Kinetics of growth of Leishmania (Leishmania) chagasi cycle in McCoy cell culture

The kinetics of growth of Leishmania performed in vitro after internalization of the promastigote form in the cell and the occurrence of the transformation of the parasite into the amastigote form have been described by several authors. They used explants of macrophages in hamster spleen cell culture or in a human macrophage lineage cell, the U937. Using microscopy, the description of morphologic inter-relationship and the analysis of the production of specific molecules, it has been possible to define some of the peculiarities of the biology of the parasite. The present study shows the growth cycle of Leishmania chagasi during the observation of kinetic analysis undertaken with a McCoy cell lineage that lasted for a period of 144 hours. During the process, the morphologic transformation was revealed by indirect immunofluorescence (IF) and the molecules liberated in the extra cellular medium were observed by SDS-PAGE at 24-hour intervals during the whole 144-hour period. It was observed that in the first 72 hours the promastigote form of L. chagasi adhered to the cell membranes and assumed a rounded (amastigote-like) form. At 96 hours the infected cells showed morphologic alterations; at 120 hours the cells had liberated soluble fluorescent antigens into the extra cellular medium. At 144 hours, new elongated forms of the parasites, similar to promastigotes, were observed. In the SDS-PAGE, specific molecular weight proteins were observed at each point of the kinetic analysis showing that the McCoy cell imitates the macrophage and may be considered a useful model for the study of the infection of the Leishmania/cell binomial.

Remediação Ambiental

A agricultura é uma das atividades mais antigas realizadas pelo Homem, sendo de grande importância para a obtenção tanto de bens alimentares como de bens para outros fins. No entanto desde o início constatou-se que as culturas eram afetadas por pragas e doenças que levavam à perda das colheitas. Este motivo deu origem à necessidade de nesses termos surgiu a aplicação de substâncias com o objetivo de proteger as colheitas. Os pesticidas são substâncias naturais ou sintéticas, aplicadas com o objetivo de proteger as plantas eliminando pragas e doenças. Para além da potencial toxicidade destas substâncias, em alguns casos a sua degradação no meio ambiente por microrganismos, hidrólise, radiação solar, etc. dá origem a produtos de degradação tanto ou mais tóxicos que os próprios pesticidas. A utilização deste tipo de substâncias acarreta problemas, visto a sua aplicação ser feita de forma a compensar perdas que ocorrem por meio de degradação, lixiviação, entre outros processos. Este tipo de aplicação leva a que haja contaminação do meio ambiente por parte dos pesticidas, pondo em risco tanto a saúde humana como os restantes seres vivos. A utilização de ciclodextrinas no encapsulamento destes compostos tem como objetivo aumentar a estabilidade do composto e promover a sua libertação de forma controlada. No presente trabalho pretende-se efetuar um estudo comparativo sobre a fotodegradação do herbicida terbutilazina e do fungicida pirimetanil livres e quando encapsulados com 2- hidroxipropil-β- ciclodextrina. De forma a quantificar os pesticidas ao longo do estudo foi utilizado o método analítico de HPLC de fase reversa. Os resultados permitiram constatar que a terbutilazina é fotoquimicamente estável, nas condições aplicadas, visto que ao fim de 75 dias de as soluções de pesticida livre em água desionizada e em água do rio apresentarem ainda 98% do pesticida inicial e as soluções de pesticida encapsulado em água desionizada e em água do rio apresentarem ainda 98% do pesticida inicial. Neste caso particular não foi possível, no intervalo de tempo considerado, avaliar a influência do encapsulamento no processo de fotodegradação da terbutilazina. Dada a baixa fotodegradação observada optou-se pela adição de peróxido de hidrogénio às soluções de controlo e 35 mM de HP-β-CD e acetona às soluções de 0 mM e 17,5 mM de HP-β-CD, para tentar promover a degradação do pesticida. Através dos resultados obtidos constatou-se que particularmente para as soluções onde foi adicionada acetona houve um aumento da velocidade de degradação no entanto esta ainda ocorria de forma lenta e muito semelhante quer para o pesticida livre quer para o encapsulado. Relativamente ao estudo da fotodegradação do pirimetanil verificou-se que ao fim de 4 dias de irradiação as soluções de pesticida livre apresentavam já alguma degradação do pesticida e tendo o período de irradiação uma duração de 53 dias foi possível para este pesticida determinar os parâmetros cinéticos em algumas das soluções. Quanto as soluções de água desionizada e água do rio com pirimetanil livre ambas apresentaram degradação do pesticida verificando-se uma cinética de reação de 1ª ordem com constantes de 0,0018 dias-1 e de 0,0060 dias-1 respetivamente. Para a solução de água desionizada com pirimetanil encapsulado não foi detetada degradação do pesticida, já para a solução com pirimetanil encapsulado em água do rio verificou-se a existência de degradação que correspondeu a uma cinética de degradação de 1ª ordem com uma constante de 0,0013 dias-1. Através dos resultados obtidos pode-se concluir que o encapsulamento do pirimetanil com 2-hidroxipropil-β-ciclodextrina é vantajoso visto diminuir a quantidade de pesticida utilizado e aumentar a eficácia do controlo das pragas.

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

J Biol Inorg Chem (2007) 12:691–698 DOI 10.1007/s00775-007-0219-9

Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases

J Biol Inorg Chem (2006) 11: 433–444 DOI 10.1007/s00775-006-0090-0

Kinetics of the cercaria-schistosomulum transformation in vivo

Sitice most studies on the cercaria-schistosomulum transformation have been carried out in vitro, the authors used the inoculation ofcercariae into the peritoneal cavity of mice tofollow the steps involved in this progressive adaptation of cercarie to the vertebmte host. The main conclusions were: 1. Most cercariae reach the schistosomular stage between 90-120 min after intraperitoneal inoculation. 2. Changes usuallystart with detachment of the tail followed by loss, rupture or changes of the glycocalix. 3. After 120 min most larvae loss their tails and present water sensitivity. 4. Acetabular grands depletion usually does not occur in cercaria-shistosomulum changes in the peritoneal cavity of mice. These steps differ in some way from those described in the kinetics of the in vitro observations performed by other investigators, and is more like those described in the penetration in the skin of living vertebrates.

Kinetics of the cercaria-schistosomul um transformation in vivo: 2. The effect of oxamniquine

To study the cercaria-schistosomulum transformation in vivo, underthe influence of an antischistosomal compound (oxamniquine), a model using cercarial infections into the abdominal cavity of mice was chosen. This procedure provided easy and reproducible recoveries of larvae from peritoneal washings with appropriate solutions for a long time (30 to 180 min) after inoculation. The results show that high doses of oxamniquine (given intramuscularly one hour before the infection) produce a marked delay in the kinetics of the cercaria-schistosomulum transformation. Cercariae, tail-less cercarial bodies and schistosomula were recovered from the peritoneal cavity ofdrug treated mice in numbers significantly different from those recovered from untreated mice.

Production and Characterization of the biological activity of peptides obtained via hydrolysis from whey proteins by cardosins

Dissertation presented to obtain the Ph.D degree in Biochemistry, Engineering and Technological Sciences

Kinetics of Trypanosoma cruzi destruction in the mouse spleen

Massive destruction of parasitized splenic macrophages was histologically observed at the height of a virulent infection caused by Trypanosoma cruzi (Y strain) in the mouse. This was coincident with a sudden drop in parasitemic curve. Most of the animals died at this point, probably due to the liberation of toxic products, such as TNF, following the massive destruction of parasitized cells. However, parasitized-cell destruction indicated the transition from susceptibility to resistance. Although it has been extensively studied in vitro, this study contributes with the morphological counterpart observed in vivo by optical and electron microscopy. When infected animals were specifically treated during early infection transition to chronic phase was immediately observed without splenic parasitism. Animals that apparently recovered from massive cell-destruction in the spleen showed evidences of a rapid restoration of splenic architecture.

Dynamics and kinetics of natural killer cell cytotoxicity in human malaria as evaluated by a novel stepwise cytotoxicity assay

Malaria causes important functional alterations of the immune system, but several of them are poorly defined. To evaluate thoroughly the natural killer cell cytotoxicity in patients with malaria, we developed a technique capable to assess both the dynamics and the kinetics of the process. For the kinetics assay, human peripheral blood mononuclear cells were previously incubated with K562 cells and kept in agarose medium, while for the dynamics assay both cells were maintained in suspension. NK activity from patients with vivax malaria presented a kinetics profile faster than those with falciparum malaria. NK cytotoxicity positively correlated with parasitemia in falciparum malaria. The dynamics of NK cytotoxicity of healthy individuals was elevated at the beginning of the process and then significantly decreased. In contrast, malaria patients presented successive peaks of NK activity. Our results confirmed the occurrence of alteration in NK cell function during malaria, and added new data about the NK cytotoxicity process.

Kinetics of IFN-gamma, TNF-alpha, IL-10 and IL-4 production by mononuclear cells stimulated with gp43 peptides, in patients cured of paracoccidioidomycosis

We analyzed the kinetics of cytokine production by mononuclear cells from 17 patients who had been treated for paracoccidioidomycosis, using the stimulus of gp43 peptide groups (43kDa glycoprotein of Paracoccidioides brasiliensis) at 0.1 and 1µM, gp43 (1µg/ml) and crude Paracoccidioides brasiliensis antigen (PbAg; 75µg/ml). IFN-gamma production was a maximum at 144 hours in relation to the G2 and G8 peptide groups at 1µM and was greatest at 144 hours when stimulated by gp43 and by PbAg. The maximum TNF-alpha production was at 144 hours for the G2 group (0.1µM) and for gp43. IL-10 production was highest after 48 and 72 hours for G7 and G6 at 1µM, respectively. We also suggest the best time for analysis of IL4 production. These results may contribute towards future studies with gp43 peptides and encourage further investigations with the aim of understanding the influence of these peptides on the production of inflammatory and regulatory cytokines.

Marine toxins in bivalves: accumulation, kinetics and subcellular responses

Sociedade Polis Litoral Ria Formosa,Projects Quasus and Project Toxigest financed by PROMAR (2007-2013)

Influence of the molecular weight in mechanical properties and degradation kinetics of chitosan inverted colloidal crystals

Tissue engineering arises from the need to regenerate organs and tissues, requiring the development of scaffolds, which can provide an optimum environment for tissue growth. In this work, chitosan with different molecular weights was used to develop biodegradable 3D inverted colloidal crystals (ICC) structures for bone regeneration, exhibiting uniform pore size and interconnected network. Moreover, in vitro tests were conducted by studying the influence of the molecular weight in the degradation kinetics and mechanical properties. The production of ICC included four major stages: fabrication of microspheres; assembly into a cohesive structure, polymeric solution infiltration and microsphere removal. Chitosan’s degree of deacetylation was determined by infrared spectroscopy and molecular weight was obtained via capillary viscometry. In order to understand the effect of the molecular weight in ICC structures, the mass loss and mechanical properties were analyzed after degradation with lysozyme. Structure morphology observation before and after degradation was performed by scanning electron microscopy. Cellular adhesion and proliferation tests were carried out to evaluate ICC in vitro response. Overall, medium molecular weight ICC revealed the best balance in terms of mechanical properties, degradation rate, morphology and biological behaviour.

Evaluation of CD4+CD25+ T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens

Introduction CD4+CD25+ T lymphocytes have been implicated in the regulation of host inflammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. Methods Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. Results It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. Conclusions Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease.

Valorization of grape pomace through hot compressed water extraction/hydrolysis

The world energy consumption is expected to increase strongly in coming years, because of the emerging economies. Biomass is the only renewable carbon resource that is abundant enough to be used as a source of energy Grape pomace is one of the most abundant agro-industrial residues in the world, being a good biomass resource. The aim of this work is the valorization of grape pomace from white grapes (WWGP) and from red grapes (RWGP), through the extraction of phenolic compounds with antioxidant activity, as well as through the extraction/hydrolysis of carbohydrates, using subcritical water, or hot compressed water (HCW). The main focus of this work is the optimization of the process for WWGP, while for RWGP only one set of parameters were tested. The temperatures used were 170, 190 and 210 °C for WWGP, and 180 °C for RWGP. The water flow rates were 5 and 10 mL/min, and the pressure was always kept at 100 bar. Before performing HCW assays, both residues were characterized, revealing that WWGP is very rich in free sugars (around 40%) essentially glucose and fructose, while RWGP has higher contents of structural sugars, lignin, lipids and protein. For WWGP the best results were achieved at 210 °C and 10 mL/min: higher yield in water soluble compounds (69 wt.%), phenolics extraction (26.2 mg/g) and carbohydrates recovery (49.3 wt.% relative to the existing 57.8%). For RWGP the conditions were not optimized (180 °C and 5 mL/min), and the values of the yield in water soluble compounds (25 wt.%), phenolics extraction (19.5 mg/g) and carbohydrates recovery (11.4 wt.% relative to the existing 33.5%) were much lower. The antioxidant activity of the HCW extracts from each assay was determined, the best result being obtained for WWGP, namely for extracts obtained at 210 °C (EC50=20.8 μg/mL; EC50 = half maximum effective concentration; EC50 = 22.1 μg/mL for RWGP, at 180 ºC).