A pesquisa de substâncias bioativas apresenta diversas etapas de grande complexidade técnico-científica e envolve uma atividade multidisciplinar. No estudo de antineoplásicos, dentre outros candidatos a fármacos, a determinação do perfil de atividade biológica é um ponto essencial para a determinação da potência, toxidez e o índice de seletividade. Neste projeto, os ensaios in vitro de compostos inibidores de catepsinas serão realizados usando células de câncer de próstata resistentes à quimioterapia (DU 145 e PC-3). Os estudos envolverão análises fenotípicas da resposta celular para determinar a viabilidade e o tipo de perturbação do ciclo celular utilizando técnicas colorimétricas e citometria de fluxo, juntamente com o estudo de sinalização celular PI3K-Akt-mTOR. Os testes de atividade citotóxica serão realizados com as células de fibroblasto de camundongo (Balb-c 3T3). Os resultados levarão a caracterização do tipo de resposta biológica e também da citotoxidez das substâncias de interesse. / The search for bioactive substances leads to molecules that need to be qualified throughout many complex steps, involving multidisciplinary activities. For antineoplastic molecules, among other classes of candidate drugs, the determination of the biological activity is the core of the whole process with the aim of determining the potency, toxicity and the selectivity index. In this project, in vitro cell-based bioassays of cathepsin inhibitors are going to be performed using prostate cancer cells resistant to chemotherapy (DU 145 and PC-3). Phenotypic studies are going to be used as end-points to observe the cell viability and the perturbation of the cell cycle by means of colorimetric, flow cytometry, coupled with the study of PI3K-Akt-mTOR signaling pathway. Cytotoxic assays are going to be performed in mouse fibroblasts (Balb-c 3T3). As a result, the biological outcome and the cytotoxic profile of the compounds of interest will be addressed.

O objetivo desse estudo foi avaliar a citotoxicidade do alendronato de sódio (ALN), em diferentes concentrações, sobre osteoblastos em cultura celular. Foi utilizado para o experimento meio sem droga (controle) e meio contendo a droga em diferentes concentrações a saber: 0,5; 1; 5; 10; 20 e 40 mg/ml. Para o estudo foi utilizado uma linhagem de osteoblastos de rato (Osteo-1). A viabilidade celular foi inferida a partir da análise da atividade mitocondrial das células através do método da redução do MTT nos tempos experimentais de 0, 24, 48 e 72 horas. Após análise estatística (teste de Mann-Whitney, p<0,05), os resultados mostraram que, com exceção da concentração de 40mg/ml, todos os grupos apresentaram células viáveis, embora todas as concentrações de ALN tenham reduzido a atividade mitocondrial em mais de 50% em relação ao grupo controle, sugerindo que o ALN nessas concentrações estudadas foi citotóxico para os osteoblastos. / The aim of this study was to analyze the cytotoxicity of a bisphosphonate (sodium alendronate) on rat osteoblasts. The experimental groups were GI (control) no sodium alendronate, and GII, GIII, GIV, GV, GVI, and GVII with sodium alendronate at the concentrations of 0.5, 1, 5, 10, 20, and 40mg/ml, respectively. The cell viability was evaluated by MTT assay in experimental times 0, 24, 48, and 72 h. Data were statistically analyzed. Cultures treated with sodium alendronate showed cell viability percentages significantly lower (p < 0.05) than those of the control groups. In GVII, no viable cell was observed in all experimental times. It could be concluded that sodium alendronate, on direct contact with rat osteoblasts, is cytotoxic in all concentrations studied.

A leishmaniose é uma infecção causada pelo protozoário do gênero Leishmania, que causa um impacto social e econômico elevado, sendo a segunda maior incidência mundial de doença parasitária. Com o intuito de encontrar novas substâncias ou medicamentos de menor toxicidade e de custos inferiores que poderiam ser utilizados nos tratamentos de casos de infecção e, principalmente em situações de resistência parasitária, tem sido averiguada a possibilidade de utilização de substâncias de origem natural, tanto animal como vegetal, e substâncias sintéticas. Entre as que recentemente apresentaram propriedades biológicas úteis, estão alguns derivados de lignanas dibenzilbutirolactônicas que apresentam significativa ação tripanocida. Desse modo, propusemos avaliar a atividade biológica dessas lignanas em sistema in vitro, sobre formas promastigotas e amastigotas de Leishmania amazonensis. Os compostos utilizados foram (1) 7-O-N,N-dimetiletilamino cubebina, (3) cubebina, (D) 6-6´dinitroinoquinina e (H) hinoquinina. Para avaliação da atividade das substâncias foram utilizadas duas metodologias colorimétricas, MTT e Alamar Blue® e a contagem microscópica em hemocitômetro. Pelos resultados obtidos verificamos que pela contagem microscópica das formas promastigotas, as substâncias (1) 7-O-N,N-dimetiletilamino cubebina, (3) cubebina e (D) 6-6´dinitroinoquinina apresentaram potencial leishmanicida, sendo encontrados os seguintes valores de IC50: (1) = 19,4M; (3) 3,6M; (D) = 15,5M. Entretanto, os resultados obtidos com as mesmas substâncias em formas amastigotas do parasita não demonstraram atividade leishmanicida. Os métodos colorimétricos não apresentaram correspondência com a contagem microscópica, mostrando-se ineficazes para essa classe de substância. / Leishmaniasis is an infection caused by a protozoan parasite of the genus Leishmania, being the second great incidence of parasitic diseases in the world. In the search for new drugs or substances with low costs and low toxicity wich could be used in case of infection and situations of parasite resistance, being evaluated the possibility to use natural or synthetic substances. Dibenzylbutirolactonic lignans are substances with useful biological properties presenting significant trypanocidal effect. In this way we proposed to evaluate the in vitro biological activity of the lignans in of Leishmania amazonensis promastigote and amastigote forms . The compounds used were: (1) 7-O-N,N-dimethylamine cubebine, (3) cubebine, (D) 6-6´dinitroinokinine e (H) hinokinin. For the evaluation of these substances, two colorimetric methods were empoyed: MTT and Alamar Blue® , followed by microscopic counting in haemocytometer. After the results obtained after the couting of promastigote forms, we could verify that the substances (1) 7-O-N,N-dimethylamine cubebine, (3) cubebine, (D) 6-6´dinitroinokinine, and (H) hinokinin showed leishmanicidal potential, being fouded the followed values of IC50: (1) = 19.4M; (3) 3.6M; (D) = 15.5M. On the other hand the results obtained of the same substances in the parasite amastigote forms showed no leishmanicidal effect. The colorimetric methods showed no correspondence with microscopic couting demonstrating inefficacy of this substance class.

This study was focused on antiviral effects of stilbenoids against a virus transmitted by ticks. The cell viability of selected cell line cultures in the presence of various concentrations of stilbenoids was determined using the MTT assay. Similarly, the mixed effect of other known antiviral substances and stilbenoids was studied using the MTT assay. Both the prophylactic effects of stilbenoids on the infected culture cell lines and the effect on viral replication were examined. The viral titres from samples were determined using plaque assay. Some of the experiments were performed also in vivo using laboratory mice.

Made available in DSpace on 2014-12-17T14:13:34Z (GMT). No. of bitstreams: 1 PauloDPS_DISSERT_partes_autorizadas.pdf: 366490 bytes, checksum: 3e5abdd24789d0e82844edffadbb1a3e (MD5) Previous issue date: 2010-06-18 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The acidic galactan (AG) was obtained by extraction and proteolysis by acetone precipitation of the eggs of the mollusc Pomacea lineata. Its structure was elucidated by a combination of chemical analysis, the intrinsic viscosity and NMR spectroscopy 1D and 2D. Biological aspects of AG were evaluated by in vivo testing of healing and peritonitis induced (anti-inflammatory activity) and in vitro assays of cytotoxicity (MTT). This polymer showed a simple structure without the presence of sulfate and uronic acids in its structure. Its intrinsic viscosity and relative were evaluated at 0.44 ? 0.05 and 1.744? 0.07 dl.g-1. Spectroscopy showed that the AG has a constitution composed predominantly of &#946;-D-galactosis, and &#946;-D-glucosamine-NAcetil that comes in a smaller proportion in chain. The character of this acidic polysaccharide is given by the presence of pyruvate in the molecule, forming a cyclic acetal of six states, located in &#946;-D-galactosis. The involvement of AG in the healing process was evaluated and the histological analysis revealed that there was so early in the process of healing, a great stimulation of macrophages with granuloma formation. Suggesting that AG may have promoted the advance of biological events required for tissue healing. In the trial of the GA-induced peritonitis showed dose dependent, demonstrating the anti-inflammatory effect at concentrations above 20 mg/kg, and confirming its inflammatory character and the concentration of 1mg/kg. In vitro tests used in the GA concentration of 1000 &#956;g/mL showed proliferative activity by stimulating the growth of 3T3 cells, corroborating the findings in vivo and demonstrating the absence of cytotoxic activity / O Galactano ac?dico (GA) foi obtido atrav?s de extra??o por prote?lise e precipita??o com acetona das ovas do molusco Pomacea lineata. Sua estrutura foi elucidada atrav?s de uma combina??o de an?lises qu?micas, da viscosidade intr?nseca e de espectroscopia de Resson?ncia Magn?tica Nuclear, mono e bidimensionais. Os aspectos biol?gicos do GA foram avaliados atrav?s de ensaios in vivo de cicatriza??o e de peritonite induzida (atividade antiinflamat?ria); e ensaios in vitro da atividade citot?xica (MTT). Este polissacar?deo apresentou uma estrutura simples, sem presen?a de sulfato e ?cidos ur?nicos. Suas viscosidades intr?nseca e relativa foram avaliadas em 0,44 ? 0,05 e 1,744 ? 0,07 dL.g-1. A espectroscopia mostrou que o GA possui uma constitui??o formada predominantemente por &#946;-D-galactoses, al?m de &#946;- D-NAcetil-glucosamina que surge em menor propor??o na cadeia. O car?ter ac?dico deste polissacar?deo ? dado pela presen?a de piruvato na mol?cula, formando um acetal c?clico de 6-membros, localizados nas &#946;-D-galactoses. A participa??o do GA na cicatriza??o foi avaliada e as an?lises histol?gicas revelaram que houve, de forma precoce ao processo de cicatriza??o, uma grande estimula??o de macr?fagos, com forma??o de granulomas. Sugerindo que o GA pode ter promovido a antecipa??o de eventos biol?gicos necess?rios ? cicatriza??o do tecido. No ensaio de peritonite induzida o GA se mostrou dose dependente, demonstrando uma a??o antiinflamat?ria em concentra??es acima de 20mg/kg, e comprovando seu car?ter inflamat?rio na concentra??o de 1mg/Kg. Em ensaios in vitro o GA utilizado na concentra??o de 1000 &#956;g/mL apresentou atividade proliferativa estimulando o crescimento de c?lulas 3T3, corroborando os achados in vivo e demonstrando aus?ncia de atividade citot?xica

A pesquisa de substâncias bioativas apresenta diversas etapas de grande complexidade técnico-científica e envolve uma atividade multidisciplinar. No estudo de antineoplásicos, dentre outros candidatos a fármacos, a determinação do perfil de atividade biológica é um ponto essencial para a determinação da potência, toxidez e o índice de seletividade. Neste projeto, os ensaios in vitro de compostos inibidores de catepsinas serão realizados usando células de câncer de próstata resistentes à quimioterapia (DU 145 e PC-3). Os estudos envolverão análises fenotípicas da resposta celular para determinar a viabilidade e o tipo de perturbação do ciclo celular utilizando técnicas colorimétricas e citometria de fluxo, juntamente com o estudo de sinalização celular PI3K-Akt-mTOR. Os testes de atividade citotóxica serão realizados com as células de fibroblasto de camundongo (Balb-c 3T3). Os resultados levarão a caracterização do tipo de resposta biológica e também da citotoxidez das substâncias de interesse. / The search for bioactive substances leads to molecules that need to be qualified throughout many complex steps, involving multidisciplinary activities. For antineoplastic molecules, among other classes of candidate drugs, the determination of the biological activity is the core of the whole process with the aim of determining the potency, toxicity and the selectivity index. In this project, in vitro cell-based bioassays of cathepsin inhibitors are going to be performed using prostate cancer cells resistant to chemotherapy (DU 145 and PC-3). Phenotypic studies are going to be used as end-points to observe the cell viability and the perturbation of the cell cycle by means of colorimetric, flow cytometry, coupled with the study of PI3K-Akt-mTOR signaling pathway. Cytotoxic assays are going to be performed in mouse fibroblasts (Balb-c 3T3). As a result, the biological outcome and the cytotoxic profile of the compounds of interest will be addressed.

Leukemias are a heterogeneous group of hematologic malignancies. The treatment of these neoplasms has been a challenge to the scientific community due to genetic diversity in leukemic cells. Acquired chromosomal abnormalities as well as modifications of gene expression are involved in the genesis of leukemia. Such facts have increased interest in the development of new effective chemotherapeutic agents that reach specific molecular targets. The compounds of the triazene class like dacarbazine and temozolomide have been used in the clinical treatment of various cancer types, including melanoma, leukemia and glioma. Two new compounds triazenes complexed with copper (CuII) were synthesized to identify new agents with antiproliferative and antibacterial activity. The Compounds 1 - Bis [ 1,3-bis (2-chlorofenyl) triazenido-N11,N13--O-methoxy-pyridine-N-copper(II)] - C36H34N8O2Cl4Cu2 and 2 - Bis [1,3-bis (2-fluorfenyl) triazenido-N11,N13--O-hidroxy-pyridine-N-copper(II)]-C34H28N8O2F4Cu2 were tested in leukaemic cells from AML, ALL, CML and MDS in vitro by MTT colorimetric assay. A higher-capacity of antiproliferative was verified in compound 1 with cytotoxicity (IC50: 3.8 to 28.6 μM) in most cell types at diagnosis, particularly in cells with chromosome abnormalities. This compound also showed in vitro cytotoxicity of relapse of ALL cells with karyotypic alterations. The cytotoxic activity was significantly higher in leukemic cells than in normal cells. The MIC values showed that compound 1 had higher activity than compound 2, showing narrow spectrum antibacterial activity against gram positive ATCCs and multiresistant bacterial strains. It was observed that Compound 1 was more promising in relation to the antiproliferative potential than the antimicrobial activity, even when compared to antineoplastic agents such as etoposide. Additional studies to understand the mechanism of action will soon be developed. / As leucemias formam um grupo heterogêneo de neoplasias hematológicas. O tratamento destas neoplasias vem sendo um desafio à comunidade científica devido à diversidade genética nas células leucêmicas. Alterações cromossômicas adquiridas e modificações da expressão gênica estão envolvidas na gênese das leucemias. Isto tem estimulado o interesse no desenvolvimento de novos agentes quimioterapêuticos eficazes que atinjam alvos moleculares específicos. Os compostos da classe triazeno, tais como a dacarbazina e temozolomida, têm sido usados no tratamento clínico de diversos tipos de câncer, incluindo glioma, leucemia e melanoma. Dois novos compostos triazenos complexados com cobre (CuII) foram sintetizados com o objetivo de identificar novos agentes com atividade antiproliferativa e antibacteriana. Os compostos 1 - Bis[1,3-bis(2-clorofenil)triazenido-N11,N13--O-metóxi-piridina-N-cobre(II)] - C36H34N8O3Cl4Cu2 e 2- Bis[1,3-bis(2-fluorfenil)triazenido-N11,N13--O-hidróxi-piridina-N-cobre(II)] - C36H34N8O3F4Cu2 foram testados em células leucêmicas de LMA, LLA, LMC e SMD in vitro pelo teste colorimétrico MTT. O composto 1 apresentou melhor atividade citotóxica frente à maioria dos tipos celulares avaliados neste estudo (IC50: de 3,8 a 28,6 μM) especialmente em células com alterações cromossômicas. Este composto ainda apresentou citotoxicidade em células de recidiva de LLA com alterações cariotípicas. Ressaltamos que sua atividade citotóxica foi significativamente mais elevada em células leucêmicas do que em células normais. Os valores de CIM demonstraram que o composto 1 apresentou maior atividade antibacteriana que o composto 2, apresentando atividade de estreito espectro frente a cepas bacterianas gram positivas ATCCs e multirresistentes. Observou-se que o composto 1 foi mais promissor em relação ao potencial antiproliferativo do que ao potencial antibacteriano, inclusive quando comparado a agentes antineoplásicos como o etoposide. Estudos adicionais para melhor compreensão do mecanismo de ação deverão ser desenvolvidos.

In recent years, tissue engineering has shown great potential in treatment of injured tissues which aims to create artificial structures for cells to regenerate new tissues for replacing the damaged and diseased ones. The selection of scaffold materials is one of the critical factors affecting tissue healing process. Among a wide range of scaffold materials, chitosan (CS) has been demonstrated as an ideal material due to its biocompatibility, nontoxicity, biodegradability, antibacterial activity and favorable strength and stiffness. However, its insufficient mechanical properties limits its feasibility and scope for clinical application, especially for bone scaffolds. The main purpose of the study is to explore the potential of incorporation of nanofillers into CS to enhance the mechanical properties for tissue engineering. In this work, nanodiamond (ND) is applied and studied due to its high surface to volume ratio, rich surface chemistry, high mechanical strength, and excellent biocompatibility. ND/CS nanocomposites with different diamond concentration from 1wt. % to 5wt. % were synthetized through a solution casting method. The microstructure and mechanical properties of the composites were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC), and nanoindentation. Compared with pristine CS, the addition of ND resulted in a dramatic improvement of mechanical properties, including a 239%, 276%, 321%, 333%, and 343% increase in Young’s modulus and 68%, 96%, 114%, 118%, and 127% increase in hardness when ND amount is 1wt. %, 2wt. %, 3wt. %, 4wt. %, and 5wt. %, respectively. The strong interaction between ND surface groups and chitosan matrix is of great importance in changing polymer structure and improving mechanical properties. The cell viability and cytotoxicity of the nanocomposite were also studied using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. The results show that the addition of ND has no negative effect on cell viability and the nanocomposites have no cytotoxicity.

This thesis presents a clustering-based approach to decrease the computational cost of data association in multi-target tracking. This is achieved by clustering the sensor tracks using approximate distance functions, thereby decreasing the number of possible associations and the need to calculate expensive statistical distances between tracks. The studied tracking problem includes passive and active sensors with built-in filters. Statistical and non-statistical distance functions were designed to account for the characteristics of the different combinations of sensors. The computational cost and accuracy of these distance functions were evaluated and compared. Analysis is done in a simulated environment with randomly positioned targets and sensors. Simulations show that there are approximate distances with a cost of calculation ten times cheaper than the true statistical distance, with only minor drops in accuracy. Spectral clustering is used on these distances to divide complex association problems into sub-problems. This algorithm is evaluated on a large number of random scenarios. The mean size of the largest sub-problem is 40 % of the original, and the mean number of errors in the clustering is 5 %.

The diploma thesis was focused on the preparation and characterization of nanoparticles and nanofibres with active substances from chosen herbs with the aim to apply this materials to the products against acne. Various types of extracts were tested on the content of polyphenols, flavonoids and antioxidation activity. These extracts were encapsulated to the liposomes and fibres of polyhydroxybutyrate. Prepared liposomes and fibres were tested mainly on antioxidation activity and antimicrobial activity against the strain Propionibacterium acnes. Then, liposomes were applied to cosmetic emulsions. These creams reported high antioxidation activity and excellent stability determined by analytical centrifugation. Prepared nanofibres also reported high antioxidation activity and antimicrobial effect as well. Finally, particles and fibres were tested in contact with human cells. In appropriate concentration, there was no cytotoxic effect and tested materials can be used in applications on problems with acne.