Estudos estruturais de uma lectina presente em sementes de Lotus tetragonolobus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Synthesis and characterization of cross-linked molecularly imprinted polyacrylamide for the extraction/preconcentration of glyphosate and aminomethylphosphonic acid from water samples

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Physicochemical and structural characteristics of rice starch modified by irradiation

This work evaluated the physicochemical and structural properties of rice starch of the cultivars IAC 202 and IRGA 417 modified by irradiation. Starch samples were irradiated by (60)Co in doses 1, 2 and 5kGy, on a rate of 0.4kGy/h. A control not irradiated was used for comparison. The granule morphology and A-type X-ray diffraction pattern were not altered by irradiation. There was an increase in amylose content, carboxyl content and acidity with irradiation. Gamma radiation did not affect the thermal properties of IAC202, but increased gelatinization temperature of IRGA417, in the higher dose (5kGy). The number of long chains of amylopectin was reduced and short chains were increased for IAC202, whereas for IRGA 417, the opposite was observed, probably due to cross-linking of starch chains. Starches had their physicochemical and structural properties modified by irradiation differently.

Insights into the swelling process and drug release mechanisms from cross-linked pectin/high amylose starch matrices

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Influence of climate changes on cross allergies: possible involvement of pollen transglutaminase

Allergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Allergic sensibilization can be of food or respiratory origin and, in particular, apple and hazelnut allergens have been identified in pollens or fruits. Allergic cross-reactivity can occur in a patient reacting to similar allergens from different origins, justifying the research in both systems as in Europe a greater number of people suffers from apple fruit allergy, but little evidence exists about pollen. Apple fruit allergies are due to four different classes of allergens (Mal d 1, 2, 3, 4), whose allergenicity is related both to genotype and tissue specificity; therefore I have investigated their presence also in pollen at different time of germination to clarify the apple pollen allergenic potential. I have observed that the same four classes of allergens found in fruit are expressed at different levels also in pollen, and their presence might support that the apple pollen can be considered allergenic as the fruit, deducing that apple allergy could also be indirectly caused by sensitization to pollen. Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies (hay fever, bronchial asthma, conjunctivitis). Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood. Transglutaminases (TGases), a class of enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, suggesting a role in allergies. Recently, a calcium-dependent TGase activity has been identified in the pollen cell wall, raising the possibility that pollen TGase may have a role in the modification of pollen allergens reported above, thus stabilizing them against proteases. This enzyme can be involved also in the transamidation of proteins present in the human mucosa interacting with surface pollen or, finally, the enzyme itself can represent an allergen, as suggested by studies on celiac desease. I have hypothesized that this pollen enzyme can be affected by climate changes and be involved in exhacerbating allergy response. The data presented in this thesis represent a scientific basis for future development of studies devoted to verify the hypothesis set out here. First, I have demonstrated the presence of an extracellular TGase on the surface of the grain observed either at the apical or the proximal parts of the pollen-tube by laser confocal microscopy (Iorio et al., 2008), that plays an essential role in apple pollen-tube growth, as suggested by the arrest of tube elongation by TGase inhibitors, such as EGTA or R281. Its involvement in pollen tube growth is mainly confirmed by the data of activity and gene expression, because TGase showed a peak between 15 min and 30 min of germination, when this process is well established, and an optimal pH around 6.5, which is close to that recorded for the germination medium. Moreover, data show that pollen TGase can be a glycoprotein as the glycosylation profile is linked both with the activation of the enzyme and with its localization at the pollen cell wall during germination, because from the data presented seems that the active form of TGase involved in pollen tube growth and pollen-stylar interaction is more exposed and more weakly bound to the cell wall. Interestingly, TGase interacts with fibronectin (FN), a putative SAMs or psECM component, inducing possibly intracellular signal transduction during the interaction between pollen-stylar occuring in the germination process, since a protein immunorecognised by anti-FN antibody is also present in pollen, in particular at the level of pollen grain cell wall in a punctuate pattern, but also along the shank of the pollen tube wall, in a similar pattern that recalls the signal obtained with the antibody anti TGase. FN represents a good substrate for the enzyme activity, better than DMC usually used as standard substrate for animal TGase. Thus, this pollen enzyme, necessary for its germination, is exposed on the pollen surface and consequently can easily interact with mucosal proteins, as it has been found germinated pollen in studies conducted on human mucus (Forlani, personal communication). I have obtained data that TGase activity increases in a very remarkable way when pollen is exposed to stressful conditions, such as climate changes and environmental pollution. I have used two different species of pollen, an aero allergenic (hazelnut, Corylus avellana) pollen, whose allergenicity is well documented, and an enthomophylus (apple, Malus domestica) pollen, which is not yet well characterized, to compare data on their mechanism of action in response to stressors. The two pollens have been exposed to climate changes (different temperatures, relative humidity (rH), acid rain at pH 5.6 and copper pollution (3.10 µg/l)) and showed an increase in pollen surface TGase activity that is not accompanied to an induced expression of TGase immunoreactive protein with AtPNG1p. Probably, climate change induce an alteration or damage to pollen cell wall that carries the pollen grains to release their content in the medium including TGase enzyme, that can be free to carry out its function as confirmed by the immunolocalisation and by the in situ TGase activity assay data; morphological examination indicated pollen damage, viability significantly reduced and in acid rain conditions an early germination of apple pollen, thus possibly enhancing the TGase exposure on pollen surface. Several pollen proteins were post-translationally modified, as well as mammalian sPLA2 especially with Corylus pollen, which results in its activation, potentially altering pollen allergenicity and inflammation. Pollen TGase activity mimicked the behaviour of gpl TGase and AtPNG1p in the stimulation of sPLA2, even if the regulatory mechanism seems different to gpl TGase, because pollen TGase favours an intermolecular cross-linking between various molecules of sPLA2, giving rise to high-molecular protein networks normally more stable. In general, pollens exhibited a significant endogenous phospholipase activity and it has been observed differences according to the allergenic (Corylus) or not-well characterized allergenic (Malus) attitude of the pollen. However, even if with a different intensity level in activation, pollen enzyme share the ability to activate the sPLA2, thus suggesting an important regulatory role for the activation of a key enzyme of the inflammatory response, among which my interest was addressed to pollen allergy. In conclusion, from all the data presented, mainly presence of allergens, presence of an extracellular TGase, increasing in its activity following exposure to environmental pollution and PLA2 activation, I can conclude that also Malus pollen can behave as potentially allergenic. The mechanisms described here that could affect the allergenicity of pollen, maybe could be the same occurring in fruit, paving the way for future studies in the identification of hyper- and hypo- allergenic cultivars, in preventing environmental stressor effects and, possibly, in the production of transgenic plants.

Photodegradation and photostabilization of weathered wood flour filled polyethylene composites

Wood plastic composites (WPCs) have gained popularity as building materials because of their usefulness in replacing solid wood in a variety of applications. These composites are promoted as being low-maintenance, high-durability products. However, it has been shown that WPCs exposed to weathering may experience a color change and/or loss in mechanical properties. An important requirement for building materials used in outdoor applications is the retention of their aesthetic qualities and mechanical properties during service life. Therefore, it is critical to understand the photodegradation mechanisms of WPCs exposed to UV radiation and to develop approaches to stabilize these composites (both unstabilized and stabilized) as well as the effect of weathering on the color fade and the retention of mechanical properties were characterized. Since different methods of manufacturing WPCs lead to different surface characteristics, which can influence weathering, the effect of manufacturing method on the photodegradation of WPCs was investigated first. Wood flour (WF) filled high-density polyethylene (HDPE) composite samples were either injection molded, extruded, or extruded and then planed. Fourier transform infrared (FTIR) spectroscopy was used to monitor the surface chemistry of the manufactured composites. The spectra showed that the surface of planed samples had more wood component than extruded and injection molded samples, respectively. After weathering, the samples were analyzed for color fade, and loss of flexural properties. The final lightness of the composites was not dependent upon the manufacturing method. However the mechanical property loss was dependent upon manufacturing method. The samples with more wood component at the surface (planed samples) experienced a larger percentage of total loss in flexural properties after weathering due to a greater effect of moisture on the samples. The change in surface chemistry of HDPE and WF/HDPE composites after weathering was studied using spectroscopic techniques. X-ray photoelectron spectroscopy (XPS) was used to characterize the occurrence of surface oxidation whereas FTIR spectroscopy was used to monitor the development of degradation products, such as carbonyl groups and vinyl groups, and to determine changes in HDPE crystallinity. Surface oxidation occurred immediately after exposure for both the neat HDPE and WF/HDPE composites. After weathering, the surface of the WF/HDPE composites was oxidized to a greater extent than the neat HDPE after weathering. This suggests that photodegradation is exacerbated by the addition of the carbonyl functional groups of the wood fibers within the HDPE atrix during composite manufacturing. While neat HDPE may undergo cross-linking in the initial stages of accelerated weathering, the WF may physically hinder the ability of the HDPE to cross-link resulting in the potential for HDPE chain scission to dominate in the initial weathering stages of the WF/HDPE composites. To determine which photostabilizers are most effective for WF/HDPE composites, factorial experimental designes were used to determine the effects of adding two hindered amine light stabilizers, an ultraviolet absorber, and a pigment on the color made and mechanical properties of both unweathered and UV weathered samples. Both the pigment and ultraviolet absorber were more effective photostabilizers for WF/HDPE composites than hinder amine light stabilizers. The ineffectiveness of hindered amine light stabilizers in protecting WPCs against UV radiation was attribuated to the acid/base reactions occurring between the WF and hindered amine light stabilizer. The efficiency of an ultraviolet absorber and/or pigment was also examined by incorporating different concentration of an ultraviolet absorber and/or pigment into WF/HDPE composites. Color change and flexural properties were determined after accelerated UV weathering. The lightness of the composite after weathering was influenced by the concentration of both the ultraviolet absorber by masking the bleaching wood component as well as blocking UV light. Flexural MOE loss was influenced by an increase in ultraviolet absorber concentration, but increasing pigment concentration from 1 to 2% had little influence on MOE loss. However, increasing both ultraviolet absorber and pigment concentration resulted in improved strength properties over the unstabilized composites after 3000 h of weather. Finally, the change in surface chemistry due to weathering of WF/HDPE composites that were either unstabilized or stabilized with an ultraviolet absorber and/or pigment was analyzed using FTIR spectroscopy. The samples were tested for loss in modulus of elasticity, carbonyl and vinyl group formation at the surface, and change in HDPE crystallinity. It was concluded that structural changes in the samples; carbonyl group formation, terminal vinyl group formation, and crystallinity changes cannot reliably be used to predict changes in modulus of elasticity using a simple linear relationship. The effect of cross-linking, chain scission, and crystallinity changes due to ultraviolet exposure as well as the interfacial degradation due to moisture exposure are inter-related factors when weathering HDPE and WF/HDPE composites.

INDUCTION OF DNA STRAND BREAKS AND CROSS-LINKS BY THE NEW ANTICANCER AGENT 3,6-DIAZIRIDINYL-2,5-BIS(CARBOETHOXYAMINO) -1,4-BENZOQUINONE

The DNA breakage effect of the anticancer agent 3,6-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone (AZQ, NSC-182986) on bacteriophage PM2 DNA was investigated using agarose gel electrophoresis. AZQ caused both single-stranded and double-stranded breaks after reduction with NaBH(,4), but it was not active in the native state. At 120 (mu)M, it degraded 50% of the closed circular form I DNA into 40% form II DNA (single-stranded break) and 10% form III DNA (double-stranded break). It produced a dose-response breakage between 1 (mu)M and 320 (mu)M. The DNA breakage exhibited a marked pH dependency. At 320 (mu)M, AZQ degraded 80% and 60% of form I DNA at pH 4 and 10 respectively, but none between pH 6 to 8. The DNA breakage at physiologic pH was greatly enhanced when 10 (mu)M cupric sulfate was included in the incubation mixture. The DNA strand scission was inhibited by catalase, glutathione, KI, histidine, Tiron, and DABCO. These results suggest that the DNA breakage may be caused by active oxygen metabolites including hydroxyl free radical. The bifunctional cross-linking activity of reduced AZQ on isolated calf thymus DNA was investigated by ethidium fluorescence assay. The cross-linking activity exhibited a similar pH dependency; highest in acidic and alkaline pH, inactive under neutral conditions. Using the alkaline elution method, we found that AZQ induced DNA single-stranded breaks in Chinese hamster ovary cells treated with 50 (mu)M of AZQ for 2 hr. The single-stranded break frequencies in rad equivalents were 17 with 50 (mu)M and 140 with 100 (mu)M of AZQ. In comparison, DNA cross-links appeared in cells treated with only 1 to 25 (mu)M of AZQ for 2 hr. The cross-linking frequencies in rad equivalents were 39 and 90 for 1 and 5 (mu)M of AZQ, respectively. Both DNA-DNA and DNa-protein cross-links were induced by AZQ in CHO cells as revealed by the proteinas K digestion assay. DNA cross-links increased within the first 4 hr of incubation in drug-free medium and slightly decreased by 12 hr, and most of the cross-links disappeared after cells were allowed to recovered for 24 hr.^ By electrochemical analysis, we found that AZQ was more readily reduced at acidic pH. However, incubation of AZQ with NaBH(,4) at pH 7.8 or 10, but not at 4, produced superoxide anion. The opening of the aziridinyl rings of AZQ at pH 4 was faster in the presence of NaBH(,4) than in its absence; no ring-opening was detected at pH 7.8 regardless of the inclusion of NaBH(,4). . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Stability and mechanical evaluation of bovine pericardium cross-linked with polyurethane prepolymer in aqueous medium

The present study investigates the potential use of non-catalyzed water-soluble blocked polyurethane prepolymer (PUP) as a bifunctional cross-linker for collagenous scaffolds. The effect of concentration (5, 10, 15 and 20%), time (4, 6, 12 and 24 h), medium volume (50, 100, 200 and 300%) and pH (7.4, 8.2, 9 and 10) over stability, microstructure and tensile mechanical behavior of acellular pericardial matrix was studied. The cross-linking index increased up to 81% while the denaturation temperature increased up to 12 °C after PUP crosslinking. PUP-treated scaffold resisted the collagenase degradation (0.167 ± 0.14 mmol/g of liberated amine groups vs. 598 ± 60 mmol/g for non-cross-linked matrix). The collagen fiber network was coated with PUP while viscoelastic properties were altered after cross-linking. The treatment of the pericardial scaffold with PUP allows (i) different densities of cross-linking depending of the process parameters and (ii) tensile properties similar to glutaraldehyde method.

The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells

The Chinese hamster ovary (CHO) mutant UV40 cell line is hypersensitive to UV and ionizing radiation, simple alkylating agents, and DNA cross-linking agents. The mutant cells also have a high level of spontaneous chromosomal aberrations and 3-fold elevated sister chromatid exchange. We cloned and sequenced a human cDNA, designated XRCC9, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and γ-radiation. The spontaneous chromosomal aberrations in XRCC9 cDNA transformants were almost fully corrected whereas sister chromatid exchanges were unchanged. The XRCC9 genomic sequence was cloned and mapped to chromosome 9p13. The translated XRCC9 sequence of 622 amino acids has no similarity with known proteins. The 2.5-kb XRCC9 mRNA seen in the parental cells was undetectable in UV40 cells. The mRNA levels in testis were up to 10-fold higher compared with other human tissues and up to 100-fold higher compared with other baboon tissues. XRCC9 is a candidate tumor suppressor gene that might operate in a postreplication repair or a cell cycle checkpoint function.

Arp2/3 Complex from Acanthamoeba Binds Profilin and Cross-links Actin Filaments

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.

Chain scission and branching in irradiated poly(tetrafluoroethylene-co-hexafluoropropylene)

The radiation chemistry of poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) with a TFE mole fraction of 0.90 has been studied under vacuum using Co-60 gamma-radiation over a range of temperatures and absorbed doses. The radiolysis temperatures were 300, 363, 423, 523 and 543 K. New structure formation in the copolymers was analysed by solid-state F-19 NMR spectroscopy. The new structures formed in the copolymers have been identified and the G-values for the formation of new chemical structures have been investigated at 363 and 523 K. These two temperatures are just above and just below the polymer T-g and T-m, respectively. At the lower temperature, there was no evidence for any chain branching and an estimate of G(S) of 1.0 was obtained. A value of G(S) of 1.3 and a minimum value of G(X)(Y) of 1.3 were obtained at 523 K. (C) 2003 Society of Chemical Industry.

Equilibrium swelling measurements of network and semicrystalline polymers in supercritical carbon dioxide using high-pressure NMR

The extent of swelling of cross-linked poly(dimethylsiloxane) and linear low-density poly(ethylene) in supercritical CO2 has been investigated using high-pressure NMR spectroscopy and microscopy. Poly(dimethylsiloxane) was cross-linked to four different cross-link densities and swollen in supercritical CO2. The Flory-Huggins interaction parameter, x, was found to be 0.62 at 300 bar and 45 degrees C, indicating that supercritical CO2 is a relatively poor solvent compared to toluene or benzene. Linear low-density poly(ethylene) was shown to exhibit negligible swelling upon exposure to supercritical CO2 up to 300 bar. The effect Of CO2 pressure on the amorphous region of the poly(ethylene) was investigated by observing changes in the H-1 T-2 relaxation times of the polymer. These relaxation times decreased with increasing pressure, which was attributed to a decrease in mobility of the polymer chains as a result of compressive pressure.

In vitro characterization of a collagen scaffold enzymatically cross-linked with a tailored elastin-like polymer

Collagen, the main structural component of the extracellular matrix (ECM), provides tensile stiffness to different structures and organs against rupture. However, collagen tissue-engineered implants are hereto still lacking in mechanical strength. Attempts to create stiffer scaffolds have resulted in increased brittleness of the material, reducing the versatility of the original component. The hypothesis behind this research is that the introduction of an elastic element in the scaffold will enhance the mechanical properties of the collagen-based scaffolds, as elastin does in the ECM to prevent irreversible deformation. In this study, an elastin-like polymer (ELP) designed and synthesized using recombinant DNA methodology is used with the view to providing increased proteolytic resistance and increased functionality to the scaffolds by carrying specific sequences for microbial transglutaminase cross-linking, endothelial cell adhesion, and drug delivery. Evaluation of the effects that cross-linking ELP-collagen has on the physicochemical properties of the scaffold such as porosity, presence of cross-linking, thermal behavior, and mechanical strength demonstrated that the introduction of enzymatically resistant covalent bonds between collagen and ELP increases the mechanical strength of the scaffolds in a dose-dependent manner without significantly affecting the porosity or thermal properties of the original scaffold. Importantly, the scaffolds also showed selective behavior, in a dose (ELP)-dependent manner toward human umbilical vein endothelial cells and smooth muscle cells when compared to fibroblasts.

Assessment of cell viability in a three-dimensional enzymatically cross-linked collagen scaffold

Microbial transglutaminase (mTGase) is an enzyme that introduces a covalent bond between peptide bound glutamine and lysine residues. Proteins cross-linked in this manner are often more resistant to proteolytic degradation and show increased tensile strength. This study evaluates the effects of mTGase mediated cross-linking of collagen on the cellular morphology, behaviour and viability of murine 3T3 fibroblasts following their seeding into collagen scaffolds. Additionally, cell mediated scaffold contraction, porosity and level of cross-linking of the scaffold has been analysed using image analysis software, scanning electron microscopy (SEM), colorimetric assays, and Fourier transform infrared spectroscopy (FTIR). We demonstrate that the biocompatibility and cellular morphology, when comparing cultures of fibroblasts integrated in mTGase cross-linked collagen scaffolds with the native collagen counterparts, remained unaffected. It has been also elicited that the structural characteristics of collagen have been preserved while introducing enzymatically resistant covalent bonds.

Characterization of a microbial transglutaminase cross-linked type II collagen scaffold

This study investigated the effect on the mechanical and physicochemical properties of type II collagen scaffolds after cross-linking with microbial transglutaminase (mTGase). It is intended to develop a collagen-based scaffold to be used for the treatment of degenerated intervertebral discs. By measuring the amount of ε-(γ-glutamyl)lysine isodipeptide formed after cross-linking, it was determined that the optimal enzyme concentration was 0.005% (w/v). From the production of covalent bonds induced by mTGase cross-linking, the degradation resistance of type II collagen scaffolds can be enhanced. Rheological analysis revealed an almost sixfold increase in storage modulus (G') with 0.005% (w/v) mTGase cross-linked scaffolds (1.31 ± 0.03 kPa) compared to controls (0.21 ± 0.01 kPa). There was a significant reduction in the level of cell-mediated contraction of scaffolds with increased mTGase concentrations. Cell proliferation assays showed that mTGase cross-linked scaffolds exhibited similar cytocompatibility properties in comparison to non-cross-linked scaffolds. In summary, cross-linking type II collagen with mTGase imparted more desirable properties, making it more applicable for use as a scaffold in tissue engineering applications. © Mary Ann Liebert, Inc.