The immunophilins are a superfamily of proteins comprising the cyclophilins, the FKBPs and the parvulin sub-families. Members are present ubiquitously in plant and animal cells, acting as both prolyl-isomerases and signalling proteins. Some also have chaperone activity. The prolyl isomerase function of the immunophilins has been identified as being central to progression of a large number of diseases, making them tempting drug targets. Whilst there are several assays which can be used to identify inhibitors of the prolyl isomerase function, they are hampered by one or more problems: multistep mechanisms, poor signal-to-noise ratios, expensive, laborious and unamenable to high throughput screening. Multiple fluorescent systems (fluorescence anisotropy, FRET, 2D-FIDA/FCS) and several technologies (solution and solid phase synthesis, solution and solid phase screening, combinatorial synthesis, and stopped-flow spectrometry) were explored to develop a system suitable for fast, efficient screening of immunophilins. The most promising of these is a prototype assay based on the design, cloning, expression and production of fluorescently labelled mutant of cyclophilin B, which shows an increase in fluorescence emission upon cyclosporin ligand binding.

Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-02-06T14:55:43Z No. of bitstreams: 2 Dissertação - Paulo de Tarso Garcia - 2014..pdf: 2337784 bytes, checksum: 4d5ad6bbe0d8446d9325ce820d3f96af (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-02-19T14:20:12Z (GMT) No. of bitstreams: 2 Dissertação - Paulo de Tarso Garcia - 2014..pdf: 2337784 bytes, checksum: 4d5ad6bbe0d8446d9325ce820d3f96af (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-19T14:20:13Z (GMT). No. of bitstreams: 2 Dissertação - Paulo de Tarso Garcia - 2014..pdf: 2337784 bytes, checksum: 4d5ad6bbe0d8446d9325ce820d3f96af (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-08-14 / This report describes the development of microfluidic paper-based analytical devices (μPADs) with chemically modified surface for clinical assays with colorimetric detection. The μPADs were fabricated by a stamping-based method with a heated metal stamp for obtain hydrophobic barriers of paraffin in paper. Before of the stamp step, the paper was oxidized to promote the conversion of hydroxyl groups in aldehyde groups for further chemical activation for the immobilization of enzymes. The μPADs were used for complexometric assays of nitrite and bovine serum albumin (BSA) and enzymatic assays of glucose and uric acid (UA). The chemical modification did provide better color uniformity inside of the detection zones of the enzymatic bioassays. After the chemical modification, the relative standard deviation (RSD) values for glucose and UA assays decreased from 40 to 10% and from 20 to 8%, respectively. Clinical assays for glucose, UA, nitrite and BSA were performed in levels which included the clinical range for each bioassay. We performed quantitative analysis of all analytes in artificial urine sample with error values ranged from 2,5 to 4,0%. The robustness tests proved the stability of the chemical modification process and the thermal stability of the μPADs when stored at different temperatures, showing the potential of the devices as trade platforms for clinical analysis. Advantages such as low cost per assay ($ 0.01), portability and easiness of fabrication enable for the use of μPADs in clinical diagnostics in places with limited resources and at the point-of-care, which is the place where the patient requires the analysis. / Este trabalho descreve o desenvolvimento de dispositivos microfluídicos de papel (μPADs, do inglês microfluidic paper-based analytical devices) com superfície quimicamente modificada para ensaios clínicos utilizando detecção colorimétrica. Os μPADs foram fabricados por um método de carimbagem com o auxílio de um carimbo metálico aquecido para delimitar barreiras hidrofóbicas de parafina no papel. Antes da etapa de carimbagem, o papel foi oxidado para promover a conversão dos grupos hidroxila em grupos aldeídos, para posterior ativação química para imobilização de enzimas. Os μPADs foram utilizados para ensaios complexométricos de nitrito e albumina bovina sérica (BSA, do inglês bovine serum albumin) e ensaios enzimáticos de glicose e ácido úrico (AU). A modificação química utilizada proporcionou uma melhora significativa na uniformidade de cor gerada no interior das zonas de detecção dos bioensaios enzimáticos. Após a modificação química, os valores do desvio padrão relativo (DPR) para os ensaios de glicose e AU diminuíram de 40 para 10% e de 20 para 8%, respectivamente. Ensaios clínicos para glicose, AU, nitrito e BSA foram realizados em concentrações que abrangem a faixa clínica de cada bioensaio. Realizou-se uma análise quantitativa de todos os analitos em uma amostra artificial de urina, obtendo valores de erro entre 2,5 e 4,0%. Os testes de robustez comprovaram a estabilidade do processo de modificação química e a estabilidade térmica dos μPADs quando estocados em diferentes temperaturas, mostrando a potencialidade dos dispositivos como plataformas comerciais para análises clínicas. Vantagens como o baixo custo por ensaio (R$ 0,01), portabilidade e facilidade de fabricação contribuem para a utilização dos μPADs em diagnósticos clínicos em locais com recursos limitados e no local de necessidade (point-of-care), que é o local onde o paciente necessita da análise.

Este trabalho apresenta o processo para obtenção de partículas de sílica amino-funcionalizadas contendo complexos de TR3+ utilizando os métodos de Stöber e por micro-ondas. Os espectros de absorção no infravermelho das partículas TR-BTC-Si preparadas pelo método de micro-ondas exibiram bandas de absorção atribuídas aos modos vibracionais dos complexos TR-BTC e da rede de sílica, indicando a incorporação destes complexos na matriz SiO2. Por outro lado, os complexos Eu-(β-dicetonatos) preparados pelo método Stöber mostraram apenas as bandas atribuídas à estrutura da rede de sílica, devido à dupla camada de revestimento de sílica. As morfologias das partículas de sílica amino-funcionalizadas contendo complexos de TR3+ foram visualizadas usando a técnica MEV. As diferenças nas morfologias entre o complexo precursor e o material amino-funcionalizado pode ser atribuído a presença da sílica na superfície do material. Além do mais, o método da ninidrina indicou a presença de grupos amina (-NH2) na superfície destes materiais. Os espectros de emissão dos materiais funcionalizados com os complexos de Eu3+ e Tb3+ apresentaram as bandas de emissão da transição intraconfiguracional dos íons Eu3+ (5D0→7FJ, J = 0-6) e Tb3+ (5D4→7FJ, J = 6-0), exibindo cores características de emissão vermelha e verde, respectivamente. É observado um decréscimo nos valores dos parâmetros Ω2 dos materiais Eu-(β-dicetonato)-Si e Eu-BTC-Si comparados com os respectivos complexos, devido a diminuição da intensidade da transição 5D0→7F2, indicando um maior caráter centrossimétrico. Conseqüentemente, os íons Eu3+ nos materiais com sílica estão em um ambiente químico menos polarizável do que nos complexos, sugerindo uma menor contribuição do mecanismo de acoplamento dinâmico. Os altos valores dos Ω4 para os sistemas com sílica Eu-(&#946-dicetonato)-Si e Eu-BTC-Si comparados com os valores de Ω2 reflete a intensidade extremamente alta da transição 5D0→7F4 observada nos espectros de emissão. Este resultado corrobora com o maior caráter centrossimétrico e evidencia a incorporação dos complexos de Eu3+ na rede de sílica. Foi realizado um fluoroimunoensaio usando os compostos amino-funcionalizados, que mostraram uma luminescência e propriedades físico-químicas eficientes para atuarem como marcadores biológicos. O marcador óptico foi conjugado com o anticorpo anti-oxLDL, que se liga a um suporte específico com o antígeno oxLDL. Portanto, estes materiais são candidatos promissores para conjugação molecular em clínicas de diagnóstico. / This work presents the process to obtain amino-functionalized silica particles containing complexes of trivalent rare earth ions (RE3+) using Stöber and microwave methods. Infrared spectra of the TR-BTC-Si particles prepared by microwave method exhibited absorption bands assigned to the vibrational modes of the TR-BTC complexes and silica network, indicating that the complexes have been incorporated in the SiO2 matrix. However, due to double coating of the silica network, the IR spectra of the Eu-(β-diketonates) complexes prepared by Stöber method showed only bands assigned to the silica structure. The morphologies of the amino-functionalized silica particles containing RE3+ complexes were examined using SEM technique. The difference in morphology between the complex precursor and amino-functionalized material can be attributed to the silica network on the material surface. Besides, the ninhydrin method confirmed the presence of amine groups (-NH2) in the functionalized materials. The emission spectra of the functionalized materials containing Eu3+ and Tb3+ complexes showed the emission bands originated from the intraconfigurational transitions of the Eu3+ (5D0→7FJ, J = 0-4) and Tb3+ (5D4→7FJ, J = 6-0), exhibiting red and green color emission, respectively. It is observed decreasing values of experimental intensity parameters (Ω2) of the Eu-(β-diketonate)-Si and Eu-BTC-Si materials when compared with the complex precursors, owing to the decreased intensity of the 5D0→7F2 transition, indicating a higher centrosymmetric character. As a result, the Eu3+ ions in silica materials are located in a chemical environment less polarizable than in the complexes, suggesting a smaller contribution of dynamic coupling mechanism. On the other hand, an abnormally high intensity of the 5D0→7F4 transition was observed, which is reflected by the high values of Ω4 of the silica systems, Eu-(β-diketonate)-Si and Eu-BTC-Si, compared with the Ω2 ones. These spectroscopic data corroborate with a higher centrosymmetric character, indicating the incorporation of the Eu3+ complexes in the silica network. A fluoroimmunoassay was developed using the amino-functionalized compounds that exhibit efficient luminescence and physical and chemical properties suitable for optical label. The biolabel was then chemically conjugated to anti-oxLDL antibody, which is linked in a specific support with oxLDL antigen. The result showed that it is a promising candidate for molecular conjugation in clinical diagnosis.

Os ensaios celulares têm ganhado relevância na gênese planejada de fármacos, devido a sua utilização nas diversas etapas envolvidas neste processo. Estes ensaios envolvem a caracterização da atividade farmacológica, propriedades farmacocinéticas e atividade tóxica para compreender a atividade biológica das moléculas de interesse. Neste trabalho, os ensaios celulares foram usados para identificar a atividade anticancerígena e a atividade tóxica de moléculas, uma vez que a morte celular é o parâmetro avaliado em ambos os casos. No presente estudo foram avaliados 34 compostos, sendo dezessete moléculas do grupo NEQUIMED, determinando-se sua atividade citotóxica na célula neoplásica de fígado (HepG2) e na célula de fibroblasto (V79-4). A determinação da atividade citotóxica dos compostos bioativos foi realizada por o método colorimétrico de triagem envolvendo o MTT (brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio), que é metabolizado pela mitocôndria da célula viva, com confirmação da atividade biológica realizada por citometria de fluxo para a linhagem de fibroblasto. As triagens iniciais foram estabelecidas para determinar a atividade biológica, sendo que as moléculas Neq256, Neq385 e Neq388 apresentaram atividade citotóxica frente à célula HepG2 (caracterizando assim a atividade anticancerígena), enquanto que Neq385 apresentou seletividade em relação a atividade nas células de fibroblasto. Dentre as moléculas de referência, YM-155 apresentou os melhores resultados de atividade citotóxica com IC50 (HepG2) de 0,094 µmol L-1 e IC50 (V79-4) > 100 µmol L-1, sendo muito seletiva para a linhagem cancerígena. Os resultados demonstraram que as moléculas Neq265, Neq385 e Neq388 são promissoras e serão usadas para o planejamento de modificações estruturais que visa obter moléculas com maior potência e seletividade frente às células cancerígenas. Outra vertente do trabalho envolve o planejamento de inibidores da survivina, que apresentam grande potencial para a descoberta e desenvolvimento de estratégias quimioterápicas seletivas. / Cell-based assays are gaining relevance in the drug discovery and development area, being in use almost throughout the whole process. These assays are applied to characterize the pharmacological, pharmacokinetic and toxic activities of new molecules. In this work, cell-based assays were performed to identify anticancer and toxic activities of novel compounds, once the cell death process is the parameter that was evaluated in both cases. In this work, 34 compounds were evaluated (17 of them from the NEQUIMED database) in which the cytotoxic activity in liver cancer cells (HepG2) and hamster fibroblast cells (V79-4) were determined by means of the MTT colorimetric screening. The biological activity in fibroblast cells was further confirmed by using flow cytometry. Out of the whole set, molecules Neq256, Neq385 e Neq388 were cytotoxic to HepG2 (having anticancer activity). Neq385 was selective towards the liver hepatocellular carcinoma when compared with the fibroblasts. Among the reference compounds, YM-155 was the most selective and potent anticancer molecule: IC50 (HepG2) 0.094 µmol L-1 and IC50 (V79-4) > 100 µmol L-1. Taken together, these results provide promissing new molecules (Neq265, Neq385 e Neq388) for further optimization of the potency and selectivity using drug design. Another important outcome for further exploration is the design of survivin inhibitors bearing a huge potential for novel selective chemotherapeutic approaches.

Os ensaios celulares têm ganhado relevância na gênese planejada de fármacos, devido a sua utilização nas diversas etapas envolvidas neste processo. Estes ensaios envolvem a caracterização da atividade farmacológica, propriedades farmacocinéticas e atividade tóxica para compreender a atividade biológica das moléculas de interesse. Neste trabalho, os ensaios celulares foram usados para identificar a atividade anticancerígena e a atividade tóxica de moléculas, uma vez que a morte celular é o parâmetro avaliado em ambos os casos. No presente estudo foram avaliados 34 compostos, sendo dezessete moléculas do grupo NEQUIMED, determinando-se sua atividade citotóxica na célula neoplásica de fígado (HepG2) e na célula de fibroblasto (V79-4). A determinação da atividade citotóxica dos compostos bioativos foi realizada por o método colorimétrico de triagem envolvendo o MTT (brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio), que é metabolizado pela mitocôndria da célula viva, com confirmação da atividade biológica realizada por citometria de fluxo para a linhagem de fibroblasto. As triagens iniciais foram estabelecidas para determinar a atividade biológica, sendo que as moléculas Neq256, Neq385 e Neq388 apresentaram atividade citotóxica frente à célula HepG2 (caracterizando assim a atividade anticancerígena), enquanto que Neq385 apresentou seletividade em relação a atividade nas células de fibroblasto. Dentre as moléculas de referência, YM-155 apresentou os melhores resultados de atividade citotóxica com IC50 (HepG2) de 0,094 µmol L-1 e IC50 (V79-4) > 100 µmol L-1, sendo muito seletiva para a linhagem cancerígena. Os resultados demonstraram que as moléculas Neq265, Neq385 e Neq388 são promissoras e serão usadas para o planejamento de modificações estruturais que visa obter moléculas com maior potência e seletividade frente às células cancerígenas. Outra vertente do trabalho envolve o planejamento de inibidores da survivina, que apresentam grande potencial para a descoberta e desenvolvimento de estratégias quimioterápicas seletivas. / Cell-based assays are gaining relevance in the drug discovery and development area, being in use almost throughout the whole process. These assays are applied to characterize the pharmacological, pharmacokinetic and toxic activities of new molecules. In this work, cell-based assays were performed to identify anticancer and toxic activities of novel compounds, once the cell death process is the parameter that was evaluated in both cases. In this work, 34 compounds were evaluated (17 of them from the NEQUIMED database) in which the cytotoxic activity in liver cancer cells (HepG2) and hamster fibroblast cells (V79-4) were determined by means of the MTT colorimetric screening. The biological activity in fibroblast cells was further confirmed by using flow cytometry. Out of the whole set, molecules Neq256, Neq385 e Neq388 were cytotoxic to HepG2 (having anticancer activity). Neq385 was selective towards the liver hepatocellular carcinoma when compared with the fibroblasts. Among the reference compounds, YM-155 was the most selective and potent anticancer molecule: IC50 (HepG2) 0.094 µmol L-1 and IC50 (V79-4) > 100 µmol L-1. Taken together, these results provide promissing new molecules (Neq265, Neq385 e Neq388) for further optimization of the potency and selectivity using drug design. Another important outcome for further exploration is the design of survivin inhibitors bearing a huge potential for novel selective chemotherapeutic approaches.

Target cell lysis is the hallmark of immune effector responses of cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and monocytes. The most commonly used assay to measure target cell lysis is the 51Cr release assay and is considered the ‘gold standard’. However, this assay has many disadvantages that limit its use by most laboratories. Thus, several alternative assays have been developed. Some of these alternative assays are more sensitive, easy to perform and do not use radioactive elements. In this study, four of these assays were evaluated for their ability to detect antigen- specific CTL responses in equine blood. Three long-term equine arteritis virus (EAV) carrier stallions, two vaccinated stallions and one naïve stallion were included in this study. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected of these stallions to be used as effector cells. The PBMCs were stimulated with EAV in vitro for 7-10 days to generate antigen-specific effector cells. The granzyme B assay, the Carboxyfluorescein succinimidyl ester (CFSE)/7-Aminoactinomycin D (7AAD) assay and the Lactate dehydrogenase (LDH) assay were performed using these effector cells and autologous equine dermal cells (isolated from each stallion) as target cells. The first two assays (i.e., granzyme B and CFSE/7AAD assays) were difficult to optimize for this study because they work well with non-adherent targets and require immediate flow cytometry analysis. The LDH assay, however detected CTL lysis in one of the two vaccinated stallions at day 99 post vaccination and no response was detected in PBMCs isolated from carrier stallions and control stallion. Based on these findings, the LDH assay is the most suitable assay since it works well with adherent target cells, it produces quantitative data, and is ideal for high-throughput screening.

Cryptosporidiosis is a common diarrheal disease caused by intestinal infection with the apicomplexan parasite Cryptosporidium, in humans usually either with C. hominis or C. parvum. Unfortunately, given a large burden of disease in children and immunocompromised people like AIDS patients, the only currently approved treatment, nitazoxanide, is unreliable for these patient populations. To address the urgent need for new drugs for the most vulnerable populations, large phenotypic screening efforts have been established to identify anti-Cryptosporidium growth inhibitors in vitro (hits). However, in the absence of a gold standard drug, the in vitro and in vivo characteristics that should be used to prioritize screening hits are not known. This thesis is focused on identifying promising anti-Cryptosporidium hits and drug leads, and using them to establish validated methods to guide hit-to-lead studies for anti-Cryptosporidium drug development. A re-analysis of our phenotypic screen of the Medicines for Malaria Venture Open Access Malaria Box identified a promising C. parvum growth inhibitor, MMV665917. It had similar in vitro activity against C. hominis, C. parvum Iowa, and C. parvum field strains, and it was amenable to preliminary structural activity relationship studies using commercially available variants, with one variant demonstrating nanomolar potency. Furthermore, MMV665917 was effective in vivo in an acute interferon-γ mouse model of cryptosporidiosis; and it appeared to cure an established infection in the chronic NOD SCID gamma (NSG) mouse model, unlike nitazoxanide, paromomycin, and clofazimine. We hypothesized that anti-Cryptosporidium activity in the highly immunocompromised chronic NSG mouse model might relate to compounds being capable of killing and eliminating parasites (cidal), rather than only preventing growth (static). To test this, we developed a novel in vitro parasite persistence assay that showed that MMV665917 was potentially cidal, whereas nitazoxanide, paromomycin and clofazimine appeared static. This pharmacodynamic assay also provided the concentration of compound required to maximize rate of parasite elimination, which could help design in vivo experiments. To further characterize compounds based on mechanism of action, we developed a range of in vitro medium-throughput life-stage assays. To validate and gain value from the assays, a “learner set” of compounds from our in-house screens and collaborations were tested in all of the in vitro assays and in the in vivo NSG mouse model. Using these assays, it was possible to group molecules based on chemical class/mechanism of action. Because compounds from distinct groups showed activity in the NSG mouse model, these methods could be used to obtain a diverse set of early-stage Cryptosporidium inhibitors for prioritization. Furthermore, compounds that appeared static in the in vitro parasite persistence assay did not have activity in the NSG mouse model. In summary, we report the identification and development of a highly promising initial lead, MMV665917, and report a range of in vitro assays that can be used to prioritize anti-Cryptosporidium hits and leads.

No description available.

The antimicrobial properties of dextrose encapsulated gold nanoparticles (dGNPs) with average diameters of 25 nm, 60 nm, and 120 nm (± 5 nm) synthesized by green chemistry principles were investigated against both Gram-negative and Gram-positive bacteria. Studies were performed involving the effect of the dGNPs on the growth, morphology and the ultrastructural properties of bacteria. dGNPs were found to have significant dose dependent antibacterial activity which was directly proportional to their size and also their concentration. The microbial assays revealed the dGNPs to be bacteriostatic as well as bactericidal. The dGNPs exhibited their bactericidal action through the disruption of the bacterial cell membrane causing leakage of cytoplasmic content. The overall outcomes of this study suggest that dGNPs hold promise as a potent antimicrobial agent against a wide range of disease causing bacteria and can control and prevent possible infections or diseases.

African sleeping sickness is a parasitic infection spread by the protozoan parasite Trypanosoma brucei, and drugs used today are toxic and painful. Galactose metabolism is essential for the survival of T. brucei and without a functional UDP galactose 4’ epimerase (GalE) galactose starvation occurs and cell death will follow. In this Master thesis project two assays observing binding of small molecules to TbGalE has been investigated in attempt to establish an assay that in the future could be used for screening for drugs. TbGalE was biotinylated through the Pinpoint Xa vector and expressed in E. coli cells. The protein was successfully immobilized to a Streptavidin chip for Surface Plasmon Resonance experiments and the binding of the substrates UDP-galactose and UDP-glucose was observed. Unfortunately, the assay was not optimal for screening due to low signal response. However, the established protocol for expressing biotinylated proteins that bind to Streptavidin surfaces could be used in further experiments with TbGalE and other drug targets for African sleeping sickness. The fluorescent sugar nucleotide analogue UDPAmNS, which is a known inhibitor for E. coli GalE, was synthesised and purified and then used to establish a displacement assay. IC50 of UDPAmNS against TbGalE was determined and a synergic effect in fluorescence between the protein and the inhibitor was proven. Further, evidence for a reduction in fluorescence by displacing UDPAmNS with UDP was obtained. This reduction in fluorescence was also shown by a predicted cofactor inhibitor. The IC50 against TbGalE for this compound was determined before the displacement assay, which showed that the cofactor inhibitor, at least partly, binds to the active site of TbGalE. The UDPAmNS displacement assay could have the potential of becoming a robust screening assay for TbGalE, in the effort to find a better drug for African sleeping sickness.