Ultrastructure of Biofilms Formed by Bacteria from Industrial Processes

Microorganisms exist predominantly as sessile multispecies communities in natural habitats. Most bacterial species can form these matrix-enclosed microbial communities called biofilms. Biofilms occur in a wide range of environments, on every surface with sufficient moisture and nutrients, also on surfaces in industrial settings and engineered water systems. This unwanted biofilm formation on equipment surfaces is called biofouling. Biofouling can significantly decrease equipment performance and lifetime and cause contamination and impaired quality of the industrial product. In this thesis we studied bacterial adherence to abiotic surfaces by using coupons of stainless steel coated or not coated with fluoropolymer or diamond like carbon (DLC). As model organisms we used bacterial isolates from paper machines (Meiothermus silvanus, Pseudoxanthomonas taiwanensis and Deinococcus geothermalis) and also well characterised species isolated from medical implants (Staphylococcus epidermidis). We found that coating of steel surface with these materials reduced its tendency towards biofouling: Fluoropolymer and DLC coatings repelled all four biofilm formers on steel. We found great differences between bacterial species in their preference of surfaces to adhere as well as their ultrastructural details, like number and thickness of adhesion organelles they expressed. These details responded differently towards the different surfaces they adhered to. We further found that biofilms of D. geothermalis formed on titanium dioxide coated coupons of glass, steel and titanium, were effectively removed by photocatalytic action in response to irradiation at 360 nm. However, on non-coated glass or steel surfaces irradiation had no detectable effect on the amount of bacterial biomass. We showed that the adhesion organelles of bacteria on illuminated TiO2 coated coupons were complety destroyed whereas on non-coated coupons they looked intact when observed by microscope. Stainless steel is the most widely used material for industrial process equipments and surfaces. The results in this thesis showed that stainless steel is prone to biofouling by phylogenetically distant bacterial species and that coating of the steel may offer a tool for reduced biofouling of industrial equipment. Photocatalysis, on the other hand, is a potential technique for biofilm removal from surfaces in locations where high level of hygiene is required. Our study of natural biofilms on barley kernel surfaces showed that also there the microbes possessed adhesion organelles visible with electronmicroscope both before and after steeping. The microbial community of dry barley kernels turned into a dense biofilm covered with slimy extracellular polymeric substance (EPS) in the kernels after steeping in water. Steeping is the first step in malting. We also presented evidence showing that certain strains of Lactobacillus plantarum and Wickerhamomyces anomalus, when used as starter cultures in the steeping water, could enter the barley kernel and colonise the tissues of the barley kernel. By use of a starter culture it was possible to reduce the extensive production of EPS, which resulted in a faster filtration of the mash.

Allergenic Cross-Reactivity between Parthenium and Ragweed Pollen Allergens

Cross-reactivity of allergens from the pollen of the Compositae weeds, Parthenium hysterophorus (American feverfew) and Ambrosia (ragweed), in 2 groups of patients with different geographic distributions was studied. Parthenium-sensitive Indian patients, who were never exposed to ragweed, elicited positive skin reactions with ragweed pollen extracts. A significant correlation in the RAST scores of Parthenium and ragweed-specific IgE was observed with the sera of Parthenium and ragweed-sensitive Indian and US patients, respectively. RAST inhibition experiments demonstrated that the binding of IgE antibodies in the sera of ragweed-sensitive patients to short (Wl) and giant (W3) ragweed allergen discs could be inhibited by up to 94% by Parthenium pollen extracts. Similar inhibition (up to 82%) was obtained when the sera of Parthenium rhinitis patients were incubated with ragweed allergen extracts. A dose-dependent proliferation of lymphocytes from a Parthenium-sensitive rhinitis patient with elevated levels of both Parthenium and ragweed-specific IgE was observed when incubated with Parthenium and ragweed pollen extracts. A 1.6-fold higher proliferation, however, was observed with Parthenium pollen extract at a concentration of 100 µg/ml. These results suggest that shared epitopes present on Parthenium and ragweed pollen allergens are recognized by both Indian and US patients sensitized by exposure to Parthenium and ragweed pollen, respectively. The high degree of cross-reactivity between Parthenium and ragweed pollen allergens suggests that individuals sensitized to Parthenium may develop type-I hypersensitivity reactions to ragweed and vice versa when they travel to regions infested with the weed to which they had not been previously exposed.

Standardization of pollen allergens of Parthenium hysterophoms and selection of an in-house reference extract

A standardized in-house reference extract from the pollen of Parthenium hysterophorus, which is responsible for the high incidence of allergic rhinitis in India, was generated and examined by skin test, radio-allergosorbent test inhibition and isoelectric focusing. Parthenium reference allergen discs and positive reference serum were also generated. These reference reagents could not only be used for the quantitation of Parthenium-specific IgE in the sera of rhinitis patients but also for the evaluation of allergenic activity (relative potency and lot-to-lot variation) of different batches of Parthenium pollen.

Altered ultrastructure of rinderpest virus and its nucleocapsids induced by 5-fluorouracil

he ultrastructure of purified rinderpest virus and intracellular viral nucleocapsids from infected vero cells treated with a subtoxic dose of 5-fluorouracil (5-Fu) (1 mug/ml), has been analysed by transmission electron microscopy, and compared with that of normal virus particle and nucleocapsids. The results reveal dramatic alterations in the structure of both virions and nucleocapsids. The surface glycoprotein projection of virions was not seen or present at a much reduced level. The intracellular nucleocapsids showed pronounced structural changes,with respect to size, shape and fine structure. The length of treated nucleocapsids is much smaller as compared to the control. The central hollow core is missing in case of drug-treated nucleocapsid and the herring bone structure is replaced by a 'beads on string' structure. The presence of N protein, which is a major structural component of nucleocapsids was seen in 5-Fu-treated cells, but it was associated with a predominantly diffused form of nucleocapsids as seen by immunoelectron microscopy. We report here the first definitive and visual evidence of altered structure of virions and their nucleocapsids after 5-Fu treatment

Immunochemical characterization of rapid and slowly released allergens from the pollen of Parthenium hysterophorus

Allergens from the pollen of Parthenium hysterophorus (American feverfew), responsible for high incidence of allergic rhinitis, were found by immunoprint analysis to be localized on the surface of the pollen grains. The allergens were released very rapidly when extracted in vitro. The allergenic activity of the rapid (10 s) and slowly (20 h) released pollen proteins was comparable by in vivo skin test and ELISA inhibition assay. The isoelectric focusing patterns of the rapid and slowly released proteins, were also identical. SDS-PAGE and Western blot analysis revealed that all the major pollen allergens with molecular weight 14, 28, 31, 37 and 45 kDa were eluted within 10 s of extraction. Periodate-Schiff staining showed that the 28, 31 and 45 kDa components of the pollen extract are glycoproteins. The pollen allergens released after different periods of extraction lost 75% of IgE binding activity when subjected to in situ sodium m-periodate oxidation under controlled conditions, while 80% of the allergenic activity was still retained after extensive proteolysis. Our results support the clinical observation of a rapid onset of symptoms of allergic rhinitis in patients sensitive to Parthenium pollen, mediated predominantly by glycoproteins.

On distribution of pollen in lakes. [Translation from: Palynology in geological research in the Baltic Soviet Republics p113-119. Vilnius, 1973.]

Systematic investigations of the distribution of pollen in stationary water bodies until now have hardly been conducted. For clarification of the problem of how the pollen of different plants which falls into a lake is deposited in relation to its physical properties, the character of the lake, wind currents and other factors, pollen analyses were carried out of surface samples of the bottom sediments of 13 Lithuanian lakes. Lakes were selected of different sizes (areas from 2333 ha. to 8 ha.) and different depths, not uniformly overgrown, situated in different physico-geographic regions of Lithuania. As a result of the investigation, it was established that in the surface layer of the sediments of the lakes of Lithuania pollen of woody species predominates.

Palinologia e taxonomia dos gêneros Elephantopus L., Orthopappus Gleason e Pseudelephantopus Rohr. (Subtribo Elephantopinae, Vernonieae- Asteraceae) ocorrentes no Brasil

A subtribo Elephantopinae tem sido alvo de poucos estudos taxonômicos e palinológicos, necessitando de uma reavaliação em seus limites. Assim, o presente estudo teve como proposta atualizar e ampliar o conhecimento de 13 espécies de Elephantopinae ocorrentes no Brasil através de um acurado estudo palinológico, da anatomia foliar e taxonômico, promovendo a delimitação das espécies dos gêneros Elephantopus (10 espécies) Orthopappus (uma espécie) e Pseudelephantopus (duas espécies)além de oferecer subsídios para posteriores análises filogenéticas. O material botânico utilizado foi obtido através de exsicatas depositadas nos herbários brasileiros e de material coletado em campo. Os grãos de pólen foram tratados pelo método acetolítico, sendo posteriormente mensurados, descritos, e analisados sob microscopia de luz e eletrônica de varredura. As eletromicrografias foram obtidas de grãos de pólen não acetolisados, as análises taxonômicas baseiam-se na metodologia clássica. A anatomia foliar deu-se através da metodologia usual. Os resultados mostram grãos de pólen médios, 3-porados, de exina equinolofada com malhas poligonais organizadas ou não em Elephantopus e Pseudelephantopus, podendo apresentar interrupção na malha poral em Elephantopus. Em Orthopapus os grãos de pólen são 3-colporados, de sexina subequinolofada. A anatomia foliar possibilitou a separação de E. hirtiflorus e E. riparius através da forma ou contorno da nervura principal (planoconvexo), nas demais espécies o contorno é biconvexo. As espécies apresentaram tricomas capitados bisseriados com 8 células, tricomas filamentosos 3-4 celulares, bem como filamentosos de célula distal globosa ou ovóide em E. biflorus, E. micropappus, E. tomentous e Orthopappus angustifolius. Registrou-se variação na ornamentação da cutícula podendo ser lisa ou estriada entre as espécies, bem como a presença de substâncias pécticas na epiderme e drusas nos parênquimas. Em relação aos microcaracteres florais, foram considerados de importância diagnóstica os lóbulos da corola, que variaram entre glandular, glabro e penicilado, este ultimo apenas em E. hirtiflorus, os ápices das anteras que variaram entre obtuso, retuso em E. hirtiflorus e apiculado, lancelolado em E. mollis e E. racemosus, a base da antera variou entre sagitada, caudada lisa em E. riparius e E. tomentosus e caudada em E. riparius. Os macrocaracteres que apresentaram maior valor taxonômico foram a cipsela e o papus, a organização das coflorescências, o número de flores por capítulo e limbo foliar. Neste trabalho aceitou-se a segregação dos três gêneros, com base nos caracteres analisados