The microvasculature has the main function of transport of dissolved gases, nutrients and waste between blood and tissue. Systematically probing transvascular transport rates in these vessels under well defined conditions is challenging. In vivo and in vitro studies are characterized, respectively, by limited optical access and control over perfusion concentrations and failure to resemble the structure and function of an intact organ. In this thesis, I present the development of a microfluidic platform for investigating molecular transport across mouse mesenteric arteries (150-300μm diameter) in a controlled physico-chemical microenvironment. Intact vessels are perfused with 4 kDa FITC-Dextran and the permeation coefficient of this molecule across the vessel wall is quantified using laser scanning confocal microscopy paired with a 2-D numerical model. Functional viability of the examined vessel, through phenylephrine and acetylcholine dose responses, is probed, and shear and phototoxic effects are reported.

The microvasculature has the main function of transport of dissolved gases, nutrients and waste between blood and tissue. Systematically probing transvascular transport rates in these vessels under well defined conditions is challenging. In vivo and in vitro studies are characterized, respectively, by limited optical access and control over perfusion concentrations and failure to resemble the structure and function of an intact organ. In this thesis, I present the development of a microfluidic platform for investigating molecular transport across mouse mesenteric arteries (150-300μm diameter) in a controlled physico-chemical microenvironment. Intact vessels are perfused with 4 kDa FITC-Dextran and the permeation coefficient of this molecule across the vessel wall is quantified using laser scanning confocal microscopy paired with a 2-D numerical model. Functional viability of the examined vessel, through phenylephrine and acetylcholine dose responses, is probed, and shear and phototoxic effects are reported.

abstract: In this work, a novel method is developed for making nano- and micro- fibrous hydrogels capable of preventing the rejection of implanted materials. This is achieved by either (1) mimicking the native cellular environment, to exert fine control over the cellular response or (2) acting as a protective barrier, to camouflage the foreign nature of a material and evade recognition by the immune system. Comprehensive characterization and in vitro studies described here provide a foundation for developing substrates for use in clinical applications. Hydrogel dextran and poly(acrylic acid) (PAA) fibers are formed via electrospinning, in sizes ranging from nanometers to microns in diameter. While "as-electrospun" fibers are continuous in length, sonication is used to fragment fibers into short fiber "bristles" and generate nano- and micro- fibrous surface coatings over a wide range of topographies. Dex-PAA fibrous surfaces are chemically modified, and then optimized and characterized for non-fouling and ECM-mimetic properties. The non-fouling nature of fibers is verified, and cell culture studies show differential responses dependent upon chemical, topographical and mechanical properties. Dex-PAA fibers are advantageously unique in that (1) a fine degree of control is possible over three significant parameters critical for modifying cellular response: topography, chemistry and mechanical properties, over a range emulating that of native cellular environments, (2) the innate nature of the material is non-fouling, providing an inert background for adding back specific bioactive functionality, and (3) the fibers can be applied as a surface coating or comprise the scaffold itself. This is the first reported work of dex-PAA hydrogel fibers formed via electrospinning and thermal cross-linking, and unique to this method, no toxic solvents or cross-linking agents are needed to create hydrogels or for surface attachment. This is also the first reported work of using sonication to fragment electrospun hydrogel fibers, and in which surface coatings were made via simple electrostatic interaction and dehydration. These versatile features enable fibrous surface coatings to be applied to virtually any material. Results of this research broadly impact the design of biomaterials which contact cells in the body by directing the consequent cell-material interaction. / Dissertation/Thesis / Ph.D. Bioengineering 2011

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.

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.

Made available in DSpace on 2014-10-09T12:33:13Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:12Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:33:13Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:12Z (GMT). No. of bitstreams: 0 / O diagnóstico precoce de tumores e metástase constitui atualmente o elemento de maior impacto dentro das políticas de saúde públicas contra o câncer. No câncer de mama e melanoma, a técnica de biópsia de linfonodo sentinela, para o diagnostico de metástase, tem sido muito utilizada evitando a dissecção total dos nodos da região anatômica afetada, e permitindo definir com precisão o procedimento terapêutico a utilizar. O objetivo principal deste trabalho centrou-se no desenvolvimento de conjugados radiomarcados de dextran-manose para diagnóstico, utilizando o núcleo de tecnécio altamente estável, [99mTc(CO)3]+. A cisteína, ligante tridentado, foi incorporada na estrutura dos conjugados, como agente quelante do Tecnécio-99m. As condições de marcação definidas para os produtos avaliados garantiram altos valores de pureza radioquímica (>90%) e atividade específica (>59,9 MBq/nmol) assim como uma alta estabilidade in vitro. Os conjugados de dextran-cisteína-manose demonstraram uma captação superior (4 vezes maior) nos nodos linfáticos em relação aos homólogos que não possuíam manose na estrutura. O conjugado de dextran-cisteína-manose de 30 kDa radiomarcado (99mTc-DCM2) foi o traçador com melhor desempenho biológico entre os avaliados à diferentes atividades injetadas. Demonstrou-se que concentrações superiores a 1 M favorecem a retenção do produto nos nodos linfáticos. As comparações com radiofármacos já utilizados no Brasil (Dextran-500 e Fitato) para detecção de linfonodo sentinela evidenciaram a superioridade do 99mTc-DCM2. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Abstract During surgery of the aortic arch and pediatric heart surgery, the blood flow to the brain has to be interrupted at times to allow a bloodless operation field and adequate conditions for surgical repair. During this no-flow period the brain is exposed to a high risk of ischaemic injury, as it will become irreversibly damaged after 5 minutes of circulatory arrest at 37°C. Additional time can be gained by cooling the patient with an extracorporeal heart-lung machine, as hypothermia reduces the cerebral metabolic rate and allows longer safe periods of circulatory standstill. This method of cerebral protection, called hypothermic circulatory arrest (HCA), is widely used in clinical practice. Thus the brain becomes susceptible to ischaemic injury after 30 minutes of HCA at 15°C. Lower temperatures than this are not practicable, however, as they require longer periods of cardiopulmonary bypass, which may further aggravate cerebral injury. To ensure a better outcome for patients undergoing these operations, additional ways of protecting the brain are required. The present work focuses on neuroprotective biochemical and fluid therapy methods for use during HCA, employing a surviving porcine model. Fructose-1,6-bisphosphate (FDP), a high-energy intermediate of glycolysis, was examined for potential neuroprotective properties in two cerebral injury settings associated with HCA. First, FDP was administered before and after a 75-minute period of HCA at a brain temperature of 18°C. This led to better survival, neurological recovery and brain histopathological findings and had supportive effects on brain metabolism (I). Second, a 25-minute period of HCA along with an iatrogenic embolic load produced by microsphere injection was used to generate a massive ischaemic injury to the brain. In this setting FDP did not affect the neurological outcome but had a clear supportive impact on cerebral metabolism (II). In addition, cerebral histopathological samples taken during the first study were analysed by electron microscopy, which revealed significant preservation of the ultrastructure in the FDP-treated animals (III). Hypertonic saline dextran (HSD) is a novel fluid therapy method which has been shown to enhance the outcome after hypovolaemic shock with or without head injury and is potentially very effective in reducing ischaemia-reperfusion injury. Its administration led to a decrease in intracranial pressure, improved brain metabolism, faster and better recovery and less histopathologically observable morphological damage (IV). The findings indicate that both FDP and HSD have significant neuroprotective properties and should be assessed in humans as well.

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.

No description available.