Use of pyrosequencing to identify streptococci and to detect mutations causing antimicrobial resistance

Rapid identification and resistance determination of pathogens in clinical specimens is vital for accurate treatment and monitoring of infectious diseases. Antimicrobial drug resistance is increasing globally and healthcare settings are facing this cost-intensive and even life-threatening problem. The incidence of resistant pathogens in Finland has remained relatively steady and manageable at least for the time being. DNA sequencing is the gold standard method for genotyping, mutation analysis, and identification of bacteria. Due to significant cost decrease in recent years, this technique is available to many research and clinical laboratories. Pyrosequencing technique, a rapid real-time DNA sequencing method especially suitable for analyzing fairly short stretches of DNA, was used in this study. Due to its robustness and versatility, pyrosequencing was applied in this study for identification of streptococci and detection of certain mutations causing antimicrobial resistance in different bacteria. Certain streptococcal species such as S. pneumoniae and S. pyogenes are significantly important clinical pathogens. S. pneumoniae causes e.g. pneumonia and otitis media and is one of the most important community-acquired pathogens. S. pyogenes, also known as group A streptococcus, causes e.g. angina and erysipelas. In contrast, the socalled alpha-haemolytic streptococci, such as S. mitis and S. oralis, belong to the normal microbiota, which are regarded to be non-pathogenic and are nearly impossible to identify by phenotypic methods. In this thesis, a pyrosequencing method was developed for identification of streptococcal species based on the 16S rRNA sequences. Almost all streptococcal species could be differentiated from one another by the developed method, including S. pneumoniae from its close relatives S. mitis and S. oralis . New resistance genes and their variants are constantly discovered and reported. In this study, new methods for detecting certain mutations causing macrolide resistance or extended spectrum beta-lactamase (ESBL) phenotype were developed. These resistance detection approaches are not only suitable for surveillance of mechanisms causing antimicrobial resistance but also for routine analysis of clinical samples particularly in epidemic settings. In conclusion, pyrosequencing was found to be an accurate, versatile, cost-effective, and rapid DNA sequencing method that is especially suitable for mutation analysis of short DNA fragments and identification of certain bacteria.

GAS EXCHANGE IN YOUNG PLANTS OF Tabebuia aurea(Bignoniaceae Juss.) SUBJECTED TO FLOODING STRESS1

ABSTRACT The Paratudo (Tabebuia aurea) is a species occurring in the Pantanal of Miranda, Mato Grosso do Sul, Brazil, an area characterized by seasonal flooding. To evaluate the tolerance of this plant to flooding, plants aged four months were grown in flooded soil and in non-flooded soil (control group). Stomatal conductance, transpiration and CO2 assimilation were measured during the stress (48 days) and recovery (11 days) period, totalling 59 days. The values of stomatal conductance of the control group and stressed plants at the beginning of the flooded were 0.33 mol m-2s-1 and reached 0.02 mol m-2 s-1 (46th day) at the end of this event. For the transpiration parameter, the initial rate was 3.1 mol m s-1, and the final rate reached 0.2 or 0.3 mol m-2 s-1 (47/48 th day). The initial photosynthesis rate was 8.9 mmol m-2s-1 and oscillated after the sixth day, and the rate reached zero on the 48th day. When the photosynthesis rate reached zero, the potted plants were dried, and the rate was analyzed (11th day). The following values were obtained for dried plants: stomatal conductance = 0.26 mol m-2 s-1, transpiration rate = 2.5 mol m-2 s-1 and photosynthesis rate = 7.8 mmol m-2 s-1. Flooded soil reduced photosynthesis and stomatal conductance, leading to the hypertrophy of the lenticels. These parameters recovered and after this period, and plants exhibited tolerance to flooding stress by reducing their physiological activities.

Analysis and validation of space averaged drag model for numerical simulations of gas-solid flows in fluidized beds

This thesis presents an approach for formulating and validating a space averaged drag model for coarse mesh simulations of gas-solid flows in fluidized beds using the two-fluid model. Proper modeling for fluid dynamics is central in understanding any industrial multiphase flow. The gas-solid flows in fluidized beds are heterogeneous and usually simulated with the Eulerian description of phases. Such a description requires the usage of fine meshes and small time steps for the proper prediction of its hydrodynamics. Such constraint on the mesh and time step size results in a large number of control volumes and long computational times which are unaffordable for simulations of large scale fluidized beds. If proper closure models are not included, coarse mesh simulations for fluidized beds do not give reasonable results. The coarse mesh simulation fails to resolve the mesoscale structures and results in uniform solids concentration profiles. For a circulating fluidized bed riser, such predicted profiles result in a higher drag force between the gas and solid phase and also overestimated solids mass flux at the outlet. Thus, there is a need to formulate the closure correlations which can accurately predict the hydrodynamics using coarse meshes. This thesis uses the space averaging modeling approach in the formulation of closure models for coarse mesh simulations of the gas-solid flow in fluidized beds using Geldart group B particles. In the analysis of formulating the closure correlation for space averaged drag model, the main parameters for the modeling were found to be the averaging size, solid volume fraction, and distance from the wall. The closure model for the gas-solid drag force was formulated and validated for coarse mesh simulations of the riser, which showed the verification of this modeling approach. Coarse mesh simulations using the corrected drag model resulted in lowered values of solids mass flux. Such an approach is a promising tool in the formulation of appropriate closure models which can be used in coarse mesh simulations of large scale fluidized beds.

Effect of pentoxifylline on lung inflammation and gas exchange in a sepsis-induced acute lung injury model

Experimental models of sepsis-induced pulmonary alterations are important for the study of pathogenesis and for potential intervention therapies. The objective of the present study was to characterize lung dysfunction (low PaO2 and high PaCO2, and increased cellular infiltration, protein extravasation, and malondialdehyde (MDA) production assessed in bronchoalveolar lavage) in a sepsis model consisting of intraperitoneal (ip) injection of Escherichia coli and the protective effects of pentoxifylline (PTX). Male Wistar rats (weighing between 270 and 350 g) were injected ip with 10(7) or 10(9) CFU/100 g body weight or saline and samples were collected 2, 6, 12, and 24 h later (N = 5 each group). PaO2, PaCO2 and pH were measured in blood, and cellular influx, protein extravasation and MDA concentration were measured in bronchoalveolar lavage. In a second set of experiments either PTX or saline was administered 1 h prior to E. coli ip injection (N = 5 each group) and the animals were observed for 6 h. Injection of 10(7) or 10(9) CFU/100 g body weight of E. coli induced acidosis, hypoxemia, and hypercapnia. An increased (P < 0.05) cell influx was observed in bronchoalveolar lavage, with a predominance of neutrophils. Total protein and MDA concentrations were also higher (P < 0.05) in the septic groups compared to control. A higher tumor necrosis factor-alpha (P < 0.05) concentration was also found in these animals. Changes in all parameters were more pronounced with the higher bacterial inoculum. PTX administered prior to sepsis reduced (P < 0.05) most functional alterations. These data show that an E. coli ip inoculum is a good model for the induction of lung dysfunction in sepsis, and suitable for studies of therapeutic interventions.

Effect of saline infusion for the maintenance of blood volume on pulmonary gas exchange during temporary abdominal aortic occlusion

We analyzed the effects of saline infusion for the maintenance of blood volume on pulmonary gas exchange in ischemia-reperfusion syndrome during temporary abdominal aortic occlusion in dogs. We studied 20 adult mongrel dogs weighing 12 to 23 kg divided into two groups: ischemia-reperfusion group (IRG, N = 10) and IRG submitted to saline infusion for the maintenance of mean pulmonary arterial wedge pressure between 10 and 20 mmHg (IRG-SS, N = 10). All animals were anesthetized and maintained on spontaneous ventilation. After obtaining baseline measurements, occlusion of the supraceliac aorta was performed by the inflation of a Fogarty catheter. After 60 min of ischemia, the balloon was deflated and the animals were observed for another 60 min of reperfusion. The measurements were made at 10 and 45 min of ischemia, and 5, 30, and 60 min of reperfusion. Pulmonary gas exchange was impaired in the IRG-SS group as demonstrated by the increase of the alveolar-arterial oxygen difference (21 ± 14 in IRG-SS vs 11 ± 8 in IRG after 60 min of reperfusion, P = 0.004 in IRG-SS in relation to baseline values) and the decrease of oxygen partial pressure in arterial blood (58 ± 15 in IRG-SS vs 76 ± 15 in IRG after 60 min of reperfusion, P = 0.001 in IRG-SS in relation to baseline values), which was correlated with the highest degree of pulmonary edema in morphometric analysis (0.16 ± 0.06 in IRG-SS vs 0.09 ± 0.04 in IRG, P = 0.03 between groups). There was also a smaller ventilatory compensation of metabolic acidosis after the reperfusion. We conclude that infusion of normal saline worsened the gas exchange induced by pulmonary reperfusion injury in this experimental model.

Characterization and classification of pequi trees (Caryocar brasiliense Camb.) based on the profile of volatile constituents using headspace solid-phase microextraction - gas chromatography - mass spectrometry and multivariate analysis

In order to determine the variability of pequi tree (Caryocar brasiliense Camb.) populations, volatile compounds from fruits of eighteen trees representing five populations were extracted by headspace solid-phase microextraction and analyzed by gas chromatography-mass spectrometry. Seventy-seven compounds were identified, including esters, hydrocarbons, terpenoids, ketones, lactones, and alcohols. Several compounds had not been previously reported in the pequi fruit. The amount of total volatile compounds and the individual compound contents varied between plants. The volatile profile enabled the differentiation of all of the eighteen plants, indicating that there is a characteristic profile in terms of their origin. The use of Principal Component Analysis and Cluster Analysis enabled the establishment of markers (dendrolasin, ethyl octanoate, ethyl 2-octenoate and β-cis-ocimene) that discriminated among the pequi trees. According to the Cluster Analysis, the plants were classified into three main clusters, and four other plants showed a tendency to isolation. The results from multivariate analysis did not always group plants from the same population together, indicating that there is greater variability within the populations than between pequi tree populations.

Electronic structure and properties of the group 4, 5 and 6 highest chlorides including elements 104, 105, and 106

Results of the Dirac-Slater discrete variational calculations for the group 4, 5, and 6 highest chlorides including elements 104, 105, and 106 have shown that the groups are not identical with respect to trends in the electronic structure and bonding. The charge density distribution data show that notwithstanding the basic increase in covalency within the groups this increase diminishes in going from group 4 to group 6. As a result, E106Cl_6 will be less stable toward thermal decomposition than WCl_6, which is confirmed by an estimated low E106-Cl bond energy. \delta H_form equal to -90.3 ± 6 kcal/rnol is obtained for E106Cl_6 in the gas phase, which is indicative of a very low stability of this compound. The stability of the maximum oxidation state is shown to decrease in the direction E104(+4) > E105(+5) > E106(+6).

Estado de salud y factores de riesgo en los estados financieros del sector de extracción de petróleo crudo y gas natural en Colombia

El objetivo de esta investigación es determinar el estado de salud y el riesgo existente en los Estados Financieros del Sector del sector de extracción de petróleo crudo y gas natural en Colombia. Esto permitirá clasificar a las compañías de dicho sector según estas variables. La importancia del sector energético en la economía colombiana y su magnitud y tamaño en la economía mundial explican la relevancia de su investigación en términos de riesgo y perdurabilidad. El proyecto estará fundamentado teóricamente en los conceptos de riesgo epidemiológico y salud financiera, así como en el uso de las razones financieras como medida y base de la gestión financiera de las empresas del sector energético en Colombia. Adicionalmente, la metodología que se llevará a cabo será cuantitativa, apoyándose en modelos de salud y epidemiológicos de las ciencias de la salud. Finalmente, esta investigación contribuirá al grupo de investigación en perdurabilidad empresarial mediante la línea de gerencia aportando conocimiento e información del sector energético teniendo en cuenta la relación entre riesgo, salud financiera y perdurabilidad. Así, al aportar los resultados de esta investigación se logrará contribuir al objetivo de la línea de gerencia el cual es identificar oportunidades gerenciales para las organizaciones que privilegien su tránsito hacia la denominada sociedad del conocimiento.

Influencia de la exploración y el almacenamiento de petróleo y gas natural en la relación de las organizaciones con las comunidades

El presente trabajo pretendía investigar y analizar el funcionamiento del mercado de hidrocarburos y gas natural, en busca de determinar la Influencia de la exploración y el almacenamiento de petróleo y gas natural en la relación de las organizaciones con las comunidades. Teniendo en cuenta el concepto de comunidad a partir del marketing relacional donde la comunidad se refiere a los consumidores y el entorno en el cual están inmersos. En este contexto se definieron los principales actores que participan en la relación comercial, el tipo de relación presente entre ellos y todos los factores que intervienen en desarrollo de esta relación que cada vez es más inestable y de corto plazo. Al finalizar esta investigación se reunió información acerca de las relaciones comerciales en el mercado de hidrocarburos, que servirán de fundamento para investigaciones futuras que permitirán plantear alternativas para sobrellevar la incertidumbre de este mercado y de esa manera lograr desarrollar una relación más confiable y duradera entre las organizaciones y las comunidades que intervienen en el proceso comercial. Debido a que aunque existe gran diversidad estrategias que pueden ser implementadas para mantener una relación estable, estas en la mayor parte de los casos no son utilizadas.

Uso del gas como recurso estratégico en la política exterior rusa. Análisis de caso Ucrania periodo 2000-2010

Estudio de caso que busca evaluar el uso del gas natural dentro de la política exterior rusa como recurso estratégico en las relaciones con Ucrania. Se describe y analiza cómo la importancia geoestratégica de Ucrania genera una percepción de seguridad y relevancia económica en Rusia, por esta razón la Federación utiliza el gas natural como mecanismo de presión frente al Estado ucraniano para satisfacer sus intereses nacionales en función de los elementos mencionados anteriormente.Todo lo anterior a través de la Teoría del Heartland desarrollada por Halford Mackinder y El Concepto de Área Pivote de Zbigniew Brzezinski para determinar cómo se genera la importancia del Estado ucraniano frente a Rusia

Utilidad de la relación estratégica comunitaria y el marketing en el sector de extracción de petróleo crudo y gas natural

El presente trabajo pretende investigar y analizar la utilidad de las relaciones estratégicas comunitarias y el marketing en el sector de extracción de petróleo crudo y gas natural. El mercadeo juega un papel importante a la hora de vender cualquier tipo de producto o servicio. Dependiendo de las características de cada sector, las estrategias de mercadeo pueden llegar a ser más o menos útiles y/o efectivas que otras. Por esta razón, esta investigación pretende analizar la utilidad de las estrategias de marketing comunitario y marketing en el sector de extracción de petróleo crudo y gas natural. La forma de llegar a esa respuesta, puede hallarse por medio de tres caminos: el primero de ellos es encontrar y analizar las características del sector, el segundo es identificar cuáles son las estrategias que están utilizando las empresas que operan este campo, y por último, consistiría en analizar la utilidad de integrar el concepto de relación estratégica comunitaria y marketing al sector de extracción de petróleo crudo y gas natural, en cuanto a los beneficios que el marketing comunitario le traería al sector de interés. El sector de petróleo crudo y gas natural es uno de los más importantes en la economía global y sus clientes son numerosos y totalmente heterogéneos. El mercadeo se vuelve muy complejo para esta industria ya que al tener tipos de clientes tan distintos se dificulta alcanzar las expectativas de cada uno de ellos con una misma estrategia y por supuesto la forma de vender un producto es más compleja. Estos factores mencionados anteriormente están generando relaciones entre empresas y clientes totalmente transaccionales de corto plazo, y es ahí donde se cree que la inclusión de mercadeo relacional y estrategias de mercadeo relacional pueden ser de utilidad para el sector, generando desarrollo y beneficios mutuos tanto para clientes como empresas.

Mortality of Migratory Birds from Marine Commercial Fisheries and Offshore Oil and Gas Production in Canada

There is an imminent need for conservation and best-practice management efforts in marine ecosystems where global-scale declines in the biodiversity and biomass of large vertebrate predators are increasing and marine communities are being altered. We examine two marine-based industries that incidentally take migratory birds in Canada: (1) commercial fisheries, through bycatch, and (2) offshore oil and gas exploration, development, and production. We summarize information from the scientific literature and technical reports and also present new information from recently analyzed data to assess the magnitude and scope of mortality. Fisheries bycatch was responsible for the highest levels of incidental take of migratory bird species; estimated combined take in the longline, gillnet, and bottom otter trawl fisheries within the Atlantic, including the Gulf of St. Lawrence, and Pacific regions was 2679 to 45,586 birds per year. For the offshore oil and gas sector, mortality estimates ranged from 188 to 4494 deaths per year due to the discharge of produced waters resulting in oil sheens and collisions with platforms and vessels; however these estimates for the oil and gas sector are based on many untested assumptions. In spite of the uncertainties, we feel levels of mortality from these two industries are unlikely to affect the marine bird community in Canada, but some effects on local populations from bycatch are likely. Further research and monitoring will be required to: (1) better estimate fisheries-related mortality for vulnerable species and populations that may be impacted by local fisheries, (2) determine the effects of oil sheens from produced waters, and attraction to platforms and associated mortality from collisions, sheens, and flaring, so that better estimates of mortality from the offshore oil and gas sector can be obtained, and (3) determine impacts associated with accidental spills, which are not included in our current assessment. With a better understanding of the direct mortality of marine birds from industry, appropriate mitigation and management actions can be implemented. Cooperation from industry for data collection, research to fill knowledge gaps, and implementation of mitigation approaches will all be needed to conserve marine birds in Canada.

Molecular structures of benzoic acid and 2-hydroxybenzoic acid, obtained by gas-phase electron diffraction and theoretical calculations

The structures of benzoic acid (C6H5COOH) and 2-hydroxybenzoic acid (C6H4OHCOOH) have been determined in the gas phase by electron diffraction using results from quantum chemical calculations to inform restraints used on the structural parameters. Theoretical methods (HF and MP2/6-311+G(d, p)) predict two conformers for benzoic acid, one which is 25.0 kJ mol(-1) (MP2) lower in energy than the other. In the low-energy form, the carboxyl group is coplanar with the phenyl ring and the O-H group eclipses the C=O bond. Theoretical calculations (HF and MP2/6-311+ G(d, p)) carried out for 2-hydroxybenzoic acid gave evidence for seven stable conformers but one low-energy form (11.7 kJ mol-1 lower in energy (MP2)) which again has the carboxyl group coplanar with the phenyl ring, the O-H of the carboxyl group eclipsing the C=O bond and the C=O of the carboxyl group oriented toward the O-H group of the phenyl ring. The effects of internal hydrogen bonding in 2-hydroxybenzoic acid can be clearly observed by comparison of pertinent structural parameters between the two compounds. These differences for 2-hydroxybenzoic acid include a shorter exocyclic C-C bond, a lengthening of the ring C-C bond between the substituents, and a shortening of the carboxylic single C-O bond.

Ag dopedWO(3)-based powder sensor for the detection of NO gas in air

WO3-based materials as sensors for the monitor of environmental gases such as NO2 (NO + NO2) have been rapidly developed for various potential applications (stationary and mobile uses). It has been reported that these materials are highly sensitive to NOx with the sensitivity further enhanced by adding precious group metals (PGM such as Pt, Pd, Au, etc.). However, there has been limited work in revealing the sensing mechanism for these gases over the WO3-based sensors. In particular, the role of promoter is not yet clear though speculations on their catalytic, electronic and structural effects have been made in the past. In parallel to these PGM promoters here we report,for the first time, that Ag promotion can also enhance WO3 sensitivity significantly. In addition, this promotion decreases the optimum sensor temperature of 300 degreesC for Most WO3-based sensors, to below 200 degreesC. Characterizations (XRD, TEM, and impedance measurement) reveal that there is no significant bulk structure change nor particle size alteration in the WO3 phases during the NO exposure. However, it is found that the Ag doping creates a high concentration of oxygen vacancies in form of coordinated crystallographic shear (CS) planes onto the underneath WO3. It is thus proposed that the Ag particle facilitates the oxidative conversion of NO to NO2 followed by a subsequent NO2 adsorption on the defective WO, sites created at the Ag-WO3 interface; hence, accounting for the high molecular sensitivity. (C) 2002 Elsevier Science B.V. All rights reserved.

Intramolecular proton transfer in [Ph2B-OH2](+). Participation of a phenyl group

Ab initio calculations at the HF/6-31+G* level on [Ph2B-OH2](+) show that in the gas phase the structure with the proton attached to an ipso C is lower in energy than the one with the proton on the oxygen atom by 8.40 kcal mol(-1). The transition states and reaction paths for intramolecular proton transfer in [Ph2B-OH2](+) have also been studied.