Frequency of insertion/deletion polymorphism in exon 8 of HLA-G and kidney allograft outcome

Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex (MHC) class Ib molecule predominantly expressed in cytotrophoblasts, where it acts as a specific immunosuppressor. Literature data have shown that grafts in some settings, such as cardiac and liver/kidney-associated transplantations, express HLA-G and this expression is associated with less severe rejection and also reduces the incidence of rejection. Fourteen-base pair deletion/insertion polymorphism has been reported in exon 8 of the 3`-untranslated region of HLA-G. This polymorphism within exon 8 of the HLA-G gene might influence transcription activity, which in turn may influence the stability of HLA-G transcripts. This influences the stability of the HLA-G protein and therefore is of potential functional relevance. In order to determine a possible correlation between the 14-bp insertion/deletion polymorphism and kidney allograft outcome, we isolated genomic DNA from 83 patients who had received isolated kidney allografts, and we classified the 83 specimens into two groups, grafts presenting Banff features of rejection group and a non-rejection group, and compared them with a control group of 97 healthy subjects. The 14-bp polymorphism at exon 8 was genotyped in all groups. There was no significant difference in allelic frequencies of 14-bp insertion/deletion polymorphism between normal controls and kidney transplant patients. In the RG, the homozygous genotype +14/+14 bp (P = 0.0238) was significantly increased in the group with acute rejection compared with the healthy control group. Analysis of other HLA-G polymorphisms and functional studies on immune regulation are essential to elucidate the role of HLA-G in kidney allografts.

Study of the effectiveness of propolis extract as a storage medium for avulsed teeth

The purpose of the present study was to evaluate the efficacy of propolis extract in maintaining the viability of human periodontal ligament (PDL) cells, and to radiographically analyze tooth replantation and the adjacent periodontium in dogs after storage in this extract. Human PDL cells were incubated with the experimental media propolis, milk, saliva, Hank`s balanced salt solution (HBSS), and Dulbecco`s modified Eagles medium (DMEM, positive controls), and distilled water (negative control). Cell viability was determined 0, 1, 3, 6, 12, and 24 h later by colorimetric MTT assay. Thirty incisors from dogs were divided into two storage time blocks (1 and 3 h) and were maintained in the experimental media. HBSS served as a positive control, and dry teeth (on gauze) as a negative control. The replanted teeth were radiographed once per month for 6 months. The radiographic images were standardized by the shortening/lengthening factor, and were both qualitatively and quantitatively analyzed. The in vitro results showed that the efficacy of propolis in maintaining functional viability of PDL cells was similar to that of milk. Propolis and milk were significantly better than controls from the 6-h time period. The in vivo results showed that teeth maintained in propolis medium exhibited replacement resorption with significant reduction in tooth length, similar to teeth maintained in saliva and dried teeth. This resorption was less intense with the 3-h storage time than the 1-h storage time. Conditions close to normal were found in teeth maintained in milk, similar to the HBSS control. Therefore, although propolis was effective in maintaining the viability of human PDL cells, resorption of the tooth replantation in dogs occurred under these experimental conditions.

beta-strand mimicking macrocyclic amino acids: Templates for protease inhibitors with antiviral activity

New amino acids are reported in which component macrocycles are constrained to mimic tripeptides locked in a beta-strand conformation. The novel amino acids involve macrocycles functionalized with both an N- and a C-terminus enabling addition of appendages at either end to modify receptor affinity, selectivity, or membrane permeability. We show that the cycles herein are effective templates within inhibitors of HIV-1 protease. Eleven compounds originating from such bifunctionalized cyclic templates are potent inhibitors of HIV-1 protease (Ki 0.3-50 nM; pH 6.5, I = 0.1 M). Unlike normal peptides comprising amino acids, five of these macrocycle-containing compounds are potent antiviral agents with sub-micromolar potencies (IC50 170-900 nM) against HIV-1 replication in human MT2 cells. The most active antiviral agents are the most lipophilic, with calculated values of LogD(6.5) greater than or equal to 4. All molecules have a conformationally constrained 17-membered macrocyclic ring that has been shown to structurally mimic a tripeptide segment (Xaa)-(Val/Ile)-(Phe/Tyr) of a peptide substrate in the extended conformation. The presence of two trans amide bonds and a para-substituted aromatic ring prevents intramolecular hydrogen bonds and fixes the macrocycle in the extended conformation. Similarly constrained macrocycles may be useful templates for the creation of inhibitors for the many other proteins and proteases that recognize peptide beta-strands.

Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translation ally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6 x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6 x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria. (C) 2003 Elsevier Science (USA). All rights reserved.

Environmental exposure to toxicants mixtures from a multi-purpose estuary: perspectives for a biomonitoring study in Portugal

This work was focused on a multi-purpose estuarine environment (river Sado estuary, SW Portugal) around which a number of activities (e.g., fishing, farming, heavy industry, tourism and recreational activities) coexist with urban centres with a total of about 200 000 inhabitants. Based on previous knowledge of the hazardous chemicals within the ecosystem and their potential toxicity to benthic species, this project intended to evaluate the impact of estuarine contaminants on the human and ecosystem health. An integrative methodology based on epidemiological, analytical and biological data and comprising several lines of evidence, namely, human contamination pathways, human health effects, consumption of local produce, estuarine sediments, wells and soils contamination, effects on commercial benthic organisms, and genotoxic potential of sediments, was used. The epidemiological survey confirmed the occurrence of direct and indirect (through food chain) exposure of the local population to estuarine contaminants. Furthermore, the complex mixture of contaminants (e.g., metals, pesticides, polycyclic aromatic hydrocarbons) trapped in the estuary sediments was toxic to human liver cells exposed in vitro, causing cell death, oxidative stress and genotoxic effects that might constitute a risk factor for the development of chronic-degenerative diseases, on the long term. Finally, the integration of data from several endpoints indicated that the estuary is moderately impacted by toxicants that affect also the aquatic biota. Nevertheless, the human health risk can only be correctly assessed through a biomonitoring study including the quantification of contaminants (or metabolites) in biological fluids as well as biomarkers of early biological effects (e.g., biochemical, genetic and omics-based endpoints) and genetic susceptibility in the target population. Data should be supported by a detailed survey to assess the impact of the contaminated seafood and local farm products consumption on human health and, particularly, on metabolic diseases or cancer development.

Modulation of osteoclastogenesis by antihypertensive drugs

Despite its rigid structure, bone is a dynamic tissue that is in constant remodeling. This process requires the action of the bone-resorbing osteoclasts and the bone-synthesing osteoblasts. One of the adverse effects attributed to some antihypertensive agents is the ability to alter normal bone metabolism. However, their effective actions on human bone cells remain to be clarified. In this work, the effects of five calcium channel blockers, a class of antihypertensive drugs (AHDs), were investigated on osteoclastic differentiation. Osteoclastic cell cultures were established from precursor cells isolated from human peripheral blood, and were maintained in the absence (control) or in the presence of 10-8-10-4 M of different AHDs (amlodipine, felodipine, diltiazem, lercanidipine and nifedipine). Cell cultures were characterized throughout a 21 day period for tartrate-resistant acid phosphatase (TRAP) activity, number of TRAP+ multinucleated cells, presence of cells with actin rings and expressing vitronectin and calcitonin receptors, and apoptosis rate. Also, the involvement of several signaling pathways on the cellular response was addressed. It was observed that the tested AHDs had the ability to differentially affect osteoclastogenesis. At low doses, amlodipine and felodipine caused an increase on osteoclastic differentiation, while the other drugs inhibited it. At higher doses, all the molecules caused a decrease on the process. The tested AHDs also showed different effects on the analysed signaling pathways. In conclusion, AHDs appeared to have a direct effect on human osteoclast precursor cells, affecting their differentiation. Interestingly, some of them increased while others inhibited the process. Unraveling the mechanisms beneath these observations might help to explain the adverse effects on bone tissue described for this drug class.

Impacto da Radioterapia no stress oxidativo em modelos celulares radiorresistentes em contexto de obesidade

Introdução: Estudos anteriores em modelos tumorais de glioma e melanoma, tumores radiorresistentes, indicaram que a obesidade pode estar relacionada com um aumento do status oxidativo e com a diminuição da resistência à radiação. Como a Radioterapia é o tratamento frequentemente utilizado para esta patologia, propomo-nos, desta forma, a explorar a influência da obesidade em células de glioma, as BC3H1, e melanoma, B16F10, submetidas a Radioterapia, na presença de agentes oxidantes e antioxidantes, para o estudo da sua influência ao nível da viabilidade celular e do impacto do stress oxidativo. Métodos: As células BC3H1 e B16F10 foram tratadas com t-BOOH (150μM e 50 μM, respetivamente), TUDCA (25μM e 1μM, respetivamente) e com a mistura de t-BOOH+TUDCA em meio DMEM sem soro e meio condicionado (CM), a partir de adipócitos 3T3-L1. Em seguida, parte das células foram irradiadas com uma dose total de 2Gy. Posteriormente avaliou-se a viabilidade celular (teste MTT) e o stress oxidativo (teste TBARS, atividade da catalase, concentração da GSH, e status antioxidante total), às 4h e 12h. Resultados: Observou-se um aumento da capacidade antioxidante total das células irradiadas, comparativamente com as células não irradiadas. O meio condicionado reduziu o stress oxidativo nas BC3H1, ao mesmo tempo que reduziu a sua viabilidade celular. O TUDCA nas células incubadas com MC e submetidas a radioterapia, tendencialmente diminuiu a viabilidade celular, nas concertações em estudo. Discussão/Conclusão: O meio condicionado e a radioterapia, por si só, aumentam a resposta antioxidante total na célula, às 4h e às 12h. O TUDCA nas células incubadas com meio condicionado e submetidas a radioterapia, teve um comportamento citotóxico para as BC3H1, nas concentrações testadas. Revelando a necessidade de aprofundar os estudos da ação deste composto como agente radiossensibilizador, neste e noutros modelos celulares de carcinogénese.

Otimização da produção de biogás com recurso a redes neuronais artificiais

Numa Estação de Tratamento de Águas Residuais (ETAR), são elevados os custos não só de tratamento das águas residuais como também de manutenção dos equipamentos lá existentes, nesse sentido procura-se utilizar processos capazes de transformar os resíduos em produtos úteis. A Digestão Anaeróbia (DA) é um processo atualmente disponível capaz de contribuir para a redução da poluição ambiental e ao mesmo tempo de valorizar os subprodutos gerados. Durante o processo de DA é produzido um gás, o biogás, que pode ser utilizado como fonte de energia, reduzindo assim a dependência energética da ETAR e a emissão de gases com efeito de estufa para a atmosfera. A otimização do processo de DA das lamas é essencial para o aumento da produção de biogás, mas a complexidade do processo constitui um obstáculo à sua otimização. Neste trabalho, aplicaram-se Redes Neuronais Artificiais (RNA) ao processo de DA de lamas de ETAR. RNA são modelos simplificados inspirados no funcionamento das células neuronais humanas e que adquirem conhecimento através da experiência. Quando a RNA é criada e treinada, produz valores de output aproximadamente corretos para os inputs fornecidos. Foi esse o motivo para recorrer a RNA na otimização da produção de biogás no digestor I da ETAR Norte da SIMRIA, usando o programa NeuralToolsTM da PalisadeTM para desenvolvimento das RNA. Para tal, efetuou-se uma análise e tratamento de dados referentes aos últimos quatro anos de funcionamento do digestor. Os resultados obtidos permitiram concluir que as RNA modeladas apresentam boa capacidade de generalização do processo de DA. Considera-se que este caso de estudo é promissor, fornecendo uma boa base para o desenvolvimento de modelos eventualmente mais gerais de RNA que, aplicado conjuntamente com as características de funcionamento de um digestor e o processo de DA, permitirá otimizar a produção de biogás em ETAR.

Effect of sialic acid loss on dendritic cell maturation

Sialic acids are key structural determinants and contribute to the functionality of a number of immune cell receptors. Previously, we demonstrated that differentiation of human dendritic cells (DCs) is accompanied by an increased expression of sialylated cell surface structures, putatively through the activity of the ST3Gal.I and ST6Gal.I sialyltransferases. Furthermore, DC endocytosis was reduced upon removal of the cell surface sialic acid residues by neuraminidase. In the present work, we evaluate the contribution of the sialic acid modifications in DC maturation. We demonstrate that neuraminidase-treated human DCs have increased expression of major histocompatibility complex (MHC) and costimulatory molecules, increased gene expression of specific cytokines and induce a higher proliferative response of T lymphocytes. Together, the data suggest that clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. This postulate is supported by mouse models, where elevated MHC class II and increased maturation of specific DC subsets were observed in DCs harvested from ST3Gal.I(-/-) and ST6Gal.I(-/-) mice. Moreover, important qualitative differences, particularly in the extent of reduced endocytosis and in the peripheral distribution of DC subsets, existed between the ST3Gal.I(-/-) and ST6Gal.I(-/-) strains. Together, the data strongly suggest not only a role of cell surface sialic acid modifications in maturation and functionality of DCs, but also that the sialic acid linkages created by different sialyltransferases are functionally distinct. Consequently, with particular relevance to DC-based therapies, cell surface sialylation, mediated by individual sialyltransferases, can influence the immunogenicity of DCs upon antigen loading.

Redes Neuronais Artificiais na Otimização do Processo de Digestão Anaeróbia - Aplicação a uma ETAR da AdCL

Numa sociedade com elevado consumo energético, a dependência de combustíveis fósseis em evidente diminuição de disponibilidades é um tema cada vez mais preocupante, assim como a poluição atmosférica resultante da sua utilização. Existe, portanto, uma necessidade crescente de recorrer a energias renováveis e promover a otimização e utilização de recursos. A digestão anaeróbia (DA) de lamas é um processo de estabilização de lamas utilizado nas Estações de Tratamento de Águas Residuais (ETAR) e tem, como produtos finais, a lama digerida e o biogás. Maioritariamente constituído por gás metano, o biogás pode ser utilizado como fonte de energia, reduzindo, deste modo, a dependência energética da ETAR e a emissão de gases com efeito de estufa para a atmosfera. A otimização do processo de DA das lamas é essencial para o aumento da produção de biogás. No presente relatório de estágio, as Redes Neuronais Artificiais (RNA) foram aplicadas ao processo de DA de lamas de ETAR. As RNA são modelos simplificados inspirados no funcionamento das células neuronais humanas e que adquirem conhecimento através da experiência. Quando a RNA é criada e treinada, produz valores de output aproximadamente corretos para os inputs fornecidos. Uma vez que as DA são um processo bastante complexo, a sua otimização apresenta diversas dificuldades. Foi esse o motivo para recorrer a RNA na otimização da produção de biogás nos digestores das ETAR de Espinho e de Ílhavo da AdCL, utilizando o software NeuralToolsTM da PalisadeTM, contribuindo, desta forma, para a compreensão do processo e do impacto de algumas variáveis na produção de biogás.

Estudo da expressão génica da Legionella pneumophila estirpe paris após co-cultura em Acanthamoeba castellanii

RESUMO: A Legionella é um bacilo Gram-negativo que replica dentro de protozoários como Acanthamoeba castellanii (A. castellanii) e no interior de macrófagos alveolares humanos, podendo resultar numa pneumonia grave. A Legionella em meio líquido tem um ciclo de vida bifásico, apresentando traços replicativos na fase exponencial e expressando factores transmissíveis na fase estacionária. Estudos recentes demonstraram que a Legionella precisa de assegurar um tempo preciso no seu ciclo de vida para efectuar com êxito a infecção das células hospedeiras. Muitos modelos de estudo foram desenvolvidos a fim de aumentar o conhecimento sobre o ciclo de vida intracelular e identificar os genes necessários para a modulação da célula hospedeira. Embora o conhecimento sobre a interacção bactéria-hospedeiro ainda seja limitado, parece que esta interacção gera um conjunto de características de virulência permitindo que a bactéria infecte células fagocíticas humanas e cause doença. O objectivo do presente projecto de investigação foi investigar e seleccionar genes críticos para a infecciosidade da Legionella pneumophila estirpe Paris (Lp Paris), desenhar e optimizar uma técnica de PCR em tempo real para o estudo da expressão génica e comparar o perfil de expressão da Lp Paris antes e depois da co-cultura em A. castellanii. Os resultados mostraram que oito dos 12 genes em estudo alteraram a sua expressão relativa após co-cultura em A. castellanii quando os ensaios foram realizados com culturas de Lp Paris na fase estacionária precoce (cinco foram induzidos e três reprimidos) Quando os ensaios foram realizados com culturas de Lp Paris na fase estacionária tardia 11 genes apresentaram repressão na sua expressão relativa. Analisando os resultados, concluímos que o perfil de expressão de Lp Paris foi modificado pela interacção com A. castellanii, no entanto essa mudança foi dependente da fase do seu ciclo de vida.-------ABSTRACT: Legionella is a pathogenic Gram-negative bacterium that replicates not only within aquatic protozoa like Acanthamoeba castellanii (A. castellanii), but also within human alveolar macrophages, which can result in a severe pneumonia. Legionella has a biphasic life cycle in broth, where exponential phase cultures display replicative traits and stationary bacteria express transmissive factors. Recent studies demonstrated that for successful infection of host cells, Legionella needs to ensure a precise timing of its life cycle. Many models of study were developed in order to learn about the intracellular life cycle and to identify the genes necessary for the host cell modulation. Although knowledge about the bacteria-host interaction is still limited, it appears that this interaction generate a pool of virulence traits, allowing the bacterium to infect human phagocytic cells and cause disease. The purpose of the present study was to investigate and select de critical genes for the infectivity of Legionella pneumophila strain Paris (Lp Paris), design and optimize a real time PCR technique for gene expression study and compare the expression profile of Lp Paris before and after co- culture of A. castellanii. The results show that eight of 12 genes in study changed its relative expression after coculture in A. castellanii when we performed the intracellular assays with early stationary phase Lp Paris cultures (five were induced and tree were repressed). When we performed the intracellular assays with late stationary phase Lp Paris cultures 11 genes showed a repressed relative expression. Analysing the results, we conclude that the expression profile of Lp Paris was modified by interaction with A. castellanii but this change was dependent of the timing of its life cycle.

Evaluation of pathogenic potential of Aeromonas spp. strains using in vitro methodologies

Dissertation to obtain a Master Degree in Molecular Genetics and Biomedicine

Echovírus 6 associado à doença exantemática

Exantema viral é considerado problema comum em regiões tropicais, afetando principalmente crianças. Diversos exantemas cutâneos estão associados a infecções por Enterovirus. Amostras biológicas provenientes de uma criança apresentando exantema generalizado foram enviadas ao Laboratório de Vírus Entéricos do Instituto Adolfo Lutz para a realização do diagnóstico laboratorial. Amostra viral isolada em RD (human rhabdomyosarcoma cells) foi submetida à reação em cadeia pela polimerase apresentando um produto de 437 pares de base, característico de gênero Enterovirus. O sorotipo echovirus 6 (E-6) foi identificado por ensaio de imunofluorescência indireta. Em adição, as amostras pareadas de soro apresentaram soroconversão para E-6. Até o momento, não há relatos do envolvimento de E-6 associado a doenças exantemáticas no Brasil, enfatizando a importância da vigilância epidemiológica para essas doenças e suas complicações.

Contribution to drug discovery and development for tauopathies using yeast as a model

This work aimed to contribute to drug discovery and development (DDD) for tauopathies, while expanding our knowledge on this group of neurodegenerative disorders, including Alzheimer’s disease (AD). Using yeast, a recognized model for neurodegeneration studies, useful models were produced for the study of tau interaction with beta-amyloid (Aβ), both AD hallmark proteins. The characterization of these models suggests that these proteins co-localize and that Aβ1-42, which is toxic to yeast, is involved in tau40 phosphorylation (Ser396/404) via the GSK-3β yeast orthologue, whereas tau seems to facilitate Aβ1-42 oligomerization. The mapping of tau’s interactome in yeast, achieved with a tau toxicity enhancer screen using the yeast deletion collection, provided a novel framework, composed of 31 genes, to identify new mechanisms associated with tau pathology, as well as to identify new drug targets or biomarkers. This genomic screen also allowed to select the yeast strain mir1Δ-tau40 for development of a new GPSD2TM drug discovery screening system. A library of unique 138 marine bacteria extracts, obtained from the Mid-Atlantic Ridge hydrothermal vents, was screened with mir1Δ-tau40. Three extracts were identified as suppressors of tau toxicity and constitute good starting points for DDD programs. mir1Δ strain was sensitive to tau toxicity, relating tau pathology with mitochondrial function. SLC25A3, the human homologue of MIR1, codes for the mitochondrial phosphate carrier protein (PiC). Resorting to iRNA, SLC25A3 expression was silenced in human neuroglioma cells, as a first step towards the engineering of a neural model for replicating the results obtained in yeast. This model is essential to understand the mechanisms of tau toxicity at the mitochondrial level and to validate PiC as a relevant drug target. The set of DDD tools here presented will foster the development of innovative and efficacious therapies, urgently needed to cope with tau-related disorders of high human and social-economic impact.