Detection and coat protein gene characterization of an isolate of Grapevine virus B from corky bark-affected grapevines in Southern Brazil

An isolate of Grapevine virus B (GVB), obtained by indexing Vitis labrusca and V. vinifera grapevines on the indicator LN33, was transmitted mechanically to several Nicotiana species. The virus was partially purified from N. cavicola and the coat protein estimated at 23 kDa by SDS-PAGE. In negatively stained leaf extracts of experimentally inoculated N. cavicola and N. occidentalis, flexuous particles with cross banding were observed, predominantly measuring 750-770 x 12 nm, with a modal length of 760 nm. Decoration indicated a clear, positive reaction against AS-GVB. In DAS-ELISA, GVB was detected in N. cavicola and grapevine extracts, and Western blots showed homologous and cross reaction of GVB and GVA antisera with GVB coat protein. Using specific primers for GVB, a fragment of 594 bp, comprising the coat protein gene coding for 197 amino acids, was amplified by RT-PCR with viral RNA extracted from GVB-infected N. occidentalis. The nucleotide and the deduced amino acid sequences of the coat protein gene showed high identities with Italian and Japanese isolates of GVB.

RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

Detection and partial characterization of a carlavirus causing stem necrosis of soybean in Brazil

Stunting and stem necrosis were noticed in soybeans (Glycine max) grown in 2000/2001 in West Central Brazil the same condition was also observed in the following year in plantations as far as 2,000 km from the initial area. Based on transmission (mechanical, graft, insect vector), purification and serology, electron microscopy and molecular studies the causal agent was determined to be a whitefly-borne carlavirus which is possibly related to Cowpea mild mottle virus (CpMMV).

Detection of Varicosavirus and Ophiovirus in lettuce associated with lettuce big-vein symptoms in Brazil

During surveys undertaken from 1998 to 2003 in the major vegetable growing areas of the city of São Paulo green belt, lettuce (Lactuca sativa) and endive (Cichorium endivia) plants were observed, which showed chlorotic thickening of foliar veins, defective growth and, in some cases, failure to form complete heads. Biological and serological [DAS-Enzyme linked immunosorbent assay (Elisa)] tests together with electron microscope observations, revealed the presence of Lettuce big-vein virus and Mirafiori lettuce virus, in these plants both responsible for the lettuce big-vein syndrome.

Electric field measurements using scanning electron microscope

The main idea of this diploma work is to study electric field distribution on the micro level. For this purpose a silicon edgeless detector was chosen as the object of investigation and scanning electron microscope as an investigation tool. Silicon edgeless detector is an important part of installation for studying proton-proton interactions in TOTEM experiment at Large Hadron Collider. For measurement of electric field distribution inside scanning electron microscope a voltage contrast method was applied.

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.

Early detection of injuries in leaves of Clusia hilariana Schltdl. (Clusiaceae) caused by particulate deposition of iron

This study aims to evaluate the prognostic value of microscopic parameters of asymptomatic leaves of Clusia hilariana Schltdl. subjected to particulate deposition of iron (2.14 mg cm-2 day-1) for 45 consecutive days. Samples of young and expanded leaves without symptoms were collected and subjected to light and scanning electron microscopy techniques. The height of the epidermal cells on both surfaces of the leaf and the thickness of the hypodermis, the chlorophyll parenchyma, and the leaf blade were measured. Micromorphological injury occurred in the abaxial surface of young leaves and on both surfaces of expanded leaves. Erosion of the epicuticular wax and cuticle rupture were frequent on the adaxial surface, while on the abaxial surface of both leaves there was a loss of sinuosity on the anticlinal wall of the epidermal cells, stomatal deformity and obstruction. Micromorphometric alterations were seen in all leaf tissues except in the height of epidermic cells, probably due to the thick cuticle and prominent cuticular flanges. The highest difference in thickness of the leaf blade was seen in young leaves of plants subjected to SPMFe, indicating greater sensibility to particulate iron in comparison to the expanded leaves. The micromorphological and micromorphometric alterations in the leaf blade of Clusia hilariana Schltdl. showed the prognostic potential of these tools on the evaluation of impacts caused by the deposition of particulate matter, especially in the 'Restinga' natural vegetation, where the exposure is increasing due to the presence of iron ore industry in their surroundings.

Ultrastructural and biochemical detection of biotin and biotinylated polypeptides in Schistosoma mansoni

Biotinylation is proposed for the identification of surface proteins in Schistosoma mansoni using the streptavidin-HRP conjugate for the detection of labeled polypeptides. However, control samples also showed several endogenous biotinylated polypeptides. In an attempt to determine the possibility of nonspecific binding between the streptavidin-HRP conjugate and polypeptides from S. mansoni, the conjugate was blocked with biotinamidecaproate-N-hydroxysuccinimide ester (BcapNHS) before biotin-streptavidin blotting. No bands were detected on the nitrocellulose sheet, demonstrating the specific recognition of biotin by the streptavidin present in the conjugate. Whole cercariae and cercarial bodies and tails showed several endogenous biotinylated polypeptides. The biotin concentration was 13 µg/190,000 cercariae. Adult worms presented less endogenous biotinylated polypeptides than cercariae. These results may be due to changes in the environment from aerobic to anaerobic conditions when cercarial bodies (schistosomula) are transformed into adult worms and a decrease in CO2 production may occur. Cercariae, cercarial bodies and adult male worms were examined by transmission electron microscopy employing an avidin-colloidal gold conjugate for the detection of endogenous biotin. Gold particles were distributed mainly on the muscle fibers, but dispersed granules were observed in the tegument, mitochondria and cytosol. The discovery of endogenous biotin in S. mansoni should be investigated in order to clarify the function of this vitamin in the parasite

A liquid phase blocking ELISA for the detection of antibodies against infectious bronchitis virus

A liquid phase blocking ELISA (LPB-ELISA) was developed for the detection and measurement of antibodies against infectious bronchitis virus (IBV). The purified and nonpurified virus used as antigen, the capture and detector antibodies, and the chicken hyperimmune sera were prepared and standardized for this purpose. A total of 156 sera from vaccinated and 100 from specific pathogen-free chickens with no recorded contact with the virus were tested. The respective serum titers obtained in the serum neutralization test (SNT) were compared with those obtained in the LPB-ELISA. There was a high correlation (r2 = 0.8926) between the two tests. The LPB-ELISA represents a single test suitable for the rapid detection of antibodies against bronchitis virus in chicken sera, with good sensitivity (88%), specificity (100%) and agreement (95.31%).

Detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in ruptured atherosclerotic plaques

This paper reports what is apparently the first observation of Mycoplasma pneumoniae in association with Chlamydia pneumoniae in thrombosed ruptured atheromas. We performed electron microscopy and in situ hybridization in specimens from three patients who died of acute myocardial infarction. These patients had typical symptoms of acute ischemic syndrome. Mycoplasmas were present mainly in the lipid core of the ruptured thrombosed plaque. Vulnerable atheromas are rich in cholesterol and may favor the growth of mycoplasmas, the only microorganisms that require cholesterol for survival. We suggest that the association of Mycoplasma pneumoniae and Chlamydia pneumoniae may increase the virulence of these microorganisms, favoring proliferation, plaque inflammation and possibly plaque rupture.

Immunocytochemical and biochemical detection of alpha-L-fucosidase in Trypanosoma cruzi

The aim of the present study was to demonstrate the presence of alpha-L-fucosidase in Trypanosoma cruzi. Immunocytochemical and biochemical techniques were used to localize and characterize a membrane-associated, neutral-pH-optimum, alpha-L-fucosidase from the parasite. Light and electron microscopy localized the alpha-L-fucosidase specifically on the surface of the parasite and on membranes in the posterior region of the epimastigote stage. Although much less intense, labeling was also detected on the surface of trypomastigotes. At least 50% of the alpha-L-fucosidase activity was associated with epimastigote membrane solubilized with 1 M NaCl or 1% Triton X-100, suggesting that alpha-L-fucosidase is peripherally associated with membranes. The enzyme from epimastigotes had a neutral pH optimum (near 7) but displayed low specific activity when p-nitrophenyl-alpha-L-fucoside was employed as substrate (0.028 U/mg protein for epimastigotes and 0.015 U/mg protein for tissue culture-derived trypomastigotes). Polyacrylamide gel electrophoresis and Western blotting analysis both showed an expected 50-kDa polypeptide which was immunoreactive with anti-alpha-L-fucosidase antibodies.

Alternative electron transfer routes involved in photoprotection of cyanobacteria

In oxygenic photosynthesis, the highly oxidizing reactions of water splitting produce reactive oxygen species (ROS) and other radicals that could damage the photosynthetic apparatus and affect cell viability. Under particular environmental conditions, more electrons are produced in water oxidation than can be harmlessly used by photochemical processes for the reduction of metabolic electron sinks. In these circumstances, the excess of electrons can be delivered, for instance, to O2, resulting in the production of ROS. To prevent detrimental reactions, a diversified assortment of photoprotection mechanisms has evolved in oxygenic photosynthetic organisms. In this thesis, I focus on the role of alternative electron transfer routes in photoprotection of the cyanobacterium Synechocystis sp. PCC 6803. Firstly, I discovered a novel subunit of the NDH-1 complex, NdhS, which is necessary for cyclic electron transfer around Photosystem I, and provides tolerance to high light intensities. Cyclic electron transfer is important in modulating the ATP/NADPH ratio under stressful environmental conditions. The NdhS subunit is conserved in many oxygenic phototrophs, such as cyanobacteria and higher plants. NdhS has been shown to link linear electron transfer to cyclic electron transfer by forming a bridge for electrons accumulating in the Ferredoxin pool to reach the NDH-1 complexes. Secondly, I thoroughly investigated the role of the entire flv4-2 operon in the photoprotection of Photosystem II under air level CO2 conditions and varying light intensities. The operon encodes three proteins: two flavodiiron proteins Flv2 and Flv4 and a small Sll0218 protein. Flv2 and Flv4 are involved in a novel electron transport pathway diverting electrons from the QB pocket of Photosystem II to electron acceptors, which still remain unknown. In my work, it is shown that the flv4-2 operon-encoded proteins safeguard Photosystem II activity by sequestering electrons and maintaining the oxidized state of the PQ pool. Further, Flv2/Flv4 was shown to boost Photosystem II activity by accelerating forward electron flow, triggered by an increased redox potential of QB. The Sll0218 protein was shown to be differentially regulated as compared to Flv2 and Flv4. Sll0218 appeared to be essential for Photosystem II accumulation and was assigned a stabilizing role for Photosystem II assembly/repair. It was also shown to be responsible for optimized light-harvesting. Thus, Sll0218 and Flv2/Flv4 cooperate to protect and enhance Photosystem II activity. Sll0218 ensures an increased number of active Photosystem II centers that efficiently capture light energy from antennae, whilst the Flv2/Flv4 heterodimer provides a higher electron sink availability, in turn, promoting a safer and enhanced activity of Photosystem II. This intertwined function was shown to result in lowered singlet oxygen production. The flv4-2 operon-encoded photoprotective mechanism disperses excess excitation pressure in a complimentary manner with the Orange Carotenoid Protein-mediated non-photochemical quenching. Bioinformatics analyses provided evidence for the loss of the flv4-2 operon in the genomes of cyanobacteria that have developed a stress inducible D1 form. However, the occurrence of various mechanisms, which dissipate excitation pressure at the acceptor side of Photosystem II was revealed in evolutionarily distant clades of organisms, i.e. cyanobacteria, algae and plants.

Evaporative respiratory cooling augments pit organ thermal detection in rattlesnakes

Rattlesnakes use their facial pit organs to sense external thermal fluctuations. A temperature decrease in the heat-sensing membrane of the pit organ has the potential to enhance heat flux between their endothermic prey and the thermal sensors, affect the optimal functioning of thermal sensors in the pit membrane and reduce the formation of thermal ‘‘afterimages’’, improving thermal detection. We examined the potential for respiratory cooling to improve strike behaviour, capture, and consumption of endothermic prey in the South American rattlesnake, as behavioural indicators of thermal detection. Snakes with a higher degree of rostral cooling were more accurate during the strike, attacking warmer regions of their prey, and relocated and consumed their prey faster. These findings reveal that by cooling their pit organs, rattlesnakes increase their ability to detect endothermic prey; disabling the pit organs caused these differences to disappear. Rattlesnakes also modify the degree of rostral cooling by altering their breathing pattern in response to biologically relevant stimuli, such as a mouse odour. Our findings reveal that low humidity increases their ability to detect endothermic prey, suggesting that habitat and ambush sites election in the wild may be influenced by external humidity levels as well as temperature.

Construction interactive de BRDFs par simulation 2D de micro-géométries en couches multiples

Les modèles de réflexion complexes, avec leurs nombreux paramètres dont certains restent non intuitifs, sont difficiles à contrôler pour obtenir une apparence désirée. De plus, même si un artiste peut plus aisément comprendre la forme de la micro-géométrie d'une surface, sa modélisation en 3D et sa simulation en 4D demeurent extrêmement fastidieuses et coûteuses en mémoire. Nous proposons une solution intermédiaire, où l'artiste représente en 2D une coupe dans un matériau, en dessinant une micro-géométrie de surface en multi-couches. Une simulation efficace par lancer de rayons en seulement 2D capture les distributions de lumière affectées par les micro-géométries. La déviation hors-plan est calculée automatiquement de façon probabiliste en fonction de la normale au point d'intersection et de la direction du rayon incident. Il en résulte des BRDFs isotropes complètes et complexes, simulées à des vitesses interactives, et permettant ainsi une édition interactive de l'apparence de réflectances riches et variées.

Etude spectroscopique d’un plasma micro-onde à la pression atmosphérique et son application à la synthèse de nanostructures

L’objectif de ce mémoire de maîtrise est de caractériser la distribution axiale des plasmas tubulaires à la pression atmosphérique créés et entretenus par une onde électromagnétique de surface ainsi que d’explorer le potentiel de ces sources pour la synthèse de matériaux et de nanomatériaux. Un précédent travail de thèse, qui avait pour objectif de déterminer les mécanismes à l’origine de la contraction radiale du plasma créé dans des gaz rares, a mis en lumière un phénomène jusque-là inconnu dans les plasmas d’onde de surface (POS). En effet, la distribution axiale varie différemment selon la puissance incidente ce qui constitue une différence majeure par rapport aux plasmas à pression réduite. Dans ce contexte, nous avons réalisé une étude paramétrique des POS à la pression atmosphérique dans l’Ar. À partir de nos mesures de densité électronique, de température d’excitation et de densité d’atomes d’Ar dans un niveau métastable (Ar 3P2), résolues axialement, nous avons conclu que le comportement axial de l’intensité lumineuse avec la puissance n’est pas lié à un changement de la cinétique de la décharge (qui est dépendante de la température des électrons et de la densité d’atomes d’Ar métastables), mais plutôt à une distribution anormale de dissipation de puissance dans le plasma (reliée à la densité d’électrons). Plus précisément, nos résultats suggèrent que ce dépôt anormal de puissance provient d’une réflexion de l’onde dans le fort gradient de densité de charges en fin de colonne, un effet plus marqué pour de faibles longueurs de colonnes à plasma. Ensuite, nous avons effectué une étude spectroscopique du plasma en présence de précurseurs organiques, en particulier le HMDSO pour la synthèse de matériaux organosiliciés et l’IPT pour la synthèse de matériaux organotitaniques. Les POS à la PA sont caractérisés par des densités de charges très élevées (>10^13 cm^-3), permettant ainsi d’atteindre des degrés de dissociation des précurseurs nettement plus élevés que ceux d'autres plasmas froids à la pression atmosphérique comme les décharges à barrière diélectrique. Dans de tels cas, les matériaux synthétisés prennent la forme de nanopoudres organiques de taille inférieure à 100 nm. En présence de faibles quantités d’oxygène dans le plasma, nous obtenons plutôt des nanopoudres à base d’oxyde de silicium (HMDSO) ou à base de titanate de silicium (IPT), avec très peu de carbone.