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Microorganisms, that evolve to acquire resistance to environmental amoeba, are likely to become human pathogens due to the physiological similarities of free-living amoebae to human macrophages. In this sense free-living amoebae can be regarded as nurseries of pathogenic microorganisms. Due to the widespread distribution of amoebae in the environment and their resistance to current disinfection procedures, they may constitute important reservoirs of pathogenic microorganisms. Striking examples are Legionella pneumophila and several mycobacteria including Mycobacterium tuberculosis. This work represents the first attempt to detect and characterize amoebae and their associated microorganisms in Portugal.Detection, isolation and in vitro culture of amoebae from several environmental sources, including lagoon and estuarine water and sediments, were performed according to previously developed methods. Identification of the isolated amoebae was done by optical microscopy based on morphological criteria and by DNA sequence analysis of PCR products amplified with one of the following two sets of primers: EUK and ITS. Transmission electron microscopy (TEM) studies and PCR/sequencing approaches were used to detect and identify amoeba-associated microorganisms (AAMs). Bacterial 16S region was amplified with the primers 616V/630R. TEM studies were performed according to standard procedures. In short, samples were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite. Thin sections contrasted with uranyl acetate and lead citrate were observed with a JEOL 100-SX electron microscope.Several species of amoeba (Acanthamoeba lenticulata, A. polyphaga, A. rhyzodes, Platyamoeba oblongata, Saccamoeba limax, Vannella simplex, Vannella sp. and other unidentified amoebae) were found and AAMs were detected both by TEM and PCR/sequencing. The bacteria species Variovorax paradoxus, previously found in association with Saccamoeba and Arcella, were isolated and identified by the PCR/sequencing approach, which also allowed the detection of an unidentified species with about 85% identity with Marivirga tractuosa, a species not yet associated with amoebae. These results point to the existence of AAMs in the environments subjected to this study and the need to evaluate their pathogenic potential. This is an important issue particularly in environments related to human recreational, nutritional and public health activities.

Helicobacter pylori is a Gram-negative microorganism that grows on microaerophilic conditions and has only one known natural reservoir: the gastric mucosa. The infection by H. pylori is very common worldwide and this bacterium is associated with the development of gastritis, peptic ulcer gastric cancer or gastric Mucosa Associated Lymphoid Tissue (MALT) lymphoma. Although its natural habitat is the acidic gastric mucosa, H. pylori is considered to be a neutralophile. The bacterium survives brief exposure to pHs of In the present work we have compared the exposure to UV and to acidic environment in the induction of the prophage into a lytic cycle. We have tested two strains, the strain B45 carrying the prophage phiHP33 and a clinical strain 1152, isolated from a patient with peptic ulcer, that was revealed to be negative for the presence of integrase gene (a prophage gene essential for genome integration of prophage) by PCR, as negative control. Since the H. pylori reservoir is the human stomach the exposition to acid is very common, and with this experiment we intended to test if acid can trigger a phage lytic cycle.The induction using UV radiation has been previously described. For acid induction we have used a protocol adapted from Karita and Blaser. A 48 hours culture of H. pylori was grown in Brucella broth (Oxoid) supplemented with 10% of fetal bovine serum (Gibco) and 1% of Polivitex (BioMérieaux) in microaerophilic conditions at 37ºC. The liquid culture was centrifuged and the cell pellet ressuspended in citrate-phosphate buffer pH 6 and incubated 15 minutes, centrifuged again and ressuspended in citrate-phosphate buffer pH 3 and incubated for 30 minutes. After centrifugation the supernatant was recovered and incubated for 3 hours in phage precipitant (33% polyethylene glycol [PEG], 3M NaCl). After centrifugation at 10000 rpm for 10 minutes at 4ºC the pellet was ressuspended in phage buffer. These samples were analysed by transmission electron microscopy (TEM) after negative staining with 1% aqueous uranyl acetate, using a JEOL 100SX electron microscope.For B45 strain the induction using UV radiation (previously reported in Lehours, 2011) and acid exposure produced similar results (Figure 1 and Figure 2) showing numerous phage-like particles of about 100 nm diameter, apparently lacking a tail, after UV or acid exposition, respectively. These particles were not observed in the control strain 1152. Currently we are analysing the samples using molecular biology techniques and fixation embedding followed by ultrathin sectioning for TEM analysis, to detect the presence of phages.These preliminary results suggest that acid also appears to induce the H. pylori prophage phiHP33. However, since the number of phage particles observed is small, we can not rule out that the observed particles were released spontaneously. The exposition to the natural acidic environment of the human stomach may induce H. pylori prophage into a lytic cycle and to the propagation of phages among different H. pylori strains colonizing the same individual. Although highly speculative, transduction may be another form of horizontal gene transfer, which has not been described for this bacterium yet.Financial support received from the Portuguese Science and Technology foundation under the contract PTDC/EBB-EBI/119860/2010.

Helicobacter pylori is a pathogenic spiral-shaped, microaerophilic, gram-negative bacterium, that inhabits the human stomach. Infection is usually acquired during childhood and always elicits an acute immune response that is, however, inefficient in bacteria clearance. Therefore, in the absence of effective treatment, infection and gastritis (non ulcer dyspepsia, NUD) persist throughout the patient’s life. Depending on its severity and pattern, in about 15% of infected adults, this silent destruction of the gastric mucosa may further progress to peptic ulcer disease (PUD) (gastric and duodenal ulcers, GU and DU respectively) and/or gastric cancer. Infection with H. pylori is also the major cause for the development of paediatric PUD, a rare event that may occur shortly after infection. In addition to the still undisclosed genetic susceptibility of these young patients, the virulence of the implicated H. pylori strain plays a crucial role in the paediatric PUD pathogenesis. Recently, we proved by in vitro infection assays that, compared with paediatric NUD-associated isolates, a group of paediatric ulcerogenic-strains present a greater ability to induce a marked decrease in the gastric cells viability and to cause them severe cytoskeleton damage and mucins’ production/secretion impairment. Moreover, we showed that their enhanced virulence result from a synergy between the ability to better adapt to the hostility of their niche and the expression of cagA, vacAs1, oipA ‘‘on’’ status, homB and jhp562 virulence factors. Accordingly, these ulcerogenic strains share a particular proteome profile, providing them with better antioxidant defences, a metabolism favouring the biosynthesis of aromatic amino acids and higher motility.We are now characterizing/comparing the cellular and flagella morphologies of H. pylori strains isolated from Portuguese children, associated with DU, GU or NUD, belonging to the vast and multiethnic collection of the Instituto Nacional de Saúde Dr. Ricardo Jorge (Portugal). For that, bacteria were grown in H. pylori selective medium (Biogerm, Maia, Portugal) at 37ºC in a microaerobic environment (Anoxomat®, MART Microbiology BV, Drachten, The Netherlands) for 24 h. For Leifson staining analysis, a drop of each bacterial suspension (in PBS) was spread in cleaned microscope slides, stained with the Leifson dye solution until a golden film developed on the dye surface and a precipitate appeared throughout the sample, and analysed by optical microscopy. For Transmission-Electronic-Microscopy (TEM) studies bacterial pellets were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite. Thin sections contrasted with uranyl acetate and lead citrate were observed with a JEOL 100-SX electron microscope.Corroborating the better swimming abilities of the PUD strains, as previously shown by motility assays, optical microscopy analysis of Leifson stained slides demonstrated marked differences in the morphology of the studied strains (Figure 1). The H. pylori strain associated with DU (Hp 1152/04) seem longer than all the others and, in contrast, that associated with GU (Hp 499/02) is the shortest one and presents a, more pronounced, spiral morphology. Moreover, our preliminary data on TEM analysis indicate the presence of more abundant and apparently more organized flagella in the GU-associated strain Hp 499/02, in contrast to the NUD control strain, Hp 655/99 (Figure 2).Work supported by Research Grant 2011 – Sociedade Portuguesa de Gastrenterologia.

O objetivo deste trabalho foi analisar fertilizantes tipo fritas comercializados no Brasil, utilizando o método prescrito para análise de fertilizantes minerais não-fritas, o que normalmente tem sido feito na rotina pelos laboratórios de análises de fertilizantes. Também foram analisados os teores de Cd, Ni e Pb nesses fertilizantes. Observou-se que o método utilizado foi adequado para a análise dos fertilizantes tipo fritas, o que indica que as fritas comercializadas no Brasil não são realmente fritas, isto é, silicatos fundidos com micronutrientes. A presença de micronutrientes metálicos nos produtos sem as respectivas garantias e a alta concentração de metais indesejáveis, principalmente Pb, são fortes indícios de utilização de resíduos industriais na fabricação desses fertilizantes. Sugere-se que sejam estudados e propostos extratores químicos para avaliar a eficiência agrícola desses produtos.

Helicobacter pylori is a human pathogen involved in gastritis and gastric cancer whose mode of transmission remains unknown. Association of H. pylori with humans is thought to date from remote antiquity and the bacterium has apparently evolved together with the human host. A few studies have shown the presence of H. pylori in aquatic environments, which might provide a route of transmission of the bacteria to humans. A recent study has also disclosed the association of the bacteria with Acantamoeba castellanii. Amoeba are known to harbor and promote the persistence of several human pathogens in the environment, representing a significant source of contamination in community and hospital acquired infections.

Amoebae graze on bacteria from the environment. In some cases, bacteria acquire resistance to the destructive action of amoebae and survive inside the host. The resistance of microorganisms to amoebae is often accompanied by resistance to the physiologically related human macrophage and displays increased pathogenic potential to humans. The association of bacteria with amoebae, that are ubiquitous in the environment, protects the bacteria from common disinfection measures. This leads to the creation of reservoirs of pathogenic microorganisms that are difficult to eradicate. This process occurs widely in the environment, but is of special concern in hospitals, where many established and emerging pathogens involved in nosocomial disease, such as Legionella pneumophila, are increasingly being identified in association with amoebae. One particularly important amoeba-associated microorganism (AAM) is the newly recognized giant mimivirus, a member of the Nucleo-Cytoplasmic Large Deoxyribovirus (NCLDV) group. The virus has been isolated from hospital sources in France and, together with other members of the NCLDV group, is known from metagenomic studies to be widespread in the Oceans, representing one of the main groups of marine viruses. Our project aims to detect amoeba and potentially pathogenic AAMs with particular relevance to the NCLDVs in the environment and in hospital facilities. In the present work we have developed methods for detection, isolation and in vitro culture of amoebae from several environmental sources including hospital dust, sea water and estuarine sediments.

****, F. Morgado***** and R. Marschang*******Centro de Estudos do Ambiente e do Mar (CESAM) – Faculdade de Ciencias da Universidadede Lisboa, Campo Grande, 1749-016, Lisboa, Portugal** Centro Hospitalar de Lisboa Central - HCC, Anatomia Patologica, R. da Beneficencia 8, 1069-166 Lisboa, Portugal*** Faculdade de Engenharia, Universidade Catolica Portuguesa, Lisboa, Portugal**** Laboratorio de Optica, Lasers e Sistemas, Faculdade de Ciencias da Universidade de Lisboa, 1749-016, Lisboa, Portugal***** CESAM, Centro de Estudos do Ambiente e do Mar, Universidade de Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, Portugal****** Institut fur Umwelt und Tierhygiene, Hohenheim University, Garbenstr. 30, 70599 Stuttgart, GermanyHeterotermic vertebrates are hosts of a variety of iridoviruses, many with recognized economical importance for aquaculture and with a role in the decline of amphibian populations, and others with no known disease associations. These include members of the Lymphocystisvirus, Magalocystisvirus and Ranavirus genera [1].Erythrocytic infections of reptiles, amphibians and fish with large icosahedral iridovirus-like viruseswere detected in the beginning of the XXth century by light microscopy, since they produce large cytoplasmic inclusions in the infected cells (Figure 1) [2, 3]. Our work has confirmed the viral nature of the inclusions (Figure 2) and identified 14 new host species worldwide [4, 5]. Our ultrastructural results detected significant morphological variations among the new viruses that suggest that it is a widespread and diverse group of potential related viruses [5]. Recent data on the sequence of a conserved region of the DNA polymerase gene from our work in

O objetivo deste trabalho foi determinar a solubilidade do zinco contido em fertilizantes (sulfato de zinco, óxido de zinco, zinco metálico e quatro "fritas" comerciais) nos extratores água, ácido cítrico 20 g L-1, citrato neutro de amônio (1 + 9) e DTPA, e correlacioná-la com a disponibilidade desse elemento para plantas de arroz e milho. O zinco presente no sulfato de zinco mostrou-se mais disponível às plantas, seguido daquele contido no óxido de zinco, enquanto as "fritas" apresentaram menor disponibilidade. O extrator citrato neutro de amônio (1 + 9), na relação 1:100 e com fervura por cinco minutos, apresentou correlação satisfatória com a absorção pelas plantas. Sua adoção contribuiria para melhorar a eficiência das adubações com zinco. Zinc solubility in fertilizers (zinc sulfate, zinc oxide, metallic zinc and four FTE) was determined using water, 20 g L-1 citric acid, neutral ammonium citrate (1 + 9) and DTPA as extractant solutions, and the correlation with the availability of this micronutrient for rice and corn plants established. Zinc was most accessible for the plants in sulfate form, followed by oxide, while FTE presented the lowest availability. Neutral ammonium citrate (1 + 9), at a ratio of 1:100 and after boiling for five minutes, demonstrated a satisfactory correlation with plant adsorption. Its adoption would contribute to improve zinc fertilizations.

Helicobacter pylori (H. pylori) successfully colonizes the human stomach of the majority of the human population. This infection always causes chronic gastritis, but may evolve to serious outcomes, such as peptic ulcer, gastric carcinoma or mucosa-associated lymphoid tissue lymphoma. H. pylori first line therapy recommended by the Maastricht-4 Consensus Report comprises the use of two antibiotics and a proton-pomp inhibitor, but in some regions failure associated with this treatment is already undesirable high. Indeed, treatment failure is one of the major problems associated with H. pylori infection and is mainly associated with bacterial antibiotic resistance. In order to counteract this situation, some effort has been allocated during the last years in the investigation of therapeutic alternatives beyond antibiotics. These include vaccines, probiotics, photodynamic inactivation and phage therapy, which are briefly revisited in this review. A particular focus on phytomedicine, also described as herbal therapy and botanical therapy, which consists in the use of plant extracts for medicinal purposes, is specifically addressed, namely considering its history, category of performed studies, tested compounds, active principle and mode of action. The herbs already experienced are highly diverse and usually selected from products with a long history of employment against diseases associated with H. pylori infection from each country own folk medicine. The studies demonstrated that many phytomedicine products have an anti-H. pylori activity and gastroprotective action. Although the mechanism of action is far from being completely understood, current knowledge correlates the beneficial action of herbs with inhibition of essential H. pylori enzymes, modulation of the host immune system and with attenuation of inflammation.

A legislação brasileira exige que os micronutrientes nos fertilizantes sejam garantidos pelo teor total presente. Isto abre um precedente para a utilização de produtos não considerados como fontes de micronutrientes na fabricação dos fertilizantes. Todavia, a eficiência agronômica desses produtos é ainda duvidosa. Objetivou-se realizar um trabalho para caracterizar a solubilidade e a disponibilidade dos micronutrientes em trinta fertilizantes comerciais, por meio do uso de cinco extratores químicos: a água e soluções de ácido cítrico a 2%, de citrato neutro de amônio na diluição 1 + 9, de DTPA 0,005 mol L-1 e de EDTA 0,005 mol L-1. Os resultados mostraram a baixa solubilidade dos micronutrientes metálicos (cobre, ferro, manganês e zinco) dos fertilizantes tipo "fritas". O ácido cítrico a 2% mostrou-se promissor na caracterização da disponibilidade de cobre, manganês e zinco para as plantas. Para o ferro não houve uma definição entre os extratores estudados. O boro teve boa solubilidade, tanto nos fertilizantes solúveis, como nos insolúveis em água, e a garantia pelo teor total mostrou-se bom indicativo da disponibilidade do elemento. Para o molibdênio a solubilidade foi maior para os fertilizantes com baixo teor do elemento. A garantia dos micronutrientes catiônicos pelo teor total, conforme exige a legislação, não indicou a sua real disponibilidade nos fertilizantes comerciais, mostrando a necessidade de uma definição de extratores para esse fim. The Brazilian fertilizer legislation requires that the presence of micronutrients be guaranteed by their total amount. This makes possible to use different products as source of micronutrients for fertilizers, but their agronomic efficiency is still doubtful. The purpose of this work was to characterize the micronutrient solubility and availability of thirty commercial fertilizers through five extractors: water, 2% citric acid, neutral ammonium citrate (1 + 9), 0.005 mol L-1 DTPA and 0.005 mol L-1 EDTA solutions. The results showed low solubility of Cu, Fe, Mn and Zn in "fritted" fertilizers. The 2% citric acid has shown to be a good extractor to identify Cu, Mn and Zn availability for plants. The B solubility was suitable in all fertilizers and its total amount showed to be a good indication of B availability. The Mo solubility presented high values for fertilizers with low amount of total Mo, but lower values for fertilizers with high amount of total Mo. The guarantee of total amount of micronutrients in fertilizers did not indicate their availability: it is necessary to establish an extractor in order to quantify micronutrient availability.

The environmental conditions experienced by hosts are known to affect their mean parasite transmission potential. How different conditions may affect the variance of transmission potential has received less attention, but is an important question for disease management, especially if specific ecological contexts are more likely to foster a few extremely infectious hosts. Using the obligate-killing bacterium Pasteuria ramosa and its crustacean host Daphnia magna , we analysed how host nutrition affected the variance of individual parasite loads, and, therefore, transmission potential. Under low food, individual parasite loads showed similar mean and variance, following a Poisson distribution. By contrast, among well-nourished hosts, parasite loads were right-skewed and overdispersed, following a negative binomial distribution. Abundant food may, therefore, yield individuals causing potentially more transmission than the population average. Measuring both the mean and variance of individual parasite loads in controlled experimental infections may offer a useful way of revealing risk factors for potential highly infectious hosts.

Consumption of contaminated drinking water heavily contributes to the burden of gastrointestinal waterborne diseases. Conventional detection methodologies present several shortcomings, such as indirect measure of indicator species, low throughput and time consume. DNA chips have the potential to serve as surveillance systems for the simultaneous detection of pathogens overcoming these limitations. We have developed a DNA chip for simultaneous detection of multiple waterborne pathogens. Species specific DNA probes were implemented on a microarray, for microorganism detection. Present study reports the results of one untreated water sample analyzed by conventional methods and by the DNA chip. The results were concordant for the mandatory organisms (total coliforms, Escherichia coli, fecal enterococci) using both methods, reinforcing the utility and proof-ofconcept of the DNA chip. However, it is necessary a prior enrichment in a culture medium in order to obtain a positive signal using the DNA chip. The DNA chip may be a valuable distinctive tool for waterborne pathogens detection

SUMMARY In this work we study the weathering resistance of samples of five types of stone used in Andalusian Cathedrals, treated with six water repellent products, by subjecting the samples to a SO2 polluted atmosphere. To evaluate the alteration of the samples, besides visual observation of macroscopic aspect, changes in weight and the quantity of sulfate that remains on the surface have been measured. Also properties related with water have been measured after the test to determine if there have been any changes in the hydrophobic characteristics., En el presente artículo se estudia el comportamiento de muestras de cinco tipos de piedra utilizados en catedrales andaluzas, tratadas con seis productos hidrófugos, sometiéndolas a un ensayo acelerado de alteración en atmósfera contaminada con SO2 . Para evaluar la alteración, además de la observación visual de las manifestaciones macroscópicas, se ha medido la variación de peso de las muestras expuestas y la cantidad de sulfato que permanece en la superficie tras el ensayo. Así mismo, se han detectado los cambios experimentados en las propiedades hidrófugas conferidas por los tratamientos, mediante la medida de propiedades relacionadas con el movimiento del agua.

SUMMARY In this work we study the weathering resistance of samples of five types of stone used in Andalusian Cathedrals, treated with six water repellent products, by subjecting the samples to a SO2 polluted atmosphere. To evaluate the alteration of the samples, besides visual observation of macroscopic aspect, changes in weight and the quantity of sulfate that remains on the surface have been measured. Also properties related with water have been measured after the test to determine if there have been any changes in the hydrophobic characteristics.En el presente artículo se estudia el comportamiento de muestras de cinco tipos de piedra utilizados en catedrales andaluzas, tratadas con seis productos hidrófugos, sometiéndolas a un ensayo acelerado de alteración en atmósfera contaminada con SO2 . Para evaluar la alteración, además de la observación visual de las manifestaciones macroscópicas, se ha medido la variación de peso de las muestras expuestas y la cantidad de sulfato que permanece en la superficie tras el ensayo. Así mismo, se han detectado los cambios experimentados en las propiedades hidrófugas conferidas por los tratamientos, mediante la medida de propiedades relacionadas con el movimiento del agua.

One of the main problems affecting monuments today is the effect of atmospheric pollution on their materials. From this arises the need to develop test methods to evaluate these effects, on the original materials (to define weathering mechanisms), on new materials for restoration and on treatment products to be applied. An installation has been designed and developed that allows the simulation of polluted atmospheres (with several pollutants) and other climatic conditions. The response of the system is studied when simulating specific conditions, by submitting samples of stone used in important Andalusian monuments, treated with various water-repellent products, to an accelerated weathering test with a sulphur dioxide atmosphere and variable humidity conditions. The installation is of a modular type and can be divided into 10 sections. In sections 1 and 2 the conditioning of air used to generate the simulated atmosphere is carried out: the air is first dried with 96% sulphuric acid and then cleansed of solid particles, sulphuric acid vapour and organic compounds. In sections 3-8, pollutants are added to the airstream and their flow is controlled. Hydrogen fluoride is added to the air by evaporation from a solution; sulphur dioxide, ammonia and propene are added from bottles of liquified gas; nitrogen dioxide is added from a heated bottle of liquified gas. The degree of humidification is achieved by evaporation. The test chamber is made of polyester reinforced with glass fibre and has a volume of one cubic metre; it is cylindrical, with a thermostatic bath to control the temperature. The gas streams go into a mixing chamber with a sampling device and then into the test chamber. Samples are placed on a rack; there are spray nozzles to simulate rain over the samples or to generate salt vapour. With the normal air flow of 25 litres per minute, the residence time is 40 minutes. When the conditions change, 99% of new conditions are reached in 147 minutes. The radiation source has to be located outside the chamber, due to the corrosive nature of the atmospheres generated. As radiation transmitter, films of Teflon FEP are used, as it has good resistance to chemical attack and excellent transmission of ultraviolet radiation of every wavelength. The sampling device has five independent lines that allow sampling simultaneously at five points in the test chamber. To obtain samples of pollutant gases, sampling ports with the appropriate absorbing solutions are used. Five types of limestone used in cathedrals of Andalucia were studied, as well as samples from quarries in the region. Their most important characteristics are as follows

São apresentados dados referentes às espécies e infecção natural de triatomíneos na Ilha de São Luis-MA. Dos triatomíneos coletados foram encontradas as seguintes espécies: Rhodnius pictipes Rhodnius neglectus, Rhodnius nasutus, Triatoma rubrofasciata, Panstrongylus lignarus e Panstrogylus geniculatus. A presença de infecção natural por Trypanosoma do tipo cruzi foi detectada em 19,7% do total de triatomíneos sendo o R. pictipes a espécie mais homogênea em distribuição na Ilha e com índice de infecção natural de 38,8%.Data is presented on the species and infection rate of reduviid bugs collected in São Luís island, Maranhão State. The following species have been collected: Rhodnius pictipes, R. neglectus, R. nasutus, R. robustus, Triatoma rubrofasciata, Panstrongylus lignarus and P. geniculatus. The infection rate with T. cruzi-like trypanosomes was 19.7% of the total bugs collected. R. pictipes shows homogeneous distribution in the island and an infection rate of 38.8%.

Measurements have been made, at temperatures between 35 and 100° C, of the viscosities and thermal conductivities of gaseous methane, ethane, ethylene, propane, propylene,cyclo-propane,n-butane,cis-2-butene,trans-2-butene, 1:3-butadiene,iso-butane,n-pentane,iso-pentane,neo-pentane,cyclo-pentane,n-hexane,neo-hexane,cyclo-hexane, benzene,n-heptane, 2:4-dimethyl-pentane,n-octane, 2:3:4-trimethyl-pentane and of carbon tetrafluoride. Viscosities were measured by observing the damping of a pendulum swinging in the vapour and by a capillary-flow viscometer. Thermal conductivities were measured by the hot-wire method. The observed values of the ratio between the transport properties are discussed in the light of the kinetic theory of gases. I t is found that highly flexible molecules show systematically lower values of the ratio,k/n, than other molecules, and it is suggested th at this is due to a special type of intermolecular collision, involving some degree of intercoiling of the two molecules. This view is relevant to the interpretation of recent work on the vibrational activation of the ethylene molecule by collision with other hydrocarbons, and on the kinetics of the thermal decomposition of gaseous hydrocarbons. Inferences can also be drawn about the ease of interchange of translational energy with vibrational and with rotational energy in collisions; these are discussed in relation to information obtained from ultrasonic dispersion measurements.

This study aimed to evaluate the variation in the levels of proline, oxidative metabolism and photosynthetic pigments in plants of Pitcairnia encholirioides grown in vitro under different conditions and after acclimatization. The analyses were performed after 150 days of in vitro cultivation in MS media supplemented with 10 µM GA3 or 0.2 µM NAA, sucrose at 15 or 30 g L–1, in test tubes which allowed gas exchange or in a hermetically sealed system, and 180 days after acclimatization. The in vitro maintenance in hermetically sealed flasks, with GA3 and 15 g L–1 sucrose had adverse metabolic effects, which was demonstrated by the lower proline and photosynthetic pigments accumulation and by the increase in antioxidant enzymes activities. After acclimatization, differences for proline and photosynthetic pigments were no longer found and the enzymatic activities ranged unevenly. The results suggest that the in vitro cultivation in media with 0.2 µM NAA and 30 g L–1 sucrose, in test tubes capped with closures which allowed gas exchange, is more suitable for micropropagation of P. encholirioides, providing a prolonged maintenance of in vitro cultures and plantlets with superior quality for ex vitro development. Resumo Este trabalho objetivou avaliar a contribuição da prolina, do metabolismo oxidativo e dos pigmentos fotossintéticos na propagação in vitro e aclimatização de Pitcairnia encholirioides, uma bromélia criticamente ameaçada de extinção. As análises foram realizadas após 150 dias de cultivo in vitro em meio MS suplementado com 10 µM de GA3 ou 0,2 µM de ANA, 15 ou 30 g L–1 de sacarose, em tubos de ensaio que permitiam trocas gasosas ou em sistema hermeticamente vedado, e também 180 dias após aclimatização. A manutenção in vitro em frascos hermeticamente fechados, com GA3 e 15 g L–1 de sacarose apresentou efeito metabólico adverso, demonstrado pelo menor acúmulo de prolina e pigmentos fotossintéticos e também pelo aumento das atividades de enzimas antioxidantes. Após aclimatização, as diferenças para prolina e pigmentos fotossintéticos não foram mais encontradas e as atividades enzimáticas variaram de maneira desuniforme. Os resultados sugerem que o cultivo in vitro em meio com 0,2 µM de ANA e 30 g L–1 de sacarose, em tubos fechados com tampas que permitem trocas gasosas, é mais adequado para a micropropagação de P. encholirioides, proporcionando uma manutenção prolongada das culturas in vitro e plântulas com qualidade superior para o desenvolvimento ex vitro.

Highlights • Understanding the maintenance of pathogen variation is central to disease control. • Classical metapopulation models make useful predictions about polymorphism. • The timing of pathogen regulation is key to pathogen polymorphism and virulence., Understanding why pathogen populations are genetically variable is vital because genetic variation fuels evolution, which often hampers disease control efforts. Here I argue that classical models of evolution in spatially variable environments – specifically, models of hard and soft selection – provide a useful framework to understand the maintenance of pathogen polymorphism and the evolution of virulence. First, the similarities between models of hard and soft selection and pathogen life cycles are described, highlighting how the type and timing of pathogen control measures impose density regulation that may affect both the level of pathogen polymorphism and virulence. The article concludes with an outline of potential lines of future theoretical and experimental work.