetivos deste estudo foram: I. Otimizar e comparar métodos de extração e amplificação de DNA antigo (aDNA) de ossos de baleia-franca-austral, em diferentes estados de degradação; II. Caracterizar geneticamente segmentos da região D-Loop do DNA mitocondrial (mtDNA) a partir de ossos de espécimes de E. australis coletados na Península Valdés na Argentina e em Santa Catarina no Brasil; e III. Comparar as sequências nucleotídicas da região recuperada nas amostras de aDNA com as descritas para as populações atuais. Este estudo comparou cinco métodos diferentes para obter material genético de amostras degradadas. O método de extração que mostrou ser mais eficiente foi o que continha o reagente Dextran Blue. Com a amplificação de 20 fragmentos curtos, obtidos pelo Nested-PCR, com clonagem e posterior sequenciamento, foi possível recuperar pequenos fragmentos, que variaram de 7 a 59 pb, da região D-Loop do mtDNA de 11 amostras de ossos de E. australis das 88 tentativas realizadas. Ao analisar o material recuperado com dados atuais da espécie, o programa Network 4.6 revelou uma rede contendo 19 haplótipos, sendo quatro haplótipos restritos as populações antigas e até então não descritos na literatura. Os resultados são preliminares e seus significados devem ser avaliados com muita cautela. Contudo, deve-se ressaltar que os quatro novos haplótipos encontrados podem sustentar a hipótese de uma maior diversidade haplotípica em E. australis quando a mesma era caçada comercialmente. / The Southern Right Whale (Eubalaena australis) was object of great commercial exploitation between the XVII and XX centuries in the Southern Hemisphere. Recent surveys suggest that the world’s population is of 12,000 specimens, which would represent 17% to 21% of its original size. Despite the supposed recovery, the drastic populational decline, resultant from 400 years of hunting activities, could have generated high levels of endogamy, low reproduction capacity and loss of genetic variability of the species, compromising their potential of adjustment, thus increasing their probability of extinction. Due to the exposed, it is of extreme importance the comparative evaluation of the genetic variability between the current E. australis population and representative samples from the hunting period, especially from Brazilian coast. Therefore, the purpose of this study was: I. To optimize and compare extraction and amplification methods of aDNA from Southern Right Whale’s bones in different stages of deterioration; II. To genetically characterize segments of the mtDNA’s D-Loop region from E. australis bones collected at Península Valdés in Argentina and Santa Catarina in Brazil, and III. To compare nucleotide sequences of the recovered region from aDNA samples with those described for the current populations. This study compared five different extraction methods in order to obtain genetic material from deteriorated samples. The extraction method containing Dextran Blue reagent showed the higher efficiency. With the amplification of 20 short fragments and overlapping of aDNA through Nested-PCR technique, and further cloning of the latter fragment, it was possible to sequence small fragments, varying from 7 to 59 pb, from the mtDNA’s D-loop region from 11 bones samples of E. australis, out of the 88 attempts performed. Analyzing the recovered material together with the updated data of the species, the Network 4.6 program revealed a network containing 19 haplotypes, 4 of them generated only with ancient population samples, which are new for the species. The results are still preliminary and their meanings should be evaluated with caution. However, it should be noted that the four new haplotypes found could support the hypothesis of a higher haplotype diversity in E. australis when it was hunted.

Objetivos: Avaliar a eficácia da injeção intraoperatória para identificação do linfonodo sentinela (LS) em câncer de mama com o uso do Dextran 500-99m-Tecnécio (Tc) e azul patente. Analisar se as doses do radiofármaco, o IMC (índice de massa corporal) e o volume da mama influenciam no tempo para migração ao LS. Metodologia: Estudo prospectivo, realizado entre abril de 2008 e junho de 2009, que incluiu 74 biópsias de LS em pacientes com câncer de mama em estádios T1N0 e T2N0. Injetou-se, após indução anestésica, de 0,5 a 1,5 mCi de Dextran 500-99m-Tc filtrado 0,22 μm na região subareolar num volume de 5 ml e 2 ml de azul patente. Resultados: Identificou-se o LS em 100% dos casos. Um LS (1,35%) estava marcado apenas com o azul patente. A taxa de identificação com o “probe” foi de 98% (73/74 casos). A dose média de radiofármaco aplicada foi 0,97 mCi + 0,22. O tempo médio para marcação do LS foi de 10,7 minutos (+ 5,7min). Identificamos em média 1,66 LS com o radioisótopo. A dose aplicada não apresentou relação com o tempo para captação (p=0,73). Quanto maior o volume da mama e IMC, maior o tempo para captação na região axilar (Pearson Correlation r=0,393 p<0,01; r=0,469 p<0,01 - respectivamente). Conclusão: A injeção intraoperatória do radiofármaco é eficaz para identificação do LS em câncer de mama. O tempo para marcação do LS é maior em pacientes com IMC elevado e mamas volumosas. Doses maiores de radiofármaco não diminuem o tempo de migração. / Objectives: To determine the identification of sentinel lymph node (SLN) in breast cancer after intraoperative injection of Dextran 500‐99mTechnetium (Tc) and blue dye. To analyze if the doses of the radioisotope, body mass index (BMI) and breast volume influence the migration time of the SLN. Methodology: Prospective study between april 2008 and june 2009, which included 74 biopsies of SLN in patients with breast cancer in stages T1N0 and T2N0. Intraoperative injection after induction of general anesthesia, 0.5 to 1.5 mCi of dextran 500‐99m‐Tc filtered 0.22 μm in the subareolar region in a volume of 5 ml and 2 ml of blue dye. Results: We identified the SLN in 100% of cases. In one case (1.35%) the SLN was marked only with the blue dye. The SLN identification rate with the probe was 98% (73/74 cases). The mean dose of radioisotope injected was 0.97 + 0.22 mCi. The average time to mark the SLN was 10.7 minutes (+ 5.7 min). We identified an average 1.66 SLN with the radioisotope. The dose had no effect on the time to capture (p = 0.73). The larger breast volume and BMI, the greater the capture time in the axillary region (Pearson Correlation r=0.393 p <0.01, r=0.469 p <0.01 - respectively). Conclusion: Intraoperative injection of the radioisotope is effective for the identification the SLN in breast cancer. Time to mark the SLN is higher in patients with high BMI and large breasts. Higher doses of radioisotope do not decrease the migration time.

Submitted by Jaqueline Silva (jtas29@gmail.com) on 2014-09-15T19:44:43Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Chelry Fernanda Alves de Jesus.pdf: 3412626 bytes, checksum: 196a769a0f857f15c408d233116a895b (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-09-15T19:45:11Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Chelry Fernanda Alves de Jesus.pdf: 3412626 bytes, checksum: 196a769a0f857f15c408d233116a895b (MD5) / Made available in DSpace on 2014-09-15T19:45:11Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Chelry Fernanda Alves de Jesus.pdf: 3412626 bytes, checksum: 196a769a0f857f15c408d233116a895b (MD5) Previous issue date: 2014-04-04 / In this study, maghemite nanoparticles associated to dextran were synthesized (crystallites with average diameters of 5.6 and 5.3 nm) from the precipitation of iron salts in an alkaline medium in the presence of dextran of molecular weights of 250 kDa and 75 kDa. Aqueous colloidal dispersions of maghemite/dextran reacted with different amounts of epichlorohydrin aiming at the crosslinking of dextran. The characteristics of the samples were evaluated by X-ray diffraction, high resolution electron microscopy, infrared spectroscopy, thermogravimetric analysis, iron determination, hydrodynamic diameter and zeta potential. One of the maghemite/dextran samples was treated with an ammonium hydroxide solution for different times, seeking the functionalization of dextran with NH2 groups, whose content was determined by potentiometric titration. The maghemite/dextran-NH2 system was conjugated to rhodamine B fluorophore (RodB) under different reaction conditions, being the content of RodB determined by absorption spectrophotometry in the visible region. Photoluminescent characteristics of maghemite/dextran-RodB systems were evaluated by fluorescence microscopy, fluorescence spectrometry and flow cytometry. The results of the analyses and experiments showed that the molecular mass and the crosslinking of dextran did not influence significantly the characteristics of the colloidal systems, since the hydrodynamic diameter values were in the range of 110 and 138 nm and zeta potential was of -4.54 the -10.76 mV. However, the profile of the thermogravimetric curves of the solids was influenced by the crosslinking of dextran, as well as by the presence of the polymer not associated to the nanoparticles. The treatment with ammonia was proved efficient, increasing the number of NH2 groups per particle from 45 to 71. The conjugation of RodB to the maghemite/dextran-NH2 system was more efficient at a pH of 8.5, using a greater amount of carbodiimide and N-hydroxysuccinimide. The maghemite/dextran-RodB system presented different photoluminescent properties according to the content of NH2 groups with in the sample used in the conjugation reaction. The sample treated with the ammonia solution, containing 71 NH2-groups/particle, although it presented less amount of conjugated RodB (2.2 RodB/particle) showed greater fluorescence intensity than the sample showing the highest amount of RodB (2.8 Rod/particle), but that was not treated with ammonia (45-NH2/particle). This result was interpreted considering that the system treated with ammonia favored the RodB conjugation via covalent bond, while the untreated system favored the formation of aggregates of RodB via electrostatic interactions. / Neste trabalho, foram sintetizadas nanopartículas de maghemita (cristalitos com diâmetros médios de 5,6 e 5,3 nm) associadas à dextrana a partir da precipitação de sais de ferro em meio alcalino na presença de dextrana de massas moleculares de 250 kDa e 75 kDa. As dispersões coloidais aquosas de maghemita/dextrana reagiram com diferentes quantidades de epicloridrina visando à reticulação da dextrana. As características das amostras obtidas foram avaliadas por difração de raios-x, microscopia eletrônica de alta resolução, espectroscopia no infravermelho, análises termogravimétricas e medidas de teor de ferro, diâmetro hidrodinâmico e potencial zeta. Uma das amostras de maghemita/dextrana foi tratada com solução de hidróxido de amônio por diferentes tempos, visando à funcionalização da dextrana com grupos -NH2, cujo teor foi determinado por titulação potenciométrica. O sistema maghemita/dextrana-NH2 foi conjugado ao fluoróforo rodamina B (RodB) em diferentes condições reacionais, sendo o teor de RodB determinado por espectrofotometria de absorção na região do visível. As características fotoluminescentes dos sistemas maghemita/dextrana-RodB foram avaliadas por microscopia de fluorescência, espectrometria de fluorescência e citometria de fluxo. Os resultados das análises e experimentos mostraram que a massa molecular e a reticulação da dextrana não influenciaram, significativamente, nas características dos sistemas coloidais, já que os valores de diâmetro hidrodinâmico ficaram na faixa de 110 a 138 nm e os de potencial zeta de -4,54 a -10,76 mV. Entretanto, o perfil das curvas termogravimétricas dos sólidos foi influenciado pela reticulação da dextrana, assim como pela presença de polímero não associado às nanopartículas. O tratamento com amônia mostrou-se eficiente, incrementando o número de grupos -NH2 por partícula de 45 para 71. A conjugação da RodB ao sistema maghemita/dextrana-NH2 foi mais eficiente em pH 8,5, empregando-se maior quantidade de carbodiimida e N-hidroxisuccinimida. O sistema maghemita/dextrana-RodB apresentou propriedades fotoluminescentes distintas em função do teor de grupos –NH2 superficiais na amostra empregada na reação de conjugação. A amostra tratada com solução de amônia, contendo 71 grupos NH2/partícula, embora tenha apresentado menor quantidade de RodB conjugada (2,2 RodB/partícula) mostrou maior intensidade de fluorescência do que a amostra com maior quantidade de RodB (2,8 Rod/partícula), mas que não foi tratada com amônia (45 –NH2/partícula). Este resultado foi interpretado considerando-se que o sistema tratado com amônia favoreceu a conjugação de RodB via ligação covalente, enquanto que o sistema não tratado favoreceu a formação de agregados de RodB via interações eletrostáticas.

Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is a polycationic, biocompatible and biodegradable polymer. In addition, chitosan has different functional groups that can be modified with a wide array of ligands. Because of its unique physicochemical properties, chitosan has great potential in a range of biomedical applications, including tissue engineering, non-viral gene delivery and enzyme immobilization. In our work, the primary amine groups of chitosan were utilized for chitosan modification through biotinylation using N-hydroxysuccinimide chemistry. This was followed by the addition of avidin which strongly binds to biotin. Biotinylated ligands such as polyethylene glycol (PEG) and RGD peptide sequence, or biotinylated enzymes such as trypsin, were then added to modify the surface properties of the chitosan for a variety of purposes. Modified chitosans were formulated into nano-sized particles or cast into films. Different factors affecting fabrication of chitosan particles, such as the pH of the preparation, the inclusion of polyanions, the charge ratios and the degree of deacetylation and the molecular weight of chitosan were studied. Similarly, parameters affecting the fabrication of chitosan films, such as cross-linking, were investigated for potential applications in tissue engineering and enzyme immobilization. It was found that the inclusion of dextran sulfate resulted in optimum interaction between chitosan and DNA, as shown by the high stability of these nanoparticles and their high in vitro transfection efficiencies in HEK293 cells. When applying these formulations as DNA vaccines in vivo, chitosan nanoparticles loaded with the ovalbumin antigen and the plasmid DNA encoding the same antigen resulted in the highest antibody response in C57BL/6 mice. Furthermore, engineering of the surface of chitosan nanoparticles was done by utilizing the avidin-biotin interaction for attaching PEG and RGD. The modified formulations were tested for their in vitro gene delivery properties and it was found that these ligands improved gene transfection efficiencies significantly. Chitosan nanoparticles were optimized further for enzyme immobilization purposes using sodium sulfate and glutaraldehyde as physical and chemical cross-linking agents, respectively. These particles and chitosan films were used for immobilizing trypsin utilizing several techniques. Enzyme immobilization via avidin-biotin interaction resulted in high immobilization efficiency and high enzymatic activity in different reaction conditions. Additionally, the immobilized trypsin systems were stable and amenable to be regenerated for multiple uses. Finally, glutaraldehyde cross-linked chitosan films were modified with PEG and RGD for their cell repellant and cell adhesion properties, respectively, using avidin-biotin interaction. This method was again effective in engineering chitosan surfaces for modulating cell adhesion and proliferation. In conclusion, using avidin-biotin technique to modify biotinylated chitosan surfaces is a facile method to attach a wide variety of ligands in mild reaction conditions, while preserving the functionality of these ligands.

Fluorescence-based techniques are widely used in bioscience, offering a high sensitivity and versatility. In this work, fluorescence electronic energy migration/ transfer is applied to measure intramolecular distances in two types of systems and under various conditions. The main part of the thesis utilizes the process of donor-acceptor energy transfer to probe distances within the ribosomal protein S16. Proteins are essential to all organisms. Therefore, it is of great interest to study protein structure and function in order to understand and prevent protein malfunction. Moreover, it is also important to try to study the proteins in an environment which resembles its natural habitat. Here two protein homologs were investigated; S16Thermo and S16Meso, isolated from a hyperthemophilic bacterium and a mesophilic bacterium, respectively. It was concluded that the chemically induced unfolded state ensemble of S16Thermo is more compact than the corresponding ensemble of S16Meso. This unfolded state compaction may be one reason for the increased thermal stability of S16Thermo as compared to S16Meso. The unfolded state of S16 was also studied under highly crowded conditions, mimicking the environment found in cells. It appears that a high degree of crowding, induced by 200 mg/mL dextran 20, forces the unfolded state ensemble of S16Thermo to become even more compact. Further, intramolecular distances in the folded state of five S16 mutants were investigated upon increasing amounts of dextran 20. We found that the probed distances in S16Thermo are unaffected by increasing degree of crowding. However, S16Meso shows decreasing intramolecular distances for all three studied variants, up to 100 mg/mL dextran. At higher concentrations, the change in distance becomes anisotropic. This suggests that marginally stable proteins like s16Meso may respond to macromolecular crowding by fine-tuning its structure. More stable proteins like S16Thermo however, show no structural change upon increasing degree of crowding. We also investigated the possibility of local specific interactions between the protein and crowding agent, by means of fluorescence quenching experiments. Upon increasing amounts of a tyrosine labelled dextran, a diverse pattern of fluorescence quantum yield and lifetime suggests that specific, local protein-crowder interactions may occur. In a second studied system, electronic energy migration between two donor-groups, separated by a rigid steroid, was studied by two-photon excitation depolarization experiments. Data were analysed by using recent advances, based on the extended Förster theory, which yield a reasonable value of the distance between the two interacting donor-groups. To the best of our knowledge, this is the first quantitative analysis of energy migration data, obtained from two-photon excited fluorescence.