Osteoarthritis (OA) is a degenerative joint disease which has yet no complete treatment with medication. Doxycycline, a well-known antibiotic, has been shown to prevent matrixmetallopreoteinases-MMPs, indicating potency on OA treatment. However, long term systemic use can cause side effects on other tissues. This study aimed to develop controlled drug delivery systems of doxycycline/doxycycline-chondroitin sulfate (D/D-CS) in the form of PCL microspheres for providing a better and new treatment approach via local application. After optimization studies for size, loading efficiency, surface/structure and release properties, microspheres of low Mw PCL (14 kDa) was decided to be more suitable than those of high Mw (65 kDa). The release profile of former was also more compatible with diffusion model than that of latter. The bio-effectiveness of the microspheres was evaluated with three-dimensional in vitro model / osteoarthritic-rabbit chondrocytes embedded in agarose and subjected to interleukin-1&beta / throughout incubations. In vitro treatments with D/D-CS microspheres showed significant reduction in MMP-13 activity compared with untreated OA controls for 15 and 24-day incubations. Although collagen and GAG analysis results showed no enhancement of synthesis with MS treatments, significant decrease in GAG and collagen release from D/D-CS MS treated groups and from D MS treated ones respectively. Overall evaluations of the efficacy using in vivo rabbit OA model showed better radiographic scores and histological outcomes for D/D-CS MS groups compared to only hyaluronan injected and/or untreated controls in 8 weeks. The ex-vivo biomechanical properties of cartilages demonstrated improved hardness with values comparable to healthy group upon application of D-CS MS.

ABSTRACT BACKGROUND: Arthritis is increasingly becoming a public health concern as it is the leading cause of disability. Glucosamine and chondroitin, which are alternative dietary supplements, are commonly marketed for persons with joint pain. The purpose of this study is to examine if self-reported 12-month and past 30-day use of glucosamine and/or chondroitin among persons with any arthritis, unspecified arthritis, and rheumatoid arthritis is associated with reports of past 12-month improved health and reports of past 30-day joint pain, aching, and stiffness, respectively. METHODS: The 2012 National Health Interview Survey (NHIS), a nationally representative cross-sectional household interview survey, was used for this study. The adult sample consisted of 34,525. Subgroup analyses were conducted on 7,654 respondents with any arthritis, 6,016 with unspecified arthritis, and 898 with rheumatoid arthritis. The independent variables were defined as the use of glucosamine only, chondroitin only, or glucosamine and chondroitin one or more times in the past 12 months and past 30 days. The dependent variables were defined as self-reported past 12 month improved health and past 30 day joint pain, aching, and stiffness. Descriptive, bivariate, and multivariate analyses were conducted using SAS 9.4 accounting for the complex survey design, computing missing values as missing completely at random for variance estimation. All multivariate logistic regression models included sociodemographics, use of other observed alternative therapies, and other chronic conditions. RESULTS: Approximately 21.8% of U.S adults had any arthritis, 17.0% had unspecified arthritis and 2.5% had rheumatoid arthritis. Among persons with any arthritis, approximately 3.7% used glucosamine, 0.4% used chondroitin, and 3.4% used both glucosamine and chondroitin within the past 12 months while approximately 5.1% used glucosamine, 0.6% used chondroitin, and 0.4% used both glucosamine and chondroitin within the past 30 days. Among persons with unspecified arthritis, approximately 3.7% used glucosamine, 0.5% used chondroitin, and 3.8% used both glucosamine and chondroitin within the past 12 months while 5.5% used glucosamine, 0.5% used chondroitin, and 0.4% used both glucosamine and chondroitin within the past 30 days. Among persons with rheumatoid arthritis, approximately 2.4% used glucosamine, 0.3% used chondroitin, and 2.1% used both glucosamine and chondroitin within the past 12 months while approximately 2.9% used glucosamine, 0.7% used chondroitin, and 0.5% used both glucosamine and chondroitin within the past 30 days. Women used more of all supplements (past 12 months and past 30 days) except past 12 month use of chondroitin among persons with any arthritis. Persons 56 to 70 years old had the highest proportion of past 12 month and 30 day supplement use among persons with unspecified arthritis. After adjusting for sex, age, race, BMI, poverty level, other health conditions, and other CAM therapies (acupuncture, energy, mind-body, and chiropractic/osteopathic therapies), the use of chondroitin only (adjusted OR= 0.6; p= <0.01) and the use of both glucosamine and chondroitin (adjusted OR= 5.7; p= <0.01) during the past 30 days was associated with self-reported past 30 day joint pain, aching, and stiffness among persons with any arthritis. After adjusting for age, BMI, poverty level, region, other health conditions, and other CAM therapies (acupuncture, energy, mind-body, and chiropractic/osteopathic therapies), the use of chondroitin only was also associated with past 30 day joint pain, aching, and stiffness among persons with unspecified arthritis (adjusted OR= 0.5; p= 0.02). CONCLUSION: Chondroitin alone was associated reports of past 30 day joint pain, aching, and stiffness among persons with any arthritis and unspecified arthritis highlighting a potential effective role and use for this supplement. In addition, the use of both glucosamine and chondroitin were associated with reports of past 30 day joint pain, aching, and stiffness among persons with any arthritis. Marketing may play a role in these relationships and should be further examined.

Proteoglycans (PGs) are composed of highly sulfated glycosaminoglycans chains (GAGs) attached to specific core proteins. They are present in extracellular matrices, on the cell surface and in storage granules of hematopoietic cells. Heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) GAGs play indispensable roles in a wide range of biological processes, where they can serve as protein carriers, be involved in growth factor or morphogen gradient formation and act as co-receptors in signaling processes. Protein binding abilities of GAGs are believed to be predominantly dependent on the arrangement of the sugar modifications, sulfation and epimerization, into specific oligosaccharide sequences. Although the process of HS and CS/DS assembly and modification is not fully understood, a set of GAG biosynthetic enzymes have been fairly well studied and several mutations in genes encoding for this Golgi machinery have been linked to human genetic disorders. This thesis focuses on the zebrafish N-deacetylase/N-sulfotransferase gene family, encoding key enzymes in HS chain modification, as well as glycosyltransferases responsible for chondroitin/dermatan sulfate elongation present in zebrafish. Our data illustrates the strict spatio-temporal expression of both the NDST enzymes (Paper I) and CS/DS glycosyltransferases (Paper II) in the developing zebrafish embryo. In Paper III we took advantage of the four preexisting zebrafish mutants with defective GAG biosynthesis. We could demonstrate a relation between HS content and the severity of the pectoral fin defects, and additionally correlate impaired HS biosynthesis with altered chondrocyte intercalation. Interestingly, altered CS biosynthesis resulted in loss of the chondrocyte extracellular matrix. One of the main findings was the demonstration of the ratio between the HS biosynthesis enzyme Extl3 and the Csgalnact1/Csgalnact2 proteins, as a main factor influencing the HS/CS ratio. In Paper IV we used the newly developed CRISPR/Cas9 technique to create a collection of zebrafish mutants with defective GAG biosynthetic machineries. Lack of phenotypes linked to null-mutations of most of the investigated genes is striking in this study.

A inexistência de um agente terapêutico único satisfatório para o tratamento do melanoma metastático e a potencialidade do quimioterápico 5FU (5-fluorouracil) motivou esta pesquisa, a qual teve por objetivo o desenvolvimento tecnológico de sistemas carreadores micro-/ e nanoestruturados contendo 5FU a fim de aumentar sua eficácia terapêutica e reduzir a toxicidade por meio da administração pulmonar. Duas formulações pulverulentas foram desenvolvidas com polímeros naturais, sulfato de condroitina e hidroxipropil-metil-celulose, denominadas 5FU-MS e 5FU-NS, utilizando as técnicas de aspersão e atomização vibracional piezoelétrica, respectivamente. Ambas as formulações foram avaliadas quanto às características físicas e químicas, perfil toxicológico in vivo (C. elegans e em ratos Wistar), e penetração e biodisponibilidade no tecido pulmonar pela quantificação da fração livre de fármaco por microdiálise pulmonar. A análise físico-química revelou a obtenção de partículas micrométricas para 5FU-MS e submicrométricas para 5FU-NS, com diâmetros médios de partícula de 2,546 ± 0,07 m e 0,652 ± 0,03 m, e fração respirável (FR%) de 55,12 ± 2,98 e 76,84 ± 0,07, respectivamente. Ambas demonstraram características e propriedades adequadas para administração pulmonar, com capacidade de deposição nas porções média e profunda. A toxicidade das formulações avaliada em C. elegans considerou o percentual de morte, desenvolvimento, DL50 e produção de ROS para os nematodos sob tratamento agudo e crônico. Os resultados evidenciaram redução significativa da toxicidade proporcionada pela redução da taxa de morte e maior desenvolvimento dos grupos tratados com as formulações 5FU-MS e 5FU-NS em comparação ao fármaco livre, sugerindo perfis de segurança satisfatórios para administração. Além disso, 5FU-MS revelou-se um agente pró-oxidante, representando um diferencial promissor deste sistema, podendo alcançar maior sensibilização das células tumorais com menores doses. A toxicidade pulmonar aguda foi avaliada pela análise de LDH e proteínas totais no fluido de lavagem bronco-alveolar (BALF) após a administração combinada das formulações 5FU-MS e 5FU-NS para administração como um sistema terapêutico único (5FU-MS/NS), e análise de dano tecidual pulmonar em ratos. Os resultados da análise bioquímica e histológica indicaram o baixo potencial de indução de lesão tecidual a partir da administração pulmonar combinada das formulações, em relação ao fármaco livre. A análise do perfil farmacocinético por microdiálise pulmonar evidenciou o êxito no desenvolvimento dos sistemas carreadores, tornando possível duplicar o t1/2 do 5FU e aumentar significativamente a biodisponibilidade no tecido pulmonar. Os resultados obtidos indicam a eficiência das formulações 5FU-MS e 5FU-NS em alcançar os benefícios terapêuticos do fármaco 5FU com menores doses e maiores intervalos de administração. Este trabalho de tese apresenta uma abordagem promissora na terapia de neoplasias com recorrência de metástase pulmonar. / The absence of a single therapeutic agent suitable for the treatment of metastatic melanoma and the potential of 5FU chemotherapy (5-fluorouracil) motivated this study, which aimed the development of carrier systems based on micro-/ and nanostructures containing 5FU to increase the therapeutic efficacy and reduce toxicity of this drug by pulmonary administration. Two different formulations of dry powders were developed with natural polymers, chondroitin sulfate and hydroxypropyl-methyl-cellulose, denomined 5FU-MS and 5FU-NS, using the spray-drying and vibrational piezoelectric atomization techniques, respectively. Both formulations were evaluated in terms of physico-chemical characteristics, in vivo toxicological behaviors (C. elegans and in Wistar rats), bioavailability and penetration in the lung tissue by quantifying of drug free fraction by lung microdialysis. The physicochemical analysis showed that were obtained as micrometric (5FU-MS) and submicron particles (5FU-NS), with average diameters of particle 2.546 ± 0.07 m and 0.652 ± 0.03 m, and respirable fraction (FR%) of 55.12 ± 2.98 and 76.84 ± 0.07, respectively. Both showed suitable characteristics and properties for pulmonary delivery, with deposition capacity in the middle and deep lung portions. The toxicity of the formulations evaluated in C. elegans considered the death rate, body development, DL50 and production of ROS to nematodes under acute and chronic treatment. The results showed significant reduction of toxicity, reducing the death rate and greater development of the groups treated with 5FU-MS and 5FU-NS formulations compared to the free drug, suggesting satisfactory safety profile for administration. In addition, 5FU-MS proved to be a pro-oxidant agent, representing a promising differential of this system which can achieve greater sensitization of tumoral cells with lower doses. Acute pulmonary toxicity was evaluated by analyzing LDH, and total protein in the bronchoalveolar lavage fluid (BALF) after combined administration of 5FU-MS formulations and 5FU-NS for administration as a single therapeutic system (5FU-MS/NS) and analysis of lung tissue damage in rats. The results of biochemical and histological analysis indicated the low potential to induce tissue damage from the pulmonary administration of combined formulations, compared to free drug. Analysis of the pharmacokinetic profile for pulmonary microdialysis showed the successful development of carrier systems, making it possible to double the t1/2 of 5FU and significantly increase bioavailability in lung tissue. The results indicate the effectiveness of the formulations 5FU-MS and 5FU-NS in achieving the therapeutic benefits of the drug 5FU at lower doses and higher dosing intervals. This thesis work presents a promising approach to cancer therapy with lung metastasis recurrence.

Made available in DSpace on 2015-03-26T13:46:56Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2724286 bytes, checksum: dfc4a98088cd85fae126679628671081 (MD5) Previous issue date: 2011-02-28 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The aim of this research was to evaluate the influence of chondroprotective veterinary supplement (nutraceutic) composed of glucosamine and chondroitin sulfate in the repair of osteochondral defects induced in femoral lateral condyle of dogs, by clinical, radiographic, macroscopic, histologic and morfometric analysis. We also aimed to test the safety of the supplement with tests of blood glucose, blood count, liver and kidney function, activated partial tromboplastine and time prothrombin time. Fortyeigth adult dogs with body weight ranging from 10 kg to 25 kg were used. They were divided into four treatments (I, II, III and IV), according to the postoperative period of evaluation (15, 30, 60 and 90 days) and each containing six animals. Within each treatment, six animals (GI) received the supplement daily, while the other six formed the control group (GII). No significant differences were observed between groups for each treatment. Therefore, the conditions in which this study was conducted, the chondroprotective did not cause adverse effects and the treated group did not differ from the control on the repair process of such defects. / O presente trabalho teve como objetivo avaliar a influência de um suplemento condroprotetor (nutracêutico) veterinário comercial, à base de sulfato de condroitina e glucosamina, na reparação de falhas osteocondrais induzidas no côndilo femoral lateral de cães, por meio de análises clínica, radiográfica, macroscópica, histológica e morfométrica. Objetivou-se ainda testar a segurança do produto, por meio dos exames de glicemia, hemograma, funções hepática e renal, tempo de tromboplastina parcial ativada e tempo de protombina. Foram utilizados 48 cães adultos, entre 10 e 25 kg de peso corporal e sem raça definida, distribuídos aleatoriamente entre quatro tratamentos (I, II, III e IV), de acordo com o período de pós-operatório (15, 30, 60 e 90 dias) e contendo cada um deles 12 animais. Dentro de cada tratamento, seis animais (GI) receberam diariamente o condroprotetor, enquanto os outros seis constituíram o grupo controle (GII). Não houve diferença significativa entre os grupos de cada tratamento e, portanto, nas condições em que o presente estudo foi realizado, o condroprotetor não ocasionou efeitos adversos e o grupo tratado não diferiu do controle quanto ao processo de reparação dos defeitos.

A inexistência de um agente terapêutico único satisfatório para o tratamento do melanoma metastático e a potencialidade do quimioterápico 5FU (5-fluorouracil) motivou esta pesquisa, a qual teve por objetivo o desenvolvimento tecnológico de sistemas carreadores micro-/ e nanoestruturados contendo 5FU a fim de aumentar sua eficácia terapêutica e reduzir a toxicidade por meio da administração pulmonar. Duas formulações pulverulentas foram desenvolvidas com polímeros naturais, sulfato de condroitina e hidroxipropil-metil-celulose, denominadas 5FU-MS e 5FU-NS, utilizando as técnicas de aspersão e atomização vibracional piezoelétrica, respectivamente. Ambas as formulações foram avaliadas quanto às características físicas e químicas, perfil toxicológico in vivo (C. elegans e em ratos Wistar), e penetração e biodisponibilidade no tecido pulmonar pela quantificação da fração livre de fármaco por microdiálise pulmonar. A análise físico-química revelou a obtenção de partículas micrométricas para 5FU-MS e submicrométricas para 5FU-NS, com diâmetros médios de partícula de 2,546 ± 0,07 m e 0,652 ± 0,03 m, e fração respirável (FR%) de 55,12 ± 2,98 e 76,84 ± 0,07, respectivamente. Ambas demonstraram características e propriedades adequadas para administração pulmonar, com capacidade de deposição nas porções média e profunda. A toxicidade das formulações avaliada em C. elegans considerou o percentual de morte, desenvolvimento, DL50 e produção de ROS para os nematodos sob tratamento agudo e crônico. Os resultados evidenciaram redução significativa da toxicidade proporcionada pela redução da taxa de morte e maior desenvolvimento dos grupos tratados com as formulações 5FU-MS e 5FU-NS em comparação ao fármaco livre, sugerindo perfis de segurança satisfatórios para administração. Além disso, 5FU-MS revelou-se um agente pró-oxidante, representando um diferencial promissor deste sistema, podendo alcançar maior sensibilização das células tumorais com menores doses. A toxicidade pulmonar aguda foi avaliada pela análise de LDH e proteínas totais no fluido de lavagem bronco-alveolar (BALF) após a administração combinada das formulações 5FU-MS e 5FU-NS para administração como um sistema terapêutico único (5FU-MS/NS), e análise de dano tecidual pulmonar em ratos. Os resultados da análise bioquímica e histológica indicaram o baixo potencial de indução de lesão tecidual a partir da administração pulmonar combinada das formulações, em relação ao fármaco livre. A análise do perfil farmacocinético por microdiálise pulmonar evidenciou o êxito no desenvolvimento dos sistemas carreadores, tornando possível duplicar o t1/2 do 5FU e aumentar significativamente a biodisponibilidade no tecido pulmonar. Os resultados obtidos indicam a eficiência das formulações 5FU-MS e 5FU-NS em alcançar os benefícios terapêuticos do fármaco 5FU com menores doses e maiores intervalos de administração. Este trabalho de tese apresenta uma abordagem promissora na terapia de neoplasias com recorrência de metástase pulmonar. / The absence of a single therapeutic agent suitable for the treatment of metastatic melanoma and the potential of 5FU chemotherapy (5-fluorouracil) motivated this study, which aimed the development of carrier systems based on micro-/ and nanostructures containing 5FU to increase the therapeutic efficacy and reduce toxicity of this drug by pulmonary administration. Two different formulations of dry powders were developed with natural polymers, chondroitin sulfate and hydroxypropyl-methyl-cellulose, denomined 5FU-MS and 5FU-NS, using the spray-drying and vibrational piezoelectric atomization techniques, respectively. Both formulations were evaluated in terms of physico-chemical characteristics, in vivo toxicological behaviors (C. elegans and in Wistar rats), bioavailability and penetration in the lung tissue by quantifying of drug free fraction by lung microdialysis. The physicochemical analysis showed that were obtained as micrometric (5FU-MS) and submicron particles (5FU-NS), with average diameters of particle 2.546 ± 0.07 m and 0.652 ± 0.03 m, and respirable fraction (FR%) of 55.12 ± 2.98 and 76.84 ± 0.07, respectively. Both showed suitable characteristics and properties for pulmonary delivery, with deposition capacity in the middle and deep lung portions. The toxicity of the formulations evaluated in C. elegans considered the death rate, body development, DL50 and production of ROS to nematodes under acute and chronic treatment. The results showed significant reduction of toxicity, reducing the death rate and greater development of the groups treated with 5FU-MS and 5FU-NS formulations compared to the free drug, suggesting satisfactory safety profile for administration. In addition, 5FU-MS proved to be a pro-oxidant agent, representing a promising differential of this system which can achieve greater sensitization of tumoral cells with lower doses. Acute pulmonary toxicity was evaluated by analyzing LDH, and total protein in the bronchoalveolar lavage fluid (BALF) after combined administration of 5FU-MS formulations and 5FU-NS for administration as a single therapeutic system (5FU-MS/NS) and analysis of lung tissue damage in rats. The results of biochemical and histological analysis indicated the low potential to induce tissue damage from the pulmonary administration of combined formulations, compared to free drug. Analysis of the pharmacokinetic profile for pulmonary microdialysis showed the successful development of carrier systems, making it possible to double the t1/2 of 5FU and significantly increase bioavailability in lung tissue. The results indicate the effectiveness of the formulations 5FU-MS and 5FU-NS in achieving the therapeutic benefits of the drug 5FU at lower doses and higher dosing intervals. This thesis work presents a promising approach to cancer therapy with lung metastasis recurrence.

Orientador: Fabio Trindade Maranhão Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T20:47:16Z (GMT). No. of bitstreams: 1 Bastos_MarceleFontenelle_M.pdf: 15905703 bytes, checksum: b7c6bc673961fdae05225ebe3ac50346 (MD5) Previous issue date: 2011 / Resumo: 0 seqüestro de eritrócitos infectados par Plasmodium falciparum (Pf-Els) na microvasculatura de varies órgãos envolve uma seqüência de eventos que acredita­se contribuir para a patogênese da malaria grave. Apesar do tratamento com drogas antimaláricas serem eficazes, mortalidade significativa ainda é vista nos casos graves da doença, particularmente no prazo de 24 horas da admissão hospitalar. Acredita-se que o uso de terapias anti-adesivas nesse período poderia minimizar as complicações causadas pelo P. falciparum. Nesse sentido, polissacarídeos sulfatados, como a heparina e a condroitina-sulfato-A (CSA), têm mostrado capacidade em inibir a citoaderência de Pf-Els a receptores endoteliais. A heparina foi usada no passado no tratamento da malaria grave, mas deixou de ser recomendada, devido a ocorrência de sérios efeitos colaterais, como hemorragias. Além disso, esses compostos são derivados de mamíferos, o que aumenta o risco de contaminação por agentes patogênicos. De fato, embora muitos compostos tenham sido testados, nenhum demonstrou evidência inequívoca de melhora nos testes clínicos para prevenção e tratamento da malária grave. 0 condroitim sulfate fucosilado (FucCS) é um polissacarídeo altamente sulfatado extraído do pepino-do-mar Ludwigothurea grisea, composto por uma base estrutural de condroitim sulfato de mamífero, e substituído na posição 3 dos resíduos de acido ß-D-glucurônico com cadeias de fucose sulfatadas. Nesse estudo, mostramos que o FucCS apresenta baixa toxicidade, e é um potente inibidor da citoaderência parasitária em células endoteliais de pulmão humano (HLEC) e da reinvasão de eritrócitos por merozoítos de P. falciparum. Em ambos os casos, a inibição ocorre de maneira dose dependente, e o composto mostrou ser eficiente na inibição de diferentes fenótipos parasitários. A remoção das cadeias de fucose sulfatadas do FucCS praticamente aboliu o efeito inibitório, sugerindo um papel central desempenhado por essas cadeias na ocorrência do processo inibitório. 0 composto também mostrou ser capaz de reverter a citoadesão parasitária em sistema que mimetiza o fluxo sangüíneo. Além disso, o tratamento com o FucCS (1 mg/kg/animal/dia) demonstrou retardar a morte de camundongos infectados com Plasmodium berghei ANKA (PbA), modelo experimental de malária cerebral. Assim, sugerimos o FucCS como um candidato promissor a terapia adjuvante no tratamento da malária grave e na prevenção ao agravamento da doença / Abstract: Sequestration of Plasmodium falciparum-infected erythrocytes (Pf-EIs) in the microvasculature of several organs involves a sequence of events that contributes to the pathogenesis of severe malaria. Despite treatment with effective antimalarial drugs, significant mortality is still observed in severe cases of disease, particularly within 24 hours of hospital admission. It is believed that the use of anti-adhesive therapies in this period could reduce complications caused by P. falciparum. Accordingly,sulfated polysaccharides such as heparin and chondroitin sufate A (CSA) have shown ability to inhibit the cytoadherence of Pf-EIs to endothelial receptors. Although heparin was used in the past as treatment for severe malaria, its use was discontinued due to the occurrence of serious side effects such as bleeding. Moreover, given that these compounds are obtained from mammals, potential risk of contamination has to be considered. In fact, although many compounds have been tested, none demonstrated unequivocal evidence of improvement in clinical trials for prevention and treatment of severe malaria. The fucosylated chondroitin sulfate (FucCS) is a highly sulfated polysaccharide extracted from sea cucumber Ludwigothurea grisea, composed of a chondroitin sulfate backbone substituted at the 3-position of the ß-D-glucuronic acid residues with sulfated fucose branches. In this study, we show that FucCS presents low toxicity and is a potent inhibitor of parasite cytoadherence in human lung endothelial cells (HLEC) and reinvasion of erythrocytes by P. falciparum merozoites. In both cases, inhibition occurs in a concentration-dependent manner, and the compound showed to be effective in inhibiting different parasite phenotypes. Removal of the sulfated fucose branches on the FucCS practically abolished the inhibitory effect, suggesting a central role played by these branches in the occurrence of the inhibitory process. The compound also showed ability to reverse parasite cytoadhesion under flow conditions. Furthermore, treatment with FucCS at 1 mg/kg/animal/day improved survival of C57BL/6 ice infected with Plasmodium berghei ANKA (PbA), an experimental model for cerebral malaria. Thus, we suggest FucCS as a promising candidate for adjunct therapy in the treatment of severe malaria and in prevention of severe malaria outcomes / Mestrado / Imunologia / Mestre em Genética e Biologia Molecular

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-12T12:43:09Z No. of bitstreams: 1 andreluizdacunha.pdf: 5996425 bytes, checksum: b2672b9464385b63d7ce5aa90c90c7eb (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-12T15:45:22Z (GMT) No. of bitstreams: 1 andreluizdacunha.pdf: 5996425 bytes, checksum: b2672b9464385b63d7ce5aa90c90c7eb (MD5) / Made available in DSpace on 2017-05-12T15:45:22Z (GMT). No. of bitstreams: 1 andreluizdacunha.pdf: 5996425 bytes, checksum: b2672b9464385b63d7ce5aa90c90c7eb (MD5) Previous issue date: 2012-07-31 / Condroitim sulfato é um glicosaminoglicano sulfatado composto por unidades dissacarídicas formadas por ácido D-glucurônico e N-acetil-galactosamina. Este polissacarídeo é utilizado no Brasil em combinação com outros fármacos para tratamento de osteoartrite. Este trabalho teve como objetivo avaliar a qualidade do insumo farmacêutico condroitim sulfato de sódio utilizado em farmácias de manipulação. Para isso foi necessário estabelecer uma técnica de eletroforese em gel de poliacrilamida para análise de açúcares (FACE), bem como produzir enzimas específicas para a digestão de condroitim sulfato. Diferentes dissacarídeos insaturados, monossacarídeos sulfatados e açúcares neutros derivatizados com 2-aminoacridona foram separados por FACE, usando dois sistemas tampão distintos. Fracionamos por cromatografia de interação hidrofóbica três condroitinases de F. heparinum – condroitinase AC, C e B. Por FACE caracterizamos a atividade dessas enzimas e identificamos contaminação de condroitinase AC por sulfatase específica para hexosamina sulfatada na posição 4, que foi inibida por fluoreto de sódio (10 mM). Glucuronidase com atividade específica para dissacarídeo não sulfatado e 6-sulfatado foi detectada em condroitinase C, e sacarolactona (10 mM) foi capaz de impedir somente a degradação de ΔDi0S. Dosagem de condroitim sulfato em amostras farmacêuticas foi realizada por eletroforese em gel de agarose e por titulação fotométrica. Teor superior a 80% foi encontrado em apenas cinco matériasprimas. O peso molecular médio do condroitim sulfato nessas amostras variou entre 16 e 26 kDa, e análise dissacarídica por FACE revelou proporções próximas entre dissacarídeo 4-sulfatado e 6-sulfatado. Onze amostras apresentaram teor inferior a 20%, e análise por FACE demonstrou presença de contaminação por lactose ou polímeros de glicose com diferentes pesos moleculares. Concluímos neste estudo que há grande distorção entre os resultados reportados no certificado de análise de onze insumos analisados e os resultados obtidos nesse trabalho. Torna-se necessário, portanto, maior fiscalização e regulação do comércio deste produto. / Chondroitin sulfate is a sulfated glycosaminoglycan composed of alternate sequences of D-glucuronic acid and N-acetyl-D-galactosamine. This polysaccharide is widely recommended for treatment of osteoarthritis in Brazil, in association with other drugs. The objective of the present study was to evaluate the quality of chondroitim sulfate raw material used for pharmaceutical recipes. A polyacrylamide gel electrophoresis method for carbohydrate analysis (FACE) was established, so as the production of specific enzymes able to digest chondroitin sulfate. Different unsaturated disaccharides, sulfated monosaccharides and neutral sugars derivatized with 2-aminoacridona were resolved by FACE, using two distinct buffer systems. F. heparinum chondroitinase AC, C and B were fractioned by hydrophobic interaction chromatography, and the activity of these enzymes was characterized by FACE. Chondroitinase AC demonstrated contamination by a sulfatase specific for sulfation at position 4 of hexosamine, which was inhibited by 10 mM sodium fluoride. Glucuronidase contamination with specific activity against non sulfated and 6-sulfated disaccharide was detected in chondroitinase C, and 10 mM D-saccharic acid-1,4-lactone inhibited the degradation of only non sulfated disaccharide. Chondroitin sulfate content in pharmaceutical raw materials was determined by agarose gel electrophoresis and by photometric titration. Only five samples showed content greater than 80%. Chondroitin sulfate average molecular weight in these samples varied from 16 to 26 kDa, and disaccharide analysis by FACE revealed similar proportions between 4-sulfated and 6-sulfated disaccharide. Eleven samples showed less than 20% of chondroitin sulfate, and contaminant analysis by FACE detected the presence of lactose or different polymers of glucose. We concluded that there is a large distortion between the data reported at the certificate of analysis of eleven products and the results obtained in this work. Stricter regulation of this raw material should be enforced.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-10-11T14:26:18Z No. of bitstreams: 1 rafaelguzelladecarvalho.pdf: 5817882 bytes, checksum: 3cecfbefd0364ef85d5c3183dcbdfc47 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-10-16T14:07:21Z (GMT) No. of bitstreams: 1 rafaelguzelladecarvalho.pdf: 5817882 bytes, checksum: 3cecfbefd0364ef85d5c3183dcbdfc47 (MD5) / Made available in DSpace on 2018-10-16T14:07:21Z (GMT). No. of bitstreams: 1 rafaelguzelladecarvalho.pdf: 5817882 bytes, checksum: 3cecfbefd0364ef85d5c3183dcbdfc47 (MD5) Previous issue date: 2015-07-23 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Glicosaminoglicanos (GAGs) são heteropolissacarídeos lineares ligados a um núcleo proteico dos proteoglicanos (PGs). Condroitim sulfato (CS) é GAG sulfatado composto por unidades dissacarídicas repetidas de ácido D-glucurônico e N-acetil-galactosamina, esse composto possui atividades biológicas associadas à interação com diferentes elementos da matriz. Estas ações têm sido relacionadas às suas características estruturais relativas ao elevado teor de grupos sulfato e carboxila que originam domínios estruturais que são conhecidos por participar de funções fisiológicas específicas. O presente trabalho teve por objetivo a identificação e caracterização estrutural dos GAGs extraídos de diferentes tecidos de lula (Doryteuthis plei) bem como a avaliação da atividade anticoagulante destes compostos. Para isso inicialmente determinamos a técnica de extração dos GAGs dos tecidos de manto, nadadeira, tentáculos e pele utilizando degradação com enzimas proteolíticas e purificação utilizando a técnica de cromatografia de troca iônica. Realizamos a identificação dos GAGs com degradações com liases específicas (chases AC e B de F. heparinum), em que ficou evidenciado que CS é o GAG majoritário dos tecidos de D. plei. Os CS encontrados tiveram peso molecular detectados na faixa de 30-50 kDa. A dosagem química de sulfato demonstrou que estes CS possuíam relação superior a 1,2 sulfato/hexosamina o que nos forneceu o indicativo que estas macromoléculas eram supersulfatadas. Este fato foi confirmado pela análise por FACE dos produtos da digestão de CS de manto e nadadeira com chase AC de A. aurescens em que a maior sulfatação foi relacionada à presença de alta proporção de resíduos di-sulfatados, ΔDi4,6S (≈55%). Técnicas espectroscópicas de Raman e RMN permitiram confirmar a maior substituição e sulfatação nas posições 4- e 6- da GalNAc, respectivamente. Os CS de manto e nadadeira ainda demonstraram atividade anticoagulante associada à inibição da via intrínseca da coagulação sendo detectada inibição dos fatores IIa e Xa. Desta forma concluímos que CS-E é o tipo de glicosaminoglicano constituinte nos tecidos de lula e que ele possui características tecido-específica, desempenhando sua atividade anticoagulante de acordo com o padrão de sulfatação. / Glycosaminoglycans (GAGs) are linear heteropolysaccharides attached to a core protein of proteoglycans (PGs). Chondroitin sulfate (CS) is sulfated GAG composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-galactosamine, this compound has biological activities associated to their interaction with different components of the extracellular matrix. These activities have been related to their structural characteristics due to the high content of sulfate and carboxyl groups originating structural domains which are known to participate in specific physiological functions. This aim of this study was the identification and structural characterization of glycosaminoglycans extracted from squid different tissues (Doryteuthis plei) as well as evaluation of the anticoagulant activity of these compounds. Initially, we determined the procedures GAGs extraction in mantle tissue, fin, tentacles and skin using degradation with proteolytic enzymes and purification using ion exchange chromatography. We carried out the identification of GAG to degradation with specific lyases (chases AC and B from F. heparinum), wherein we evidenced that CS is the principal GAG of the D. plei tissues. Molecular weight of CS had a range of 30-50 kDa. Quantification of sulfate groups demonstrated that these CS had higher ratio of sulfate/hexosamine (1.2), a indicative that these macromolecules were oversulfated. This fact was confirmed by FACE analysis of CS from mantle and fin after digestion with chase AC from A. aurescens where most sulfation was related to the presence of high proportion of di-sulfated residues, ΔDi4,6S (≈55%). Raman spectroscopic and NMR techniques confirmed the increased substitution and sulfation in positions 4 and 6 of GalNAc, respectively. CS of the mantle and fin demonstrated anticoagulant activity associated with inhibition of the intrinsic coagulation pathway by inhibition of factors Xa and IIa. Thus, we conclude that CS-E is the kind of glycosaminoglycan constituent in squid tissues and it has tissue-specific characteristics, playing their anticoagulant activity according to the sulfation pattern.

M.Tech. (Homoeopathy) / Osteoarthritis (OA) is a chronic and debilitating condition, characterized by irreversible damage to the joint space, most commonly affecting the knees, hips, hands and spine (Colledge et al., 2010). OA is the leading cause of joint pain and disability in middle-aged and elderly persons (Long et al., 2001). The prevalence of OA of the knee in adults living in the United Sates has grown from a reported 21 million in 1990 to a total estimate of 26.9 million in 2005 (CDC, 2011). By the age of 65 years, 80% of the total population has been reported as showing radiographic evidence of OA; while a 20-30% of the total population is symptomatic with radiographic evidence of OA (Doherty et al., 2006). Conventional treatment for OA of the knee is aimed at pain management by use of analgesics and non-steroidal anti-inflammatory drugs (NSAIDs). Some negative effects of these drugs include drug dependency, liver and kidney damage, cardiovascular pathologies, gastric upset and depression. Corticosteroid injections are also used to alleviate chronic inflammation and joint pain, but may lead to further joint destruction (Shamoon and Hochberg, 2000; Mayo Foundation for Medical Education and Research, 2011). OsteoEze Gold™ is a nutraceutical product that contains chondroitin sulphate, glucosamine sulphate, vitamin C and manganese. In combination, the constituents of OsteoEze Gold™ have been shown to be useful in the treatment for OA of the knee (Clegg et al., 2006). In addition, studies have shown that these ingredients prove effective in reducing moderate to severe pain in sufferers of OA of the knee (Vidyasagar et al., 2004). The aim of this study was to determine the effect of OsteoEze Gold™ on the inflammatory marker C-reactive protein (CRP) and quality of life in OA of the knee using blood tests and the Arthritic Impact Measurement Scales (AIMS2SF) respectively. This was a 16-week, double blind, placebo-controlled study using matched pairs according to age, gender and severity of symptoms, and formed part of a group study, with another researcher, who utilized the Intermittent and Constant Osteoarthritis Pain scale (ICOAP) Short Physical Performance Battery (SPPB) and the same sample...