Atividade antiviral de galactomananas modificadas por oxidação e/ou sulfatação

Orientadora : Maria Rita SierakowskiTese(doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica. Defesa: Curitiba, 2004Inclui bibliografiaResumo: Galactomananas extraídas de sementes de 3 espécies de leguminosas (Mimosa scabrella, Leucaena leucocephala e Cassia fastuosa), constituídas por uma cadeia principal de unidades de D-manopiranose unidas por ligações do tipo -(1 4) e com diferentes graus de substituição em O-6 por -D-galactose (Man/Gal 1,1, 1,4 e 2,9, respectivamente), foram derivatizadas para serem avaliadas quanto ao potencial antiviral frente ao HSV-1 (vírus Herpes simplex), em experimentos in vitro. Foram obtidos derivados sulfatados, urônicos (através de oxidação específica de C-6 com o reagente TEMPO - 2,2,6,6 tetrametilpiperidina1-oxil) e urônico/sulfatados. Os derivados sulfatados e urônico/sulfatados das galactomananas mais substituídas (de M. scabrella, respectivamente BR-S e BROS e de L. leucocephala, respectivamente, LL-S e LLOS) foram os que apresentaram melhor potencial antiviral, promovendo 50% de inibição do número de UFP, nas concentrações de 0,38 g.mL-1 para BR-S (DS:1,4), 14,6 g.mL-1 para BROS (DS:0,80), 4 g.mL-1 para LL-S (DS 0,6), e de 3,2 g.mL-1 para LLOS (DS: 0,33), enquanto CFS (DS: 0,22) e CFOS (DS: 0,30), promoveram, respectivamente, 91 e 94% de inibição nas concentrações de 1131 e 1218 g.mL-1 . A partir desses resultados, testou-se a atividade antiviral dos derivados sulfatados de M. scabrella (BRS) e de L. leucocephala (LLS) contra os vírus da febre amarela (YFV) e da dengue (DEN1), em experimentos in vitro e in vivo. BRS e LLS apresentaram 15,3 e 14,3% de sulfato, respectivamente, e perfil homogêneo por HPSEC-MALLS. LLS apresentou proteção média de 96,5% e BRS, 87,8% contra o YFV in vivo na concentração de 1 g.L-1 , apesar das concentrações necessárias para reduzir em 100 vezes o título viral in vitro terem sido relativamente altas (BRS: 1055 g.mL-1 e LLS: 386 g.mL-1 para YFV; BRS: 347 g.mL-1 e LLS: 37 g.mL-1 para DEN-1). Os camundongos inoculados com polissacarídeo + vírus desenvolveram anticorpos neutralizantes contra o YFV em títulos comparáveis aos animais inoculados com vírus inativados pelo formaldeído e, portanto, a atividade antiviral poderia estar sendo exercida por imunoestimulação. BRS e LLS previamente despolimerizadas por oxidação com H2O2-Cu++, utilizando análise estrutural por técnicas uni e bidimensionais (COSY, TOCSY, HMQC e HMBC) de RMN1H e de 13C, mostraram diferenças na posição dos substituintes sulfatos nos dois derivados: BRS estaria preferencialmente sulfatada no C-6 Gal ( 13C: 67,5 ppm, 1H: 4,21 ppm) e LLS, no C-6 Man ( 13C: 66,7 ppm, 1H: 4,21 ppm). Portanto, diferenças estruturais na posição do substituinte sulfato e no grau de substituição por galactose poderiam explicar um potencial antiviral maior de LLS in vivo (contra o YFV) e in vitro (contra YFV e DEN-1). Assim, foram obtidos derivados sulfatados de galactomananas de fontes renováveis com bons rendimentos de extração (entre 13 e 22%) e homogeneidade, e que mostraram, após sulfatação, características interessantes como substâncias candidatas a drogas antivirais.Abstract: Galactomannans extracted from 3 leguminous seeds (Mimosa scabrella, Leucaena leucocephala and Cassia fastuosa) consisted of a -D-(1 4) mannan backbone substituted at O-6 by different proportions of single D-galactosyl units (Man/Gal ratio of 1.1, 1.4 and 2.9, respectively) were derivatized so that the products were evaluated for their in vitro antiviral potencial against HSV-1 (herpes simplex virus). Sulfated, uronic (through specific oxidation of CH2OH-6 with TEMPO - 2,2,6,6-tetramethylpiperidine-1-oxyl) and uronic/sulfated derivatives were synthesized. The sulfated and uronic/sulfated derivatives with the highest Gal:Man ratio galactomannans had the best antiviral potential against HSV-1 (BR-S and BROS from M. scabrella, respectively and LL-S and LLOS from L. leucocephala, respectively), promoting higher than 50% inhibition of the number of PFUs at concentrations of 0.38 g.mL-1 for BR-S (DS:1.4), 14.6 g.mL-1 for BROS (DS:0.80), 4 g.mL-1 for LL-S (DS 0.6) and 3.2 g.mL-1 for LLOS (DS: 0.33). CFS (DS 0.22) and CFOS (DS 0.30), promoted 91 and 94% inhibition at concentrations of 1131 and 1218 g.mL-1 , respectively. The sulfated derivatives of M. scabrella (BRS) and L leucocephala (LLS) were then tested against yellow fever (YFV) and dengue viruses (DEN-1), in vitro and in vivo. The sulfate content was determined to be 15.3 and 14.3%, respectively, and showed homogeneous HPSEC-MALLS profiles. At a dose of 1 g.L-1 , BRS gave rise to a 87.7% death protection and 96.5% for LLS in vivo against YFV, despite the concentration necessary to make a 100-fold reduction in the in vitro viral titer being relatively high (BRS: 1055 g.mL-1 and LLS: 386 g.mL-1 for YFV; BRS: 347 g.mL-1 and LLS: 37 g.mL-1 for DEN-1). Mice inoculated with polysaccharide + virus developed neutralizing antibodies against YFV, with titers comparable to those of animals inoculated with the formaldehyde inactivated virus, so that the antiviral activity could be exerted through a immunostimulatory mechanism. For the structural characterization of BRS and LLS, the positions of the sulfate groups were verified by 1D and 2D (COSY, TOCSY, HMQC and HMBC) 1H and 13C NMR spectroscopy. The polysaccharide with a low molecular weight produced by hydrogen peroxide - cupric redox system showed a 13C signal at 67.5 ppm, which correlated with an 1H signal at 4.21 ppm in its HMQC spectrum, corresponding to sulfation at C-6 of galactose residues in the BRS spectrum. Interestingly, LLS did not contain sulfate groups in this position, probably being sulfated at C-6 of its mannosyl residues, deduced by the observation of a signal at 66.7 ppm, which correlated with one at 4.21 ppm in the HMQC spectrum. The relation structure/activity thus shows that BRS and LLS have differences in their sulfate substitution positions that could explain the differences in their antiviral activities against YFV and DEN-1. Galactomannans from renewable sources obtained in good yields (13-22%) and with homogeneity, could be an interesting source as antiviral drug candidates after sulfation

CONHECENDO MELHOR OS MICRORGANISMOS – PROPOSTA DE SEQUÊNCIA DIDÁTICA PARA O ENSINO MÉDIO

Esta proposta de sequência didática tem o intuito de promover a reflexão pelos estudantes sobre a relevância dos microrganismos, sua diversidade, importância e usos, frente à percepção frequentemente relatada de que todos ou a maioria dos microrganismos seriam danosos aos humanos. Para tanto serão explorados os recursos de debates, pesquisa bibliográfica, preparo e apresentação de seminário

The scattering of LyA radiation in the intergalactic medium: numerical methods and solutions

Two methods are developed for solving the steady-state spherically symmetric radiative transfer equation for resonance line radiation emitted by a point source in the Intergalactic Medium. One method is based on solving the ray and moment equations using finite differences. The second uses a Monte Carlo approach incorporating methods that greatly improve the accuracy compared with previous approaches in this context. Several applications are presented serving as test problems for both a static medium and an expanding medium, including inhomogeneities in the density and velocity fields. Solutions are obtained in the coherent scattering limit and for Doppler RII redistribution with and without recoils. We find generally that the radiation intensity is linear in the cosine of the azimuthal angle with respect to radius to high accuracy over a broad frequency region across the line centre for both linear and perturbed velocity fields, yielding the Eddington factors f(nu) = 1/3 and g(nu) = 3/5. We show the radiation field produced by a point source divides into three spatial regimes for a uniformly expanding homogeneous medium: at radii r small compared with a characteristic radius r*, the mean intensity near line centre varies as 1/ r^(7/3), while at r > r* it approaches 1/ r^2; for r << r* it is modified by frequency redistribution. Before the reionization epoch, r* takes on the universal value 1.1 Mpc, independent of redshift. The mean intensity and scattering rate are found to be very sensitive to the gradient of the velocity field, growing exponentially with the amplitude of the perturbation as the limit of a vanishing velocity gradient is approached near the source. We expect the 21cm signal from the Epoch of Reionization to thus be a sensitive probe of both the density and the peculiar velocity fields.Comment: 27 pages, 26 figures, 10 supplementary tables; submitted to MNRA

Experimental investigation on the controls of clumped isotopologue and hydrogen isotope ratios in microbial methane

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Geochimica et Cosmochimica Acta 237 (2018): 339-356, doi:10.1016/j.gca.2018.06.029.The abundance of methane isotopologues with two rare isotopes (e.g., 13CH3D) has been proposed as a tool to estimate the temperature at which methane is formed or thermally equilibrated. It has been shown, however, that microbial methane from surface environments and from laboratory cultures is characterized by low 13CH3D abundance, corresponding to anomalously high apparent 13CH3D equilibrium temperatures. We carried out a series of batch culture experiments to investigate the origin of the non-equilibrium signals in microbial methane by exploring a range of metabolic pathways, growth temperatures, and hydrogen isotope compositions of the media. We found that thermophilic methanogens (Methanocaldococcus jannaschii, Methanothermococcus thermolithotrophicus, and Methanocaldococcus bathoardescens) grown on H2+CO2 at temperatures between 60 and 80°C produced methane with Δ13CH3D values (defined as the deviation from stochastic abundance) of 0.5 to 2.5‰, corresponding to apparent 13CH3D equilibrium temperatures of 200 to 600°C. Mesophilic methanogens (Methanosarcina barkeri and Methanosarcina mazei) grown on H2+CO2, acetate, or methanol produced methane with consistently low Δ13CH3D values, down to -5.2‰. Closed system effects can explain part of the non-equilibrium signals for methane from thermophilic methanogens. Experiments with M. barkeri using D-spiked water or D-labeled acetate (CD3COO-) indicate that 1.6 to 1.9 out of four H atoms in methane originate from water, but Δ13CH3D values of product methane only weakly correlate with the D/H ratio of medium water. Our experimental results demonstrate that low Δ13CH3D values are not specific to the metabolic pathways of methanogenesis, suggesting that they could be produced during enzymatic reactions common in the three methanogenic pathways, such as the reduction of methyl-coenzyme M. Nonetheless C-H bonds inherited from precursor methyl groups may also carry part of non-equilibrium signals.Grants from the National Science Foundation (EAR-1250394 to S.O.), N. Braunsdorf and D. Smit of Shell PTI/EG (to S.O.), the Deep Carbon Observatory (to S.O., M.K., K.-U.H., D.S.G.), the Gottfried Wilhelm Leibniz Program of the Deutsche Forschungsgemeinschaft (HI 616-14-1 to K.- U.H.), and the Heisenberg Program (KO3651-3-1 to M.K.) of the Deutsche Forschungsgemeinschaft supported this study. D.S.G. was also supported by a National Science Foundation Graduate Research Fellowship, the Neil and Anna Rasmussen Foundation Fund, the Grayce B. Kerr Fellowship, and a Shell-MIT Energy Initiative Graduate Fellowship. D.T.W. was supported by a National Defense Science and Engineering Graduate Fellowship. L.C.S. was supported by a NASA Earth and Space Science Fellowship (grant NNX11AP78H)

ART^2 : Coupling Lyman-alpha Line and Multi-wavelength Continuum Radiative Transfer

Narrow-band Lya line and broad-band continuum have played important roles in the discovery of high-redshift galaxies in recent years. Hence, it is crucial to study the radiative transfer of both Lya and continuum photons in the context of galaxy formation and evolution in order to understand the nature of distant galaxies. Here, we present a three-dimensional Monte Carlo radiative transfer code, All-wavelength Radiative Transfer with Adaptive Refinement Tree (ART^2), which couples Lya line and multi-wavelength continuum, for the study of panchromatic properties of galaxies and interstellar medium. This code is based on the original version of Li et al., and features three essential modules: continuum emission from X-ray to radio, Lya emission from both recombination and collisional excitation, and ionization of neutral hydrogen. The coupling of these three modules, together with an adaptive refinement grid, enables a self-consistent and accurate calculation of the Lya properties. As an example, we apply ART^2 to a cosmological simulation that includes both star formation and black hole growth, and study in detail a sample of massive galaxies at redshifts z=3.1 - 10.2. We find that these galaxies are Lya emitters (LAEs), whose Lya emission traces the dense gas region, and that their Lya lines show a shape characteristic of gas inflow. Furthermore, the Lya properties, including photon escape fraction, emergent luminosity, and equivalent width, change with time and environment. Our results suggest that LAEs evolve with redshift, and that early LAEs such as the most distant one detected at z ~ 8.6 may be dwarf galaxies with a high star formation rate fueled by infall of cold gas, and a low Lya escape fraction.Comment: 20 pages, 16 figures, accepted for publication in MNRA

Monoallelic maternal expression of STAT5A affects embryonic survival in cattle

<p>Abstract</p> <p>Background</p> <p>Reproductive disorders and infertility are surprisingly common in the human population as well as in other species. The decrease in fertility is a major cause of cow culling and economic loss in the dairy herd. The conception rate has been declining for the past 30–50 years. Conception rate is the product of fertilization and embryonic survival rates. In a previous study, we have identified associations of several single nucleotide polymorphisms (SNPs) in the signal transducer and activator 5A (<it>STAT5A</it>) with fertilization and survival rates in an <it>in </it>vitro experimental system. The objectives of this study are to fine map the <it>STAT5A </it>region in a search for causative mutations and to investigate the parent of origin expression of this gene.</p> <p>Results</p> <p>We have performed a total of 5,222 fertilizations and produced a total of 3,696 in vitro fertilized embryos using gametes from 440 cows and eight bulls. A total of 37 SNPs were developed in a 63.4-kb region of genomic sequence that includes <it>STAT5A</it>, <it>STAT3</it>, and upstream and downstream sequences of these genes. SNP153137 (G/C) in exon 8 of <it>STAT5A </it>was associated with a significant variability in embryonic survival and fertilization rate compared to all other examined SNPs. Expression analysis revealed that <it>STAT5A </it>is primarily monoallelically expressed in early embryonic stages but biallelically expressed in later fetal stages. Furthermore, the occurrence of monoallelic maternal expression of <it>STAT5A </it>was significantly higher in blastocysts, while paternal expression was more frequent in degenerative embryos.</p> <p>Conclusion</p> <p>Our results imply that <it>STAT5A </it>affects embryonic survival in a manner influenced by developmental stage and allele parent of origin.</p