Memoria para optar al Título Profesional de Médico Veterinario. / El quitosano (Q) es un polisacárido de origen natural que se obtiene a partir de la desacetilación parcial de la quitina y que se caracteriza por tener un carácter catiónico debido a la presencia de un grupo funcional amino en su estructura el cual le brinda, entre otras propiedades la capacidad de poder adherirse a matrices biológicas en un proceso denominado bioadhesión. Cuando este fenómeno ocurre en epitelios cubiertos por una capa de mucina se denomina mucoadhesión. Esta propiedad ha generado gran interés en el área farmacéutica donde se busca utilizar este polímero como potenciador de la absorción de macromoléculas hidrofílicas (HFMM) como proteínas o antígenos mediante su utilización como micropartículas (MP). Por otra parte se buscan distintos métodos que permitan potenciar esta capacidad bioadhesiva, principalmente la funcionalización del Q, proceso que consiste en la conjugación del polímero con algún grupo funcional. En este proyecto se propone la conjugación del Q con grupos tiol (-SH) y sulfato (-SO3), los cuales permitirían la formación de enlaces más fuertes durante el proceso de mucoadhesión entre las micropartículas de quitosano y los residuos de cisteína presentes en la capa de mucina que recubre distintos epitelios como el intestinal. De esta forma se busca comprobar la adhesión de las MP de Q sin funcionalizar (MPQC) y MP de Q funcionalizado con grupos tiol y sulfato (MPQT y MPQS respectivamente) utilizando isotiocianato de fluoresceína (FITC) como fluoróforo, el cual se une covalentemente al quitosano, permitiendo observar e identificar la cantidad de micropartículas que se unen a monocapas celulares diferenciadas y sin diferenciar de la línea Caco-2 la cual es similar al epitelio que compone el tracto intestinal, y que además se caracteriza por presentar uniones intercelulares estrechas, trás un proceso de diferenciación en una matriz de pocillos Transwell los cuales tienen una capa microporosa en su base. De acuerdo a esto, se determinó la fluorescencia de los distintos tipos de MPQ previa administración en los cultivos y tras un periodo de incubación de 90 minutos se removió el sobrenadante y se midió su fluorescencia determinando la capacidad bioadhesiva de cada tipo de micropartículas, las cuales no presentaron diferencias estadísticamente significativas / Chitosan (C) is a natural polysaccharide obtained from deacetylated chitin, known by its cationic character due to the presence of an amine functional group in its structure. This amine group determine some chitosan properties as his capacity of adhesion to biological surfaces, process known as bioadhesion. If this union takes place on a mucin-covered epithelium it is called mucoadhesion. This property generates interest and attention from the pharmaceutical industry for its use as a controled delivery system as well as absorption enhancer of hydrophilic macromolecules (HFMM) drugs such as proteins or antigens when used as microparticles (MP). On the other hand, several efforts are focused on improving this bioadhesion capacity, mainly by chemical functionalization of the polymer. In this thesis, is proposed the chemical conjugation with tiol (-SH) and sulfate (-SO3) groups of C chains, which would allow to form stronger bonds between chitosan microparticles (CMP) and cysteine residues presents in the mucin layer that cover several epithelial during the mucoadhesion process. Thus, the objective is to test the adhesion capacity of functionalized CMP with tiol and sulfate groups (CTMP and CSMP respectively) and non-functionalized CMP (CCMP) by marking them with Fluorescein isothiocyanate (FITC) which is covalently bound to chitosan chains in order to evidence microparticles adhesion on differentiated and non-differentiated Caco-2 cell line monolayers, which has been described to be similar to the epithelium composing the intestinal tract and is also characterized by expresing tight junctions after a differentiation process on Tranwell inserts that have a microporous layer at their base. According to this, it was determined the fluorescence of each type of CMP prior and after incubation period of 90 minutes, thus the supernatants were removed and their fluorescenses were measured in order to determine the bioadhesive capacity of the microparticles. No statiscally significant differences were registered / Financiamiento: Proyecto Fondef IT13I20021.

Dissertação (mestrado) - Pontifícia Universidade Católica do Paraná, Curitiba, 2005 / Bibliografia: f.40-49 / Micropartículas são pequenas vesículas (geralmente <1m diâmetro), liberadas da membrana plasmática de células ativadas ou apoptóticas. Já foram descritas micropartículas derivadas de vários tipos celulares, como leucócitos, células vasculares, plaquetas e / Microparticles are small vesicles, produced during cell activation or apoptosis. They can be derived from several cell types, such as leukocytes, vascular cells, platelets and hepatocytes. An increase in circulating microparticles number has been document

Made available in DSpace on 2015-07-22T20:50:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-25 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introducao: Pela efetividade da TARV, a infeccao pelo HIV/aids adquiriu um carater de maior cronicidade e menor letalidade. Se por um lado as mortes por doencas relacionadas a imunodepressao estao diminuindo, a proporcao de obitos por causas nao relacionadas a aids, incluindo por doenca cardiovascular esta aumentando, parte como consequencia de eventos adversos relacionados ao tratamento. Objetivos: Quantificar o numero de celulas progenitoras endoteliais (CEP) e microparticulas (MP) em pacientes infectados pelo HIV, sem previo tratamento antirretroviral e comparar com um grupo controle. Alem do perfil bioquimico, examinar a aterosclerose subclinica por meio do espessamento medio intimal carotideo e funcao endotelial pela dilatacao mediada pelo fluxo. Metodos: Estudo tipo coorte transversal, pareado por idade e sexo, com analise cega dos objetivos dos biomarcadores. Quantificacao de CEP e MP por citometria de fluxo, aterosclerose subclinica e funcao endotelial por eco - Doppler com transdutor linear de alta resolucao. Foram incluidos 68 individuos (35 HIV+ e 33 controles), com mediana de idade de 33 anos, sendo 80% do sexo masculino e da raca branca. Resultados: O perfil lipidico diferiu entre os grupos para o colesterol total 156 mg/dL e 173 mg/dL (p = 0,02) e HDL-c 37 mg/dL e 47 mg/dL (p = 0,001), para os pacientes dos grupos caso e controle, respectivamente. A contagem das CEP foi menor nos pacientes infectados pelo HIV quando comparado aos controles para CD34+/KDR+ [0.02% vs. 0.09%, p=0.045]; mas nao para CD34+/CD133+ ou KDR+/CD133+. O numero medio de MP plaquetarias foi semelhante entre os grupos sendo 24 992 por ƒÊL de plasma no grupo infectado pelo HIV e 26 642 ƒÊL de plasma no controle (p = 0.83). A contagem das MP endoteliais/ƒÊL de plasma diferiu entre os grupos, sendo maior no grupo infectado pelo HIV em relacao ao controle (1963 versus 436, p = 0.003). A medida do espessamento medio intimal foi similar entre os grupos, mas foi observada menor dilatacao mediada pelo fluxo [media (DP)] para o grupo caso [9% (5%) versus 16% (10%), p= 0,03]. Conclusoes: Pacientes HIV+, previamente a TARV apresentaram menor quantidade de CEP e maior de MP endoteliais, ao lado de disfuncao endotelial. / Introduction: After the introduction of antiretroviral therapy, aids has acquired an aspect of chronic disease and less deaths has occurred. The deaths related to aids are diminished but the proportion of deaths unrelated to aids,including cardiovascular disease are increasing, in part due to the side effects from the antiretroviral therapy. Objectives: To quantify the number of endothelial progenitor cells (EPC) and microparticles (MP) in HIV-infected naïve patients and compared with a control group. Besides the biochemical profile we evaluated subclinical atherosclerosis through carotid intimal medial thickening and endothelial function by flow mediated dilation. Methods: A case-control study, matched for age and gender, cross-sectional with blind analysis of the goals of biomarkers. Quantification of EPC and MP was done with flow cytometry, subclinical atherosclerosis and endothelial function by echo-Doppler with high resolution linear transducer. We included 68 subjects (35 HIV + and 33 controls) with a median age of 33 years, 80% were male and Caucasian. Results: The lipid profile differ between the groups for total cholesterol 156 mg / dL and 173 mg / dL (p = 0.02) and HDL-C 37 mg / dL and 47 mg / dL (p = 0.001) for case and control groups, respectively. The count of the CEP was lower in HIV-infected patients compared with control CD34 + / KDR + [0.02% vs. 0.09%, p = 0.045], but not to CD34 + / CD133 + and KDR + / CD133 +. The average number of platelet MP was similar between the groups 24 992 per mL of plasma in HIV-infected group and 26 642 mL plasma in control (p = 0.83). The counting of endothelial MP / mL of plasma differed between groups, being higher in HIV-infected group compared to control (1963 vs. 436, p = 0.003). The measurement of intimal medial thickening was similar between groups, but was found lower flow-mediated dilation [mean (SD)] in the HIV patients [9% (5%) versus 16% (10%), p = 0.03]. Conclusions: HIV + patients, before HAART had lower amounts of EPC and increased endothelial MP, along with endothelial dysfunction. / FAPESP: 2008/55223-6 / TEDE / BV UNIFESP: Teses e dissertações