Efficient P(3HB) extraction from Burkholderia sacchari cells using non-chlorinated solvents

© 2015. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

Descripción del análisis microbológico realizado mediante maldi-tof en muestras de la fundación santa fe de Bogotá.

Introducción: La rápida detección e identificación bacteriana es fundamental para el manejo de los pacientes críticos que presentan una patología infecciosa, esto requiere de métodos rápidos para el inicio de un correcto tratamiento. En Colombia se usan pruebas microbiología convencional. No hay estudios de espectrofotometría de masas en análisis de muestras de pacientes críticos en Colombia. Objetivo general: Describir la experiencia del análisis microbiológico mediante la tecnología MALDI-TOF MS en muestras tomadas en la Fundación Santa Fe de Bogotá. Materiales y Métodos: Entre junio y julio de 2013, se analizaron 147 aislamientos bacterianos de muestras clínicas, las cuales fueron procesadas previamente por medio del sistema VITEK II. Los aislamientos correspondieron a 88 hemocultivos (60%), 28 urocultivos (19%), y otros cultivos 31 (21%). Resultados: Se obtuvieron 147 aislamientos con identificación adecuada a nivel de género y/o especie así: en el 88.4% (130 muestras) a nivel de género y especie, con una concordancia del 100% comparado con el sistema VITEK II. El porcentaje de identificación fue de 66% en el grupo de bacilos gram negativos no fermentadores, 96% en enterobacterias, 100% en gérmenes fastidiosos, 92% en cocos gram positivos, 100% bacilos gram negativos móviles y 100% en levaduras. No se encontró ninguna concordancia en bacilos gram positivos y gérmenes del genero Aggregatibacter. Conclusiones: El MALDI-TOF es una prueba rápida para la identificación microbiológica de género y especie que concuerda con los resultados obtenidos de manera convencional. Faltan estudios para hacer del MALDI-TOF MS la prueba oro en identificación de gérmenes.

Diferenciación molecular de Lemna aequinoctialis y L. Valdiviana (lemnaceae) por patrones proteicos

El estudio se llevó a cabo en el Laboratorio de Genética Molecular de la Universidad Nacional, Heredia, Costa Rica. Se utilizaron plantas acuáticas macrófitas de Lemna valdiviana y L. aequinoctialis previamente identificadas. Estas especies habitan en aguas ricas en sales disueltas y aguas servidas. Recientemente se les consideró como un indicador de la contaminación ocasionada por pesticidas y herbicidas en el agua. La composición proteica llega a niveles entre 38 y 43% de materia seca, importante para diversos usos. La extracción de proteínas totales, por medio de electroforesis discontinua en gel de poliacrilamida, se realizó en condiciones reductoras con SDS (SDS-PAGE). Los geles separadores empleados fueron al 8 y 10%, gel espaciador al 4% y la tinción se efectuó con azul commassie. Los diversos patrones proteicos reflejaron diferencias genéticas entre ambas especies que se pueden deber a las diferencias de hábitat y de reproducción.

Molecular modelling of organic functional materials

The investigation of the mechanisms lying behind the (photo-)chemical processes is fundamental to address and improve the design of new organic functional materials. In many cases, dynamics simulations represent the only tool to capture the system properties emerging from complex interactions between many molecules. Despite the outstanding progresses in calculation power, the only way to carry out such computational studies is to introduce several approximations with respect to a fully quantum mechanical (QM) description. This thesis presents an approach that combines QM calculations with a classical Molecular Dynamics (MD) approach by means of accurate QM-derived force fields. It is based on a careful selection of the most relevant molecular degrees of freedom, whose potential energy surface is calculated at QM level and reproduced by the analytic functions of the force field, as well as by an accurate tuning of the approximations introduced in the model of the process to be simulated. This is made possible by some tools developed purposely, that allow to obtain and test the FF parameters through comparison with the QM frequencies and normal modes. These tools were applied in the modelling of three processes: the npi* photoisomerisation of azobenzene, where the FF description was extended to the excited state too and the non-adiabatic events were treated stochastically with Tully fewest switching algorithm; the charge separation in donors-acceptors bulk heterojunction organic solar cells, where a tight-binding Hamiltonian was carefully parametrised and solved by means of a code, also written specifically; the effect of the protonation state on the photoisomerisation quantum yield of the aryl-azoimidazolium unit of the axle molecule of a rotaxane molecular shuttle. In each case, the QM-based MD models that were specifically developed gave noteworthy information about the investigated phenomena, proving to be a fundamental key for a deeper comprehension of several experimental evidences.

Interference mitigation techniques for cloud-RAN deployments in 5G and beyond systems

The deployment of ultra-dense networks is one of the most promising solutions to manage the phenomenon of co-channel interference that affects the latest wireless communication systems, especially in hotspots. To meet the requirements of the use-cases and the immense amount of traffic generated in these scenarios, 5G ultra-dense networks are being deployed using various technologies, such as distributed antenna system (DAS) and cloud-radio access network (C-RAN). Through these centralized densification schemes, virtualized baseband processing units coordinate the distributed access points and manage the available network resources. In particular, link adaptation techniques are shown to be fundamental to overall system operation and performance enhancement. The core of this dissertation is the result of an analysis and a comparison of dynamic and adaptive methods for modulation and coding scheme (MCS) selection applied to the latest mobile telecommunications standards. A novel algorithm based on the proportional-integral-derivative (PID) controller principles and block error rate (BLER) target has been proposed. Tests were conducted in a 4G and 5G system level laboratory and, by means of a channel emulator, the performance was evaluated for different channel models and target BLERs. Furthermore, due to the intrinsic sectorization of the end-users distribution in the investigated scenario, a preliminary analysis on the joint application of users grouping algorithms with multi-antenna and multi-user techniques has been performed. In conclusion, the importance and impact of other fundamental physical layer operations, such as channel estimation and power control, on the overall end-to-end system behavior and performance were highlighted.

AGV Safety Areas Obstacle Detection and Interaction with Point Cloud Derived Map

This thesis project aims to the development of an algorithm for the obstacle detection and the interaction between the safety areas of an Automated Guided Vehicles (AGV) and a Point Cloud derived map inside the context of a CAD software. The first part of the project focuses on the implementation of an algorithm for the clipping of general polygons, with which has been possible to: construct the safety areas polygon, derive the sweep of this areas along the navigation path performing a union and detect the intersections with line or polygon representing the obstacles. The second part is about the construction of a map in terms of geometric entities (lines and polygons) starting from a point cloud given by the 3D scan of the environment. The point cloud is processed using: filters, clustering algorithms and concave/convex hull derived algorithms in order to extract line and polygon entities representing obstacles. Finally, the last part aims to use the a priori knowledge of possible obstacle detections on a given segment, to predict the behavior of the AGV and use this prediction to optimize the choice of the vehicle's assigned velocity in that segment, minimizing the travel time.

Dna Extraction From Filter-paper Spots Of Vaginal Samples Collected After Sexual Violence.

To detect the presence of male DNA in vaginal samples collected from survivors of sexual violence and stored on filter paper. A pilot study was conducted to evaluate 10 vaginal samples spotted on sterile filter paper: 6 collected at random in April 2009 and 4 in October 2010. Time between sexual assault and sample collection was 4-48hours. After drying at room temperature, the samples were placed in a sterile envelope and stored for 2-3years until processing. DNA extraction was confirmed by polymerase chain reaction for human β-globin, and the presence of prostate-specific antigen (PSA) was quantified. The presence of the Y chromosome was detected using primers for sequences in the TSPY (Y7/Y8 and DYS14) and SRY genes. β-Globin was detected in all 10 samples, while 2 samples were positive for PSA. Half of the samples amplified the Y7/Y8 and DYS14 sequences of the TSPY gene and 30% amplified the SRY gene sequence of the Y chromosome. Four male samples and 1 female sample served as controls. Filter-paper spots stored for periods of up to 3years proved adequate for preserving genetic material from vaginal samples collected following sexual violence.

Molecular And Biochemical Characterization Of Caffeine Synthase And Purine Alkaloid Concentration In Guarana Fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

Advancing Bag-of-visual-words Representations For Lesion Classification In Retinal Images.

Diabetic Retinopathy (DR) is a complication of diabetes that can lead to blindness if not readily discovered. Automated screening algorithms have the potential to improve identification of patients who need further medical attention. However, the identification of lesions must be accurate to be useful for clinical application. The bag-of-visual-words (BoVW) algorithm employs a maximum-margin classifier in a flexible framework that is able to detect the most common DR-related lesions such as microaneurysms, cotton-wool spots and hard exudates. BoVW allows to bypass the need for pre- and post-processing of the retinographic images, as well as the need of specific ad hoc techniques for identification of each type of lesion. An extensive evaluation of the BoVW model, using three large retinograph datasets (DR1, DR2 and Messidor) with different resolution and collected by different healthcare personnel, was performed. The results demonstrate that the BoVW classification approach can identify different lesions within an image without having to utilize different algorithms for each lesion reducing processing time and providing a more flexible diagnostic system. Our BoVW scheme is based on sparse low-level feature detection with a Speeded-Up Robust Features (SURF) local descriptor, and mid-level features based on semi-soft coding with max pooling. The best BoVW representation for retinal image classification was an area under the receiver operating characteristic curve (AUC-ROC) of 97.8% (exudates) and 93.5% (red lesions), applying a cross-dataset validation protocol. To assess the accuracy for detecting cases that require referral within one year, the sparse extraction technique associated with semi-soft coding and max pooling obtained an AUC of 94.2 ± 2.0%, outperforming current methods. Those results indicate that, for retinal image classification tasks in clinical practice, BoVW is equal and, in some instances, surpasses results obtained using dense detection (widely believed to be the best choice in many vision problems) for the low-level descriptors.

On The Distinct Molecular Architectures Of Dipping- And Spray-lbl Films Containing Lipid Vesicles.

The introduction of spraying procedures to fabricate layer-by-layer (LbL) films has brought new possibilities for the control of molecular architectures and for making the LbL technique compliant with industrial processes. In this study we show that significantly distinct architectures are produced for dipping and spray-LbL films of the same components, which included DODAB/DPPG vesicles. The films differed notably in their thickness and stratified nature. The electrical response of the two types of films to aqueous solutions containing erythrosin was also different. With multidimensional projections we showed that the impedance for the DODAB/DPPG spray-LbL film is more sensitive to changes in concentration, being therefore more promising as sensing units. Furthermore, with surface-enhanced Raman scattering (SERS) we could ascribe the high sensitivity of the LbL films to adsorption of erythrosin.

A New Extraction Approach To Correct The Effect Of Apparent Increase In The Secoiridoid Content After Filtration Of Virgin Olive Oil.

In the current study, a new approach has been developed for correcting the effect that moisture reduction after virgin olive oil (VOO) filtration exerts on the apparent increase of the secoiridoid content by using an internal standard during extraction. Firstly, two main Spanish varieties (Picual and Hojiblanca) were submitted to industrial filtration of VOOs. Afterwards, the moisture content was determined in unfiltered and filtered VOOs, and liquid-liquid extraction of phenolic compounds was performed using different internal standards. The resulting extracts were analyzed by HPLC-ESI-TOF/MS, in order to gain maximum information concerning the phenolic profiles of the samples under study. The reduction effect of filtration on the moisture content, phenolic alcohols, and flavones was confirmed at the industrial scale. Oleuropein was chosen as internal standard and, for the first time, the apparent increase of secoiridoids in filtered VOO was corrected, using a correction coefficient (Cc) calculated from the variation of internal standard area in filtered and unfiltered VOO during extraction. This approach gave the real concentration of secoiridoids in filtered VOO, and clarified the effect of the filtration step on the phenolic fraction. This finding is of great importance for future studies that seek to quantify phenolic compounds in VOOs.

Polyphenol Extraction Optimisation From Ceylon Gooseberry (dovyalis Hebecarpa) Pulp.

Originally from Asia, Dovyalis hebecarpa is a dark purple/red exotic berry now also produced in Brazil. However, no reports were found in the literature about phenolic extraction or characterisation of this berry. In this study we evaluate the extraction optimisation of anthocyanins and total phenolics in D. hebecarpa berries aiming at the development of a simple and mild analytical technique. Multivariate analysis was used to optimise the extraction variables (ethanol:water:acetone solvent proportions, times, and acid concentrations) at different levels. Acetone/water (20/80 v/v) gave the highest anthocyanin extraction yield, but pure water and different proportions of acetone/water or acetone/ethanol/water (with >50% of water) were also effective. Neither acid concentration nor time had a significant effect on extraction efficiency allowing to fix the recommended parameters at the lowest values tested (0.35% formic acid v/v, and 17.6 min). Under optimised conditions, extraction efficiencies were increased by 31.5% and 11% for anthocyanin and total phenolics, respectively as compared to traditional methods that use more solvent and time. Thus, the optimised methodology increased yields being less hazardous and time consuming than traditional methods. Finally, freeze-dried D. hebecarpa showed high content of target phytochemicals (319 mg/100g and 1,421 mg/100g of total anthocyanin and total phenolic content, respectively).

The Crystal-t Algorithm: A New Approach To Calculate The Sle Of Lipidic Mixtures Presenting Solid Solutions.

Lipidic mixtures present a particular phase change profile highly affected by their unique crystalline structure. However, classical solid-liquid equilibrium (SLE) thermodynamic modeling approaches, which assume the solid phase to be a pure component, sometimes fail in the correct description of the phase behavior. In addition, their inability increases with the complexity of the system. To overcome some of these problems, this study describes a new procedure to depict the SLE of fatty binary mixtures presenting solid solutions, namely the Crystal-T algorithm. Considering the non-ideality of both liquid and solid phases, this algorithm is aimed at the determination of the temperature in which the first and last crystal of the mixture melts. The evaluation is focused on experimental data measured and reported in this work for systems composed of triacylglycerols and fatty alcohols. The liquidus and solidus lines of the SLE phase diagrams were described by using excess Gibbs energy based equations, and the group contribution UNIFAC model for the calculation of the activity coefficients of both liquid and solid phases. Very low deviations of theoretical and experimental data evidenced the strength of the algorithm, contributing to the enlargement of the scope of the SLE modeling.

Development And Characterization Of Microsatellite Loci For Ocotea Species (lauraceae) Threatened With Extinction.

The Atlantic rainforest species Ocotea catharinensis, Ocotea odorifera, and Ocotea porosa have been extensively harvested in the past for timber and oil extraction and are currently listed as threatened due to overexploitation. To investigate the genetic diversity and population structure of these species, we developed 8 polymorphic microsatellite markers for O. odorifera from an enriched microsatellite library by using 2 dinucleotide repeats. The microsatellite markers were tested for cross-amplification in O. catharinensis and O. porosa. The average number of alleles per locus was 10.2, considering all loci over 2 populations of O. odorifera. Observed and expected heterozygosities for O. odorifera ranged from 0.39 to 0.93 and 0.41 to 0.92 across populations, respectively. Cross-amplification of all loci was successfully observed in O. catharinensis and O. porosa except 1 locus that was found to lack polymorphism in O. porosa. Combined probabilities of identity in the studied Ocotea species were very low ranging from 1.0 x 10-24 to 7.7 x 10-24. The probability of exclusion over all loci estimated for O. odorifera indicated a 99.9% chance of correctly excluding a random nonparent individual. The microsatellite markers described in this study have high information content and will be useful for further investigations on genetic diversity within these species and for subsequent conservation purposes.

Albumin Is Synthesized In Epididymis And Aggregates In A High Molecular Mass Glycoprotein Complex Involved In Sperm-egg Fertilization.

The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.