Cuticular and Suberin Polymers of Edible Plants. Analysis by Gas Chromatographic-Mass Spectrometric and Solid State Spectroscopic Methods

Cutin and suberin are structural and protective polymers of plant surfaces. The epidermal cells of the aerial parts of plants are covered with an extracellular cuticular layer, which consists of polyester cutin, highly resistant cutan, cuticular waxes and polysaccharides which link the layer to the epidermal cells. A similar protective layer is formed by a polyaromatic-polyaliphatic biopolymer suberin, which is present particularly in the cell walls of the phellem layer of periderm of the underground parts of plants (e.g. roots and tubers) and the bark of trees. In addition, suberization is also a major factor in wound healing and wound periderm formation regardless of the plants’ tissue. Knowledge of the composition and functions of cuticular and suberin polymers is important for understanding the physiological properties for the plants and for nutritional quality when these plants are consumed as foods. The aims of the practical work were to assess the chemical composition of cuticular polymers of several northern berries and seeds and suberin of two varieties of potatoes. Cutin and suberin were studied as isolated polymers and further after depolymerization as soluble monomers and solid residues. Chemical and enzymatic depolymerization techniques were compared and a new chemical depolymerization method was developed. Gas chromatographic analysis with mass spectrometric detection (GC-MS) was used to assess the monomer compositions. Polymer investigations were conducted with solid state carbon-13 cross polarization magic angle spinning nuclear magnetic resonance spectroscopy (13C CP-MAS NMR), Fourier transform infrared spectroscopy (FTIR) and microscopic analysis. Furthermore, the development of suberin over one year of post-harvest storage was investigated and the cuticular layers from berries grown in the North and South of Finland were compared. The results show that the amounts of isolated cuticular layers and cutin monomers, as well as monomeric compositions vary greatly between the berries. The monomer composition of seeds was found to differ from the corresponding berry peel monomers. The berry cutin monomers were composed mostly of long-chain aliphatic ω-hydroxy acids, with various mid-chain functionalities (double-bonds, epoxy, hydroxy and keto groups). Substituted α,ω-diacids predominated over ω-hydroxy acids in potato suberin monomers and slight differences were found between the varieties. The newly-developed closed tube chemical method was found to be suitable for cutin and suberin analysis and preferred over the solvent-consuming and laborious reflux method. Enzymatic hydrolysis with cutinase was less effective than chemical methanolysis and showed specificity towards α,ω-diacid bonds. According to ₁₃C CP-MAS NMR and FTIR, the depolymerization residues contained significant amounts of aromatic structures, polysaccharides and possible cutan-type aliphatic moieties. Cultivation location seems to have effect on cuticular composition. The materials studied contained significant amounts of different types of biopolymers that could be utilized for several purposes with or without further processing. The importance of the so-called waste material from industrial processes of berries and potatoes as a source of either dietary fiber or specialty chemicals should be further investigated in detail. The evident impact of cuticular and suberin polymers, among other fiber components, on human health should be investigated in clinical trials. These by-product materials may be used as value-added fiber fractions in the food industry and as raw materials for specialty chemicals such as lubricants and emulsifiers, or as building blocks for novel polymers.

Leaf-Targeted Ferredoxin-NADP+-Oxidoreductase (FNR): Electron Transfer Properties and Thylakoid Association in Arabidopsis thaliana

Photosynthetic reactions are divided in two parts: light-driven electron transfer reactions and carbon fixation reactions. Electron transfer reactions capture solar energy and split water molecules to form reducing energy (NADPH) and energy-carrying molecules (ATP). These end-products are used for fixation of inorganic carbon dioxide into organic sugar molecules. Ferredoxin-NADP⁺ oxidoreductase (FNR) is an enzyme that acts at the branch point between the electron transfer reactions and reductive metabolism by catalyzing reduction of NADP+ at the last step of the electron transfer chain. In this thesis, two isoforms of FNR from A rabidopsis thaliana, FNR1 and FNR2, were characterized using the reverse genetics approach. The fnr1 and fnr2 mutant plants resembled each other in many respects. Downregulation of photosynthesis protected the single fnr mutant plants from excess formation of reactive oxygen species (ROS), even without significant upregulation of antioxidative mechanisms. Adverse growth conditions, however, resulted in phenotypic differences between fnr1 and fnr2. While fnr2 plants showed downregulation of photosynthetic complexes and upregulation of antioxidative mechanisms under low-temperature growth conditions, fnr1 plants had the wild-type phenotype, indicating that FNR2 may have a specific role in redistribution of electrons under unfavorable conditions. The heterozygotic double mutant (fnr1xfnr2) was severely devoid of chloroplastic FNR, which clearly restricted photosynthesis. The fnr1xfnr2 plants used several photoprotective mechanisms to avoid oxidative stress. In wild-type chloroplasts, both FNR isoforms were found from the stroma, the thylakoid membrane, and the inner envelope membrane. In the absence of the FNR1 isoform, FNR2 was found only in the stroma, suggesting that FNR1 and FNR2 form a dimer, by which FNR1 anchors FNR2 to the thylakoid membrane. Structural modeling predicted formation of an FNR dimer in complex with ferredoxin. In this thesis work, Tic62 was found to be the main protein that binds FNR to the thylakoid membrane, where Tic62 and FNR formed high molecular weight complexes. The formation of such complexes was shown to be regulated by the redox state of the chloroplast. The accumulation of Tic62-FNR complexes in darkness and dissociation of complexes from the membranes in light provide evidence that the complexes may have roles unrelated to photosynthesis. This and the high viability of fnr1 mutant plants lacking thylakoid-bound FNR indicate that the stromal pool of FNR is photosynthetically active.

Magnetic and transport properties of LaMnO3+δ, La1-xCaxMnO3, La1-xCaxMn1-yFeyO3 and La1-xSrxMn1-yFeyO3

Interest to hole-doped mixed-valence manganite perovskites is connected to the ‘colossal’ magnetoresistance. This effect or huge drop of the resistivity, ρ, in external magnetic field, B, attains usually the maximum value near the ferromagnetic Curie temperature, TC. In this thesis are investigated conductivity mechanisms and magnetic properties of the manganite perovskite compounds LaMnO3+, La1-xCaxMnO3, La1-xCaxMn1-yFeyO3 and La1- xSrxMn1-yFeyO3. When the present work was started the key role of the phase separation and its influence on the properties of the colossal magnetoresistive materials were not clear. Our main results are based on temperature dependencies of the magnetoresistance and magnetothermopower, investigated in the temperature interval of 4.2 - 300 K in magnetic fields up to 10 T. The magnetization was studied in the same temperature range in weak (up to 0.1 T) magnetic fields. LaMnO3+δ is the parent compound for preparation of the hole-doped CMR materials. The dependences of such parameters as the Curie temperature, TC, the Coulomb gap, Δ, the rigid gap, γ, and the localization radius, a, on pressure, p, are observed in LaMnO3+δ. It has been established that the dependences above can be interpreted by increase of the electron bandwidth and decrease of the polaron potential well when p is increased. Generally, pressure stimulates delocalization of the electrons in LaMnO3+δ. Doping of LaMnO3 with Ca, leading to La1-xCaxMnO3, changes the Mn3+/Mn4+ ratio significantly and brings an additional disorder to the crystal lattice. Phase separation in a form of mixture of the ferromagnetic and the spin glass phases was observed and investigated in La1- xCaxMnO3 at x between 0 and 0.4. Influence of the replacement of Mn by Fe is studied in La0.7Ca0.3Mn1−yFeyO3 and La0.7Sr0.3Mn1−yFeyO3. Asymmetry of the soft Coulomb gap and of the rigid gap in the density of localized states, small shift of the centre of the gaps with respect to the Fermi level and cubic asymmetry of the density of states are obtained in La0.7Ca0.3Mn1−yFeyO3. Damping of TC with y is connected to breaking of the double-exchange interaction by doping with Fe, whereas the irreversibility and the critical behavior of the magnetic susceptibility are determined by the phase separation and the frustrated magnetic state of La0.7Sr0.3Mn1−yFeyO3.

Distribution of tree species in a geomorphological and pedological gradient of submontane semidecidual seasonal forest in the vicinity of Rio Doce state park, Minas Gerais

The objective of this study was to test the hypothesis that the distribution of tree species in a fragment of submontane seasonal semideciduous forest, a buffer zone in the Parque Estadual do Rio Doce, Minas Gerais, is influenced by geomorphological and weather and soil variables, therefore it can represent a source of information for the restoration of degraded areas where environmental conditions are similar to those of the study area. A detailed soil survey was conducted in the area by sampling three soil profiles per slope segment, totaling 12 profiles. To sample the topsoil, four composite samples were collected from the 10-20 cm layers in each topographic range totaling 16 composite samples. In the low ramp and the lower and upper concave slopes, the texture ranged from clay to sandy-clay. The soil and topographic gradient was characterized by changes in the soil physical-chemical properties. The soil in the 10-20 cm sampled layer was sandier, slightly more fertile and less acid in the low ramp than the clayer soil, nutrient-poor and highly acid soil at the top. The soil conditions in the lower and upper slope of the sampled layers, in turn, were intermediate. The P levels were limiting in all soils. The species distribution along the topographic gradient was associated with variations in chemical fertility, acidity and soil texture. The distribution of Pera leandri, Astronium fraxinifolium, Pouteria torta, Machaerium brasiliense and Myrcia rufipes was correlated with high aluminum levels and to low soil fertility and these species may be indicated for restoration of degraded areas on hillsides and hilltops in regions where environmental conditions are similar. The distribution of Pouteria venosa, Apuleia leiocarpa and Acacia polyphylla was correlated with the less acid and more fertile soil in the environment of the low ramps, indicating the potential for the restoration of similar areas.

FRUIT MORPHOLOGY AND PRODUCTIVITY OF BABASSU PALMS IN NORTHERN MINAS GERAIS STATE, BRAZIL1

ABSTRACT The objectives of this study were to morphologically characterize fruits of the babassu palm tree (Attalea vitrivir) and to estimate their productivity in the north of Minas Gerais State, Brazil. Twenty mature fruits were collected from 10 plants in three different areas in Januária, Minas Gerais. Eighteen biometric parameters of the fruits were measured, the oil contents of the seeds was determined, the adherence to normal distribution was evaluated, distribution frequencies were evaluated and the effects of individuals and areas on the variables and the correlations between them were analyzed. The production of fruit bunches per plant and the number of fruits per bunch from 10 plants were quantified in three areas and the potential production under both natural harvesting and cultivation conditions were estimated. Significant differences were found among all of the biometric parameters examined between the different individuals and the different areas, which shows wide morphological variability in the fruits. The average oil content was 45.7%, but with significant differences among individuals. The observed variability favors the selection of productive individuals in genetic improvement programs. The potential productivity of endocarps and oil based on a density of 400/plants per hectare would be respectively 6.4 and 1.2 tons/ha, which indicates the possibility of using A. vitrivir for producing charcoal, bio fuels, and for carbon fixation.

Sensibility analysis of the pearl model for pesticide leaching in the State of Mato Grosso do Sul, Brazil

For an accurate use of pesticide leaching models it is necessary to assess the sensitivity of input parameters. The aim of this work was to carry out sensitivity analysis of the pesticide leaching model PEARL for contrasting soil types of Dourados river watershed in the state of Mato Grosso do Sul, Brazil. Sensitivity analysis was done by carrying out many simulations with different input parameters and calculating their influence on the output values. The approach used was called one-at-a-time sensitivity analysis, which consists in varying independently input parameters one at a time and keeping all others constant with the standard scenario. Sensitivity analysis was automated using SESAN tool that was linked to the PEARL model. Results have shown that only soil characteristics influenced the simulated water flux resulting in none variation of this variable for scenarios with different pesticides and same soil. All input parameters that showed the greatest sensitivity with regard to leached pesticide are related to soil and pesticide properties. Sensitivity of all input parameters was scenario dependent, confirming the need of using more than one standard scenario for sensitivity analysis of pesticide leaching models.

Environmental comfort in constructions for Sindi and Guzera calves in the agreste region of the State of Paraiba, Brazil

The objective of this study was to evaluate the quality of air, bioclimatic indexes of facilities and physiological indices of Guzera and Sindhi calves, reared in climatic conditions of Agreste. The study was conducted at the experimental station of Alagoinha, PB, Brazil, using 16 calves of Sindi and Guzerá races. The average concentration of oxygen (20.85%), ammonia (1.99 ppm), carbon monoxide (<0.01 ppm), methane (0.13 ppm) and hydrogen sulfide (<0.01) within facilities, were within the limits established by the Brazilian and international standards, for both animals and workers. The bioclimatic index of temperature and humidity and the temperature of the black globe and humidity index were within the thermal comfort zone for cattle in most of the experimental period, the mean values of respiratory frequency (26.0 min mov-1) and skin temperature (32.3 °C) were higher in the hottest time of the day (1 pm) and rectal temperature (39.3 ºC) in the late afternoon (5 pm), but remained within normal ranges for the studied races. The races have good adaptability to climatic conditions in the region of the Paraibano Agreste, Brazil.

Spatial variability of soil physical properties in two management systems in sugarcane crop

The aim of this study was to characterize the spatial variability of soil bulk density (Bd), soil moisture content (θ) and total porosity (Tp) in two management systems of sugarcane harvesting, with or without burning, in a Haplustox soil, in the 0-0.20 m layer. The study area is located in Rio Brilhante, state of Mato Grosso do Sul, Brazil, in Eldorado Sugar Mill. The plots have presented 180 m length, and 145.6 m width, totaling 90 points distributed in the form of a grid of nine rows by ten columns, with points spaced 20 m from its neighbor. Soil samples were collected at 0-0.20 m layer in 2007/2008 and 2008/2009 crops. The harvest with burning system had a higher density compared to mechanized harvest, in the two study periods. The moisture content as well as the porosity increased proportionally with the decrease of the density of the harvest burning system compared to the mechanized.

Quasiclassical Approach to the Vortex State in Iron-based Superconductors

The quasiclassical approach was applied to the investigation of the vortex properties in the ironbased superconductors. The special attention was paid to manifestation of the nonlocal effects of the vortex core structure. The main results are as follows: (i) The effects of the pairing symmetries (s+ and s₊₊) on the cutoff parameter of field distribution, ξh, in stoichiometric (like LiFeAs) and nonstoichiometric (like doped BaFe₂As₂) iron pnictides have been investigated using Eilenberger quasiclassical equations. Magnetic field, temperature and impurity scattering dependences of ξh have been calculated. Two opposite behavior have been discovered. The ξh /ξc2 ratio is less in s+ symmetry when intraband impurity scattering (Γ₀) is much larger than one and much larger than interband impurity scattering (Γπ), i.e. in nonstoichiometric iron pnictides. Opposite, the value ξh /ξc2 is higher in s+ case and the field dependent curve is shifted upward from the "clean" case (Γ₀ = Γπ = 0) for stoichiometric iron pnictides (Γ₀ = Γπ ≪ 1). (ii) Eilenberger approach to the cutoff parameter, ξh, of the field distribution in the mixed state of high

Senecio spp. POISONING OF HORSES IN SOUTHERN BRAZIL

Cases of seneciosis in horses occurring in four farms in the state of Santa Catarina and in another in the state of Rio Grande do Sul, southern Brazil, are reported. S. brasiliensis or S. oxyphyllus or both were detected in four of the five properties. Five horses (one on each property) were necropsied, and tissues for histopathological examination were collected from four horses. Neurological signs, such as depression, ataxia, aimeless walking, circling, head pressing, faulty prehension of food, dysphagia and blindness were consistently observed. Other signs included inappetence, loss of weight, colic, subcutaneous edema, icterus and photodermatitis. At necropsy the livers were firmer and darker than normal and had accentuation of lobular pattern. Edema of the mesentery and ascites were observed in one horse. Main histopathological changes consisted of hepatic chiefly periportal fibrosis, hepatomegalocytosis and biliary hyperplasia. Marked cholestasis and morphological evidence of hepatic encephalopathy were seen respectively in the liver and brain of one of the horses.

Neospora caninum seropositivity in cattle breeds in the South Fluminense Paraíba Valley, state of Rio de Janeiro

The infection by Neospora caninum of different cattle breeds in dairy properties in two municipalities of the South Fluminense Paraíba Valley, state of Rio de Janeiro, was evaluated. Considering a sampling universe of 2,491 cows, blood samples were collected from 563 dairy cows in 57 farms, which were randomically selected in proportion to the number of animals, using a random stratified sampling system. For each property the number of selected cows was proportional to the herd size. Abortion or other reproductive disorders were not considered as criteria for selecting the animals, and seropositivity was determined by indirect enzyme-linked immunosorbent assay (ELISA). A high association (p=0.006) between seropositivity and racial patterns was found. The analysis revealed that in comparison of pure black-and-white Holstein cows versus Zebu (p=0.0028), Holstein cows showed 2.65 times greater odds for seropositivity. In the same way, by comparing black-and-white Holstein versus Zebu + crossbreed Zebu/Holstein (p=0.01), it was noted that there is 2.23 times more chance for seropositivity in Holstein cattle. There were no significant differences concerning the comparison of Holstein cattle versus crossbreed Zebu/Holstein (p=0.08) or Zebu versus crossbreed Zebu/Holstein (p=0.11). This study supports the hypothesis that there is a close association between cattle breeds and the frequency of infection by N. caninum.

Membrane properties of structurally modified ceramides : effects on lipid lateral distribution and sphingomyelin-interactions in artificial bilayer membranes

Ceramides comprise a class of sphingolipids that exist only in small amounts in cellular membranes, but which have been associated with important roles in cellular signaling processes. The influences that ceramides have on the physical properties of bilayer membranes reach from altered thermodynamical behavior to significant impacts on the molecular order and lateral distribution of membrane lipids. Along with the idea that the membrane physical state could influence the physiological state of a cell, the membrane properties of ceramides have gained increasing interest. Therefore, membrane phenomena related to ceramides have become a subject of intense study both in cellular as well as in artificial membranes. Artificial bilayers, the so called model membranes, are substantially simpler in terms of contents and spatio-temporal variation than actual cellular membranes, and can be used to give detailed information about the properties of individual lipid species in different environments. This thesis focuses on investigating how the different parts of the ceramide molecule, i.e., the N-linked acyl chain, the long-chain sphingoid base and the membrane-water interface region, govern the interactions and lateral distribution of these lipids in bilayer membranes. With the emphasis on ceramide/sphingomyelin(SM)-interactions, the relevance of the size of the SMhead group for the interaction was also studied. Ceramides with methylbranched N-linked acyl chains, varying length sphingoid bases, or methylated 2N (amide-nitrogen) and 3O (C3-hydroxyl) at the interface region, as well as SMs with decreased head group size, were synthesized and their bilayer properties studied by calorimetric and fluorescence spectroscopic techniques. In brief, the results showed that the packing of the ceramide acyl chains was more sensitive to methyl-branching in the mid part than in the distal end of the N-linked chain, and that disrupting the interfacial structure at the amide-nitrogen, as opposed to the C3-hydroxyl, had greater effect on the interlipid interactions of ceramides. Interestingly, it appeared that the bilayer properties of ceramides could be more sensitive to small alterations in the length of the long-chain base than what was previously reported for the N-linked acyl chain. Furthermore, the data indicated that the SM-head group does not strongly influence the interactions between SMs and ceramides. The results in this thesis illustrate the pivotal role of some essential parts of the ceramide molecules in determining their bilayer properties. The thesis provides increased understanding of the molecular aspects of ceramides that possibly affect their functions in biological membranes, and could relate to distinct effects on cell physiology.

Influence of soil properties on the abundance of plant species in ferruginous rocky soils vegetation, southeastern Brazil

Ferruginous "campos rupestres" are a particular type of vegetation growing on iron-rich primary soils. We investigated the influence of soil properties on plant species abundance at two sites of ferruginous "campos rupestres" and one site of quartzitic "campo rupestre", all of them in "Quadrilátero Ferrífero", in Minas Gerais State, southeastern Brazil. In each site, 30 quadrats were sampled to assess plant species composition and abundance, and soil samples were taken to perform chemical and physical analyses. The analyzed soils are strongly acidic and presented low fertility and high levels of metallic cations; a principal component analysis of soil data showed a clear segregation among sites due mainly to fertility and heavy metals content, especially Cu, Zn, and Pb. The canonical correspondence analysis indicated a strong correlation between plant species abundance and soil properties, also segregating the sites.

Effect of gamma irradiation on the behavioral properties of crotoxin

Crotoxin has been detoxified with gamma radiation in order to improve crotalic antiserum production. Nevertheless, present knowledge of the biological characteristics of irradiated crotoxin is insufficient to propose it as an immunizing agent. Crotoxin is known to increase the emotional state of rats and to decrease their exploratory behavior (Moreira EG, Nascimento N, Rosa GJM, Rogero JR and Vassilieff VS (1996) Brazilian Journal of Medical and Biological Research, 29: 629-632). Therefore, we decided 1) to evaluate the effects of crotoxin in the social interaction test, which has been widely used for the evaluation of anxiogenic drugs, and 2) to determine if irradiated crotoxin induces behavioral alterations similar to those of crotoxin in the social interaction, open-field and hole-board tests. Male Wistar rats (180-220 g) were used. Crotoxin (100, 250, and 500 µg/kg) was injected intraperitoneally 2 h before the social interaction test. Similarly, irradiated crotoxin (2000 Gy gamma radiation from a 60Co source) was administered at the doses of 100, 250, and 500 µg/kg for the hole-board test, and at the doses of 1000 and 2500 µg/kg for the open-field and social interaction tests. ANOVA complemented with the Dunnett test was used for statistical analysis (P<0.05). Crotoxin decreased the social interaction time (s) at the doses of 100, 250 and 500 µg/kg (means ± SEM) from 51.6 ± 4.4 to 32.6 ± 3.7, 28.0 ± 3.6 and 31.6 ± 4.4, respectively. Irradiated crotoxin did not induce behavioral alterations. These results indicate that 1) crotoxin may be an anxiogenic compound, and 2) in contrast to crotoxin, irradiated crotoxin was unable to induce behavioral alterations, which makes it a promising compound for the production of crotalic antiserum

Abnormal proliferative response of the carotid artery of spontaneously hypertensive rats after angioplasty may be related to the depolarized state of its smooth muscle cells

Hypertension is one of the major precursors of atherosclerotic vascular disease, and vascular smooth muscle abnormal cell replication is a key feature of plaque formation. The present study was conducted to examine the relationship between hypertension and smooth muscle cell proliferation after balloon injury and to correlate neointima formation with resting membrane potential of uninjured smooth muscle cells, since it has been suggested that altered vascular function in hypertension may be related to the resetting of the resting membrane potential in spontaneously hypertensive rats (SHR). Neointima formation was induced by balloon injury to the carotid arteries of SHR and renovascular hypertensive rats (1K-1C), as well as in their normotensive controls, i.e., Wistar Kyoto (WKY) and normal Wistar (NWR) rats. After 14 days the animals were killed and the carotid arteries were submitted to histomorphometric and immunohistochemical analyses. Resting membrane potential measurements showed that uninjured carotid arteries from SHR smooth muscle cells were significantly depolarized (-46.5 ± 1.9 mV) compared to NWR (-69 ± 1.4 mV), NWR 1K-1C (-60.8 ± 1.6 mV), WKY (-67.1 ± 3.2 mV) and WKY 1K-1C (-56.9 ± 1.2 mV). The SHR arteries responded to balloon injury with an enhanced neointima formation (neo/media = 3.97 ± 0.86) when compared to arteries of all the other groups (NWR 0.93 ± 0.65, NWR 1K-1C 1.24 ± 0.45, WKY 1.22 ± 0.32, WKY 1K-1C 1.15 ± 0.74). Our results indicate that the increased fibroproliferative response observed in SHR is not related to the hypertensive state but could be associated with the resetting of the carotid smooth muscle cell resting membrane potential to a more depolarized state.