Analysis of output properties of the Peroxiredoxin/Thioredoxin/Thioredoxin reductase system

Dissertação de mestrado em Bioinformática

Study of grapevine solute transporters involved in berry quality. A biochemical and molecular approach

Tese de Doutoramento em Biologia de Plantas

Lipid based nanocarriers for the delivery of the bioactive compound resveratrol

Dissertação de mestrado em Biofísica e Bionanossistemas

Contributos para a elucidação do modo de ação de própolis português: o caso do própolis do Pereiro

Dissertação de mestrado em Genética Molecular

Evaluation of bioactivities of a propolis sample (Gerês) of Portuguese origin

Dissertação de mestrado em Genética Molecular

Enzymatic hydrophobic modification of jute fibers via grafting to reinforce composites

Horseradish peroxidase (HRP)/H2O2 system catalyzes the free-radical polymerization of aromatic compounds such as lignins and gallate esters. In this work, dodecyl gallate (DG) was grafted onto the surfaces of lignin-rich jute fabrics by HRP-mediated oxidative polymerization with an aim to enhance the hydrophobicity of the fibers. The DG-grafted jute fibers and reaction products of their model compounds were characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The results clearly indicated the grafting of DG to the jute fiber by HRP. Furthermore, the hydrophobicity of jute fabrics was determined by measuring the wetting time and static contact angle. Compared to the control sample, the wetting time and static contact angle of the grated fabrics changed from ~1 s to 1 h and from ~0° to 123.68°, respectively. This clearly proved that the hydrophobicity of jute fabrics improved considerably. Conditions of the HRP-catalyzed DG-grafting reactions were optimized in terms of the DG content of modified jute fabrics. Moreover, the results of breaking strength and elongation of DG-grafted jute/ polypropylene (PP) composites demonstrated improved reinforcement of the composite due to enzymatic hydrophobic modification of jute fibers.

Development of a new method for the characterisation of bioreactors concerning heat and mass transfer

Since the specific heat transfer coefficient (UA) and the volumetric mass transfer coefficient (kLa) play an important role for the design of biotechnological processes, different techniques were developed in the past for the determination of these parameters. However, these approaches often use imprecise dynamic methods for the description of stationary processes and are limited towards scale and geometry of the bioreactor. Therefore, the aim of this thesis was to develop a new method, which overcomes these restrictions. This new approach is based on a permanent production of heat and oxygen by the constant decomposition of hydrogen peroxide in continuous mode. Since the degradation of H2O2 at standard conditions only takes place by the support of a catalyst, different candidates were investigated for their potential (regarding safety issues and reaction kinetic). Manganese-(IV)-oxide was found to be suitable. To compensate the inactivation of MnO2, a continuous process with repeated feeds of fresh MnO2 was established. Subsequently, a scale-up was successfully carried out from 100 mL to a 5 litre glass bioreactor (UniVessel®)To show the applicability of this new method for the characterisation of bioreactors, it was compared with common approaches. With the newly established technique as well as with a conventional procedure, which is based on an electrical heat source, specific heat transfer coefficients were measured in the range of 17.1 – 24.8 W/K for power inputs of about 50 – 70 W/L. However, a first proof of concept regarding the mass transfer showed no constant kLa for different dilution rates up to 0.04 h-1.Based on this, consecutive studies concerning the mass transfer should be made with higher volume flows, due to more even inflow rates. In addition, further experiments are advisable, to analyse the heat transfer in single-use bioreactors and in larger common systems.

Degradació en planta pilot de colorants reactius mitjançant el procés de foto-Fenton assistit amb llum solar: aplicació del procés com a pre-tractament d'un procés biològic

Estudi elaborat a partir d’una estada a la Plataforma Solar de Almería entre desembre del 2006 i gener del 2007. S’ha dut a terme la degradació en planta pilot dels colorants reactius Procion Red H-E7B i Cibacron Red FN-R mitjançant el procés de foto-Fenton aplicat com a tractament únic i com a pretractament d’un procés biològic. El procés de foto-Fenton, assistit amb llum solar, es va realitzar en un fotoreactor solar tipus Col•lector Parabòlic Compost (CPC) i el tractament biològic en un Reactor de Biomassa Immobilitzada (RBI). Com a punt de partida, i amb l’objectiu d’estudiar la reproductibilitat del sistema, es van prendre resultats obtinguts d’experiments realitzats prèviament a escala de laboratori i amb llum artificial. El paràmetre Carboni Orgànic Total (COT) es va emprar com a indicador de l’eliminació dels colorants i dels seus intermedis. En aplicar únicament el procés de foto-Fenton com a tractament, concentracions de 10 mg•l-1 de Fe (II) i 250 mg•l-1 de H2O2 per degradar 250 mg•l-1 Procion Red H-E7B, i de 20 mg•l-1 de Fe (II) i 500 mg•l-1 de H2O2 per degradar 250 mg•l-1 Cibacron Red FN-R, van reproduir els resultants obtinguts al laboratori, amb uns nivells d’eliminació de COT del 82 i 86%, respectivament. A més, l’ús beneficiós de la llum solar en el procés de foto-Fenton, juntament amb la configuració del CPC, van incrementar la velocitat de degradació respecte als resultats previs, permetent la reducció de la concentració de Fe (II) de 10 a 2 mg•l-1 (Procion Red H-E7B) i de 20 a 5 mg•l-1 (Cibacron Red FN-R) sense pèrdues d’efectivitat. D’altre banda, el sistema combinat foto-Fenton/tractament biològic en planta pilot, unes concentracions d’oxidant de 225 mg•l-1 H2O2 per Cibacron Red FN-R i 65 mg•l-1 H2O2 per Procion Red H-E7B van ser suficients per generar solucions intermèdies biodegradables i alimentar així el RBI, millorant inclús els resultats obtinguts prèviament al laboratori.

Reduced capacity of peritoneal exudate cells for oxidative killing of Schistosoma mansoni schistosomula

Peritoneal exudate cells from mice infected with Schistosoma mansoni (S-PEC) can kill schistosomula in vitro in the presence of immune serum. S-PEC produce a low level of respiratory burst, and schistosomula mortality in their presence is not reduced when exogenous antioxidants are added, suggesting that with S-PEC, oxidative killing is not important. Hydrogen peroxide (H2O2) and superoxide production by S-PEC, and cells from BCG and thioglycollate (THGL) injected non-infected mice, non-specifically stimulated with opsonized zymosan, were measured. Levels of H2O2 produced by S-PEC were significantly lower than BCG or THGL PEC, and were below the H2O2 threshold for schistosomula killing. This resulted in lower levels of cell-mediated killing of schistosomula in vitro by S-PEC than by BCG or THGL PEC. Superoxide levels, however, were similar between the three cell populations. The efficiency of PEC to kill schistosomules in vitro correlated with H2O2 rather than superoxide levels. The lower tolerance of schistosomula, compared to adult S. mansoni to GSH depleting agents increases their sensitivity to oxidative attack and resulted in higher levels of cell-mediated killing in vitro.

Absorbance enhancement in microplate wells for improved-sensitivity biosensors

A generic optical biosensing strategy was developed that relies on the absorbance enhancement phenomenon occurring in a multiple scattering matrix. Experimentally, inserts made of glass fiber membrane were placed into microplate wells in order to significantly lengthen the trajectory of the incident light through the sample and therefore increase the corresponding absorbance. Enhancement factor was calculated by comparing the absorbance values measured for a given amount of dye with and without the absorbance-enhancing inserts in the wells. Moreover, the dilution of dye in solutions with different refractive indices (RI) clearly revealed that the enhancement factor increased with the ΔRI between the membrane and the surrounding medium, reaching a maximum value (EF>25) when the membranes were dried. On this basis, two H2O2-biosensing systems were developed based on the biofunctionalization of the glass fiber inserts either with cytochrome c or horseradish peroxidase (HRP) and the analytical performances were systematically compared with the corresponding bioassay in solution. The efficiency of the absorbance-enhancement approach was particularly clear in the case of the cytochrome c-based biosensor with a sensitivity gain of 40 folds and wider dynamic range. Therefore, the developed strategy represents a promising way to convert standard colorimetric bioassays into optical biosensors with improved sensitivity.

Macrophage activation and histopathological findings in Calomys callosus and Swiss mice infected with several strains of Trypanosoma cruzi

Peritoneal macrophage activation as measured by H2O2 release and histopathology was compared between Swiss mice and Calomys callosus, a wild rodent, reservoir of Trypanosoma cruzi, during the course of infection with four strains of this parasite. In mice F and Y strain infections result in high parasitemia and mortality while with silvatic strains Costalimai and M226 parasitemia is sub-patent, with very low mortality. H2O2 release peaked at 33,6 and 59 nM/2 x 10(elevado a sexta potência) cells for strains Y and F, respectively, 48 and 50 nM/2 x 10 (elevado a sexta potência) for strains Costalimai and M226, at different days after infection. Histopathological findings of myositis, myocarditis, necrotizing artheritis and abscence of macrophage parasitism were foud for strains F and Costalimai. Y strain infection presented moderate myocarditis and myositis, with parasites multiplying within macrophages. In C. callosus all four strains resulted in patent parasitemia wich was eventually overcome, with scarce mortality. H2O2 release for strains Y or F was comparable to that of mice-peaks of 27 and 53 nM/2 x 10 (elevado a sexta potência) cells, with lower values for strains Costalimai and M226 - 16.5 and 4.6 nM/2 x 10(elevado a sexta potência)cells, respectively. Histopathological lesions with Y and F strain injected animals were comparable to those of mice at the onset of infections; they subsided completely at the later stages with Y strain and partially with F strain infected C. callosus. In Costalimai infected C. callosus practically no histopathological alterations were observed.

Role of the stress sigma factor RpoS in GacA/RsmA-controlled secondary metabolism and resistance to oxidative stress in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens biocontrol strain CHA0, the two-component system GacS/GacA positively controls the synthesis of extracellular products such as hydrogen cyanide, protease, and 2,4-diacetylphloroglucinol, by upregulating the transcription of small regulatory RNAs which relieve RsmA-mediated translational repression of target genes. The expression of the stress sigma factor sigmaS (RpoS) was controlled positively by GacA and negatively by RsmA. By comparison with the wild-type CHA0, both a gacS and an rpoS null mutant were more sensitive to H2O2 in stationary phase. Overexpression of rpoS or of rsmZ, encoding a small RNA antagonistic to RsmA, restored peroxide resistance to a gacS mutant. By contrast, the rpoS mutant showed a slight increase in the expression of the hcnA (HCN synthase subunit) gene and of the aprA (major exoprotease) gene, whereas overexpression of sigmaS strongly reduced the expression of these genes. These results suggest that in strain CHA0, regulation of exoproduct synthesis does not involve sigmaS as an intermediate in the Gac/Rsm signal transduction pathway whereas sigmaS participates in Gac/Rsm-mediated resistance to oxidative stress.

Immunological and microbiological activity of Davilla elliptica St. Hill. (Dilleniaceae) against Mycobacterium tuberculosis

Mycobacterium tuberculosis is responsible for over 8 million cases of tuberculosis (TB) annually. Natural products may play important roles in the chemotherapy of TB. The immunological activity of Davilla elliptica chloroform extract (DECE) was evaluated in vitro by the determination of hydrogen peroxide (H2O2), nitric oxide (NO), and tumor necrosis factor-alpha (TNF-alpha) release in peritoneal macrophages cultures. DECE was also tested for its antimycobacterial activity against M. tuberculosis using the microplate alamar blue assay. DECE (50, 150, 250 µg/ml) stimulated the production of H2O2 (from 1,79 ± 0,23 to 7,27 ± 2,54; 15,02 ± 2,86; 20,5 ± 2,1 nmols) (means ± SD), NO (from 2,64 ± 1,02 to 25,59 ± 2,29; 26,68 ± 2,41; 29,45 ± 5,87 µmols) (means ± SD) and TNF-alpha (from 2,44 ± 1,46 to 30,37 ± 8,13; 38,68 ± 1,59; 41,6 ± 0,90 units/ml) (means ± SD) in a dose-dependent manner and also showed a promising antimycobacterial activity with a minimum inhibitory concentration of 62,5 µg/ml. This plant may have therapeutic potential in the immunological and microbiological control of TB.

Polysaccharide-rich fraction of Agaricus brasiliensis enhances the candidacidal activity of murine macrophages

A polysaccharide-rich fraction (ATF) of medicinal mushroom Agaricus brasiliensis was evaluated on the candidacidal activity, H2O2 and nitric oxide (NO) production, and expression of mannose receptors by murine peritoneal macrophages. Mice received three intraperitoneal (i.p.) injections of ATF and after 48 h their peritoneal resident macrophages were assayed against Candida albicans yeast forms. The treatment increased fungicidal activity and it was associated with higher levels of H2O2, whereas NO production was not affected. We also found that the treatment enhances mannose receptor expression by peritoneal macrophages, which are involved in the attachment and phagocytosis of non-opsonized microorganisms. Treatment of animals with ATF was able to enhance the clearance of C. albicans during the first 6 h after the experimental i.p. infection. Our results suggest that this extract can increase host resistance against some infectious agents through the stimulation of microbicidal activity of macrophages.

Resistance of melanized yeast cells of Paracoccidioides brasiliensis to antimicrobial oxidants and inhibition of phagocytosis using carbohydrates and monoclonal antibody to CD18

Paracoccidioides brasiliensis, a thermal dimorphic fungal pathogen, produces a melanin-like pigment in vitro and in vivo. We investigated the involvement of carbohydrates and monoclonal antibody to CD18, on phagocytosis inhibition, involving macrophage receptors and the resistance of melanized fungal cells to chemically generated nitric oxide (NO), reactive oxygen species (ROS), hypochlorite and H2O2. Our results demonstrate that melanized yeast cells were more resistant than nonmelanized yeast cells to chemically generated NO, ROS, hypochlorite and H2O2, in vitro. Phagocytosis of melanized yeast cells was virtually abolished when mannan, N-acetyl glucosamine and anti-CD18 antibody were added together in this system. Intratracheal infection of BALB/c mice, with melanized yeast cells, resulted in higher lung colony forming units, when compared to nonmelanized yeast cells. Therefore, melanin is a virulence factor of P. brasiliensis.