Polyol transport in yeast: studying transport proteins and manipulating osmotic responses

The Saccharomyces cerevisiae MIP channel Fps1p plays an important role in yeast osmoregulation by exporting glycerol. Glycerol accumulates in the cell as a compatible osmolyte during hyperosmotic conditions and is exported once conditions become hypotonic. A gpd1 gpd2 mutant is unable to produce glycerol and is therefore very sensitive to high concentrations of polyols in the growth medium. The sensitivity to C3, C4 and C5, but not C6 polyols, is suppressed by expression of truncated, hyperactive Fps1p. This is because the polyols can then equilibrate over the membrane and hence the concentration gradient collapses. This experiments reveals the substrate spectrum of Fps1p. The system can be used in different ways. For instance, growth assays on different polyols elucidate the substrate range of heterologous channels such as that of the rat aquaglyceroporin AQP9. In addition, the same system is used to search for novel hyperactive mutants of Fps1p, which provide additional information on the mechanism underlying channel regulation. Finally we illustrate that the gpd1 gpd2 double mutant expressing hyperactive Fps1p can be used to manipulate activation and deactivation of the HOG pathway, contributing to our understanding of the control of this osmoregulatory system.

Formulation of topical non-steroidal anti-inflammatory agents

Reversed-pahse high-performance liquid chromatographic (HPLC) methods were developed for the assay of indomethacin, its decomposition products, ibuprofen and its (tetrahydro-2-furanyl)methyl-, (tetrahydro-2-(2H)pyranyl)methyl- and cyclohexylmethyl esters. The development and application of these HPLC systems were studied. A number of physico-chemical parameters that affect percutaneous absorption were investigated. The pKa values of indomethacin and ibuprofen were determined using the solubility method. Potentiometric titration and the Taft equation were also used for ibuprofen. The incorporation of ethanol or propylene glycol in the solvent resulted in an improvement in the aqueous solubility of these compounds. The partition coefficients were evaluated in order to establish the affinity of these drugs towards the stratum corneum. The stability of indomethacin and of ibuprofen esters were investigated and the effect of temperature and pH on the decomposition rates were studied. The effect of cetyltrimethylammonium bromide on the alkaline degradation of indomethacin was also followed. In the presence of alcohol, indomethacin alcoholysis was observed and the kinetics of decomposition were subjected to non-linear regression analysis and the rate constants for the various pathways were quantified. The non-isothermal, sufactant non-isoconcentration and non-isopH degradation of indomethacin were investigated. The analysis of the data was undertaken using NONISO, a BASIC computer program. The degradation profiles obtained from both non-iso and iso-kinetic studies show that there is close concordance in the results. The metabolic biotransformation of ibuprofen esters was followed using esterases from hog liver and rat skin homogenates. The results showed that the esters were very labile under these conditions. The presence of propylene glycol affected the rates of enzymic hydrolysis of the ester. The hydrolysis is modelled using an equation involving the dielectric constant of the medium. The percutaneous absorption of indomethacin and of ibuprofen and its esters was followed from solutions using an in vitro excised human skin model. The absorption profiles followed first order kinetics. The diffusion process was related to their solubility and to the human skin/solvent partition coefficient. The percutaneous absorption of two ibuprofen esters from suspensions in 20% propylene glycol-water were also followed through rat skin with only ibuprofen being detected in the receiver phase. The sensitivity of ibuprofen esters to enzymic hydrolysis compared to the chemical hydrolysis may prove valuable in the formulation of topical delivery systems.

Conditional osmotic stress in yeast:a system to study transport through aquaglyceroporins and osmostress signaling

The accumulation and transport of solutes are hallmarks of osmoadaptation. In this study we have employed the inability of the Saccharomyces cerevisiae gpd1Δ gpd2Δ mutant both to produce glycerol and to adapt to high osmolarity to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling. High levels of different polyols, including glycerol, inhibited growth of the gpd1Δ gpd2Δ mutant. This growth inhibition was suppressed by expression of the hyperactive allele Fps1-AΔ of the osmogated yeast aquaglyceroporin, Fps1. The degree of suppression correlated with the relative rate of transport of the different polyols tested. Transport studies in secretory vesicles confirmed that Fps1-Δ1 transports polyols at increased rates compared with wild type Fps1. Importantly, wild type Fps1 and Fps1-Δ1 showed similarly low permeability for water. The growth defect on polyols in the gpd1Δ gpd2Δ mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9. We surmised that this suppression was due to polyol influx, causing the cells to passively adapt to the stress. Indeed, when aquaglyceroporin-expressing gpd1Δ gpd2Δ mutants were treated with glycerol, xylitol, or sorbitol, the osmosensing HOG pathway was activated, and the period of activation correlated with the apparent rate of polyol uptake. This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation. Taken together, our "conditional" osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

LCD-OpenPACS: sistema integrado de telerradiologia com auxílio ao diagnóstico de nódulos pulmonares em exames de tomografia computadorizada

Lung cancer is one of the most common types of cancer and has the highest mortality rate. Patient survival is highly correlated with early detection. Computed Tomography technology services the early detection of lung cancer tremendously by offering aminimally invasive medical diagnostic tool. However, the large amount of data per examination makes the interpretation difficult. This leads to omission of nodules by human radiologist. This thesis presents a development of a computer-aided diagnosis system (CADe) tool for the detection of lung nodules in Computed Tomography study. The system, called LCD-OpenPACS (Lung Cancer Detection - OpenPACS) should be integrated into the OpenPACS system and have all the requirements for use in the workflow of health facilities belonging to the SUS (Brazilian health system). The LCD-OpenPACS made use of image processing techniques (Region Growing and Watershed), feature extraction (Histogram of Gradient Oriented), dimensionality reduction (Principal Component Analysis) and classifier (Support Vector Machine). System was tested on 220 cases, totaling 296 pulmonary nodules, with sensitivity of 94.4% and 7.04 false positives per case. The total time for processing was approximately 10 minutes per case. The system has detected pulmonary nodules (solitary, juxtavascular, ground-glass opacity and juxtapleural) between 3 mm and 30 mm.

A COMUNICAÇÃO MERCADOLÓGICA E A APROPRIAÇÃO DE UM ESTILO DE VIDA – ESTUDO DE CASO DA MARCA HARLEY DAVIDSON NO BRASIL

Este estudo busca entender como um processo de comunicação mercadológica pode vincular na mente de seus consumidores elementos simbólicos sobre estilo de vida e pertencimento a uma comunidade da marca alimentando o imaginário de seus consumidores por meio de uma filosofia de vida e forma de viver muito particular. A argumentação da marca é regida em relação ao pertencimento de seus consumidores a uma "tribo", e o seu discurso aparenta ser semelhante em todos os cantos do mundo. O caso escolhido para estudo foi a Harley Davidson, pois é uma marca que integra todos esses elementos. Os procedimentos metodológicos escolhidos para esta dissertação foram pesquisa bibliográfica e documental, que contribuíram para a formação do conhecimento teórico e informacional. Além disso, uma pesquisa de campo subdividida em: observação participante com os consumidores da cidade de Sorocaba; participantes do HOG, que é o moto clube exclusivo da marca, coleta de entrevistas semiestruturadas com os membros do HOG de Sorocaba, concessionário Sorocaba e funcionários da empresa no Brasil. Por meio desse trabalho percebeu-se que a comunicação mercadológica e suas ações vêm ao encontro das aspirações, sensações e desejos dos consumidores e como eles se identificam com a marca e com o estilo de vida proporcionado por ela. Os consumidores Harley Davidson são grandes entusiastas da marca e da sua filosofia de vida.

Learning and Exploiting Camera Geometry for Computer Vision

This work explores the use of statistical methods in describing and estimating camera poses, as well as the information feedback loop between camera pose and object detection. Surging development in robotics and computer vision has pushed the need for algorithms that infer, understand, and utilize information about the position and orientation of the sensor platforms when observing and/or interacting with their environment.

The first contribution of this thesis is the development of a set of statistical tools for representing and estimating the uncertainty in object poses. A distribution for representing the joint uncertainty over multiple object positions and orientations is described, called the mirrored normal-Bingham distribution. This distribution generalizes both the normal distribution in Euclidean space, and the Bingham distribution on the unit hypersphere. It is shown to inherit many of the convenient properties of these special cases: it is the maximum-entropy distribution with fixed second moment, and there is a generalized Laplace approximation whose result is the mirrored normal-Bingham distribution. This distribution and approximation method are demonstrated by deriving the analytical approximation to the wrapped-normal distribution. Further, it is shown how these tools can be used to represent the uncertainty in the result of a bundle adjustment problem.

Another application of these methods is illustrated as part of a novel camera pose estimation algorithm based on object detections. The autocalibration task is formulated as a bundle adjustment problem using prior distributions over the 3D points to enforce the objects' structure and their relationship with the scene geometry. This framework is very flexible and enables the use of off-the-shelf computational tools to solve specialized autocalibration problems. Its performance is evaluated using a pedestrian detector to provide head and foot location observations, and it proves much faster and potentially more accurate than existing methods.

Finally, the information feedback loop between object detection and camera pose estimation is closed by utilizing camera pose information to improve object detection in scenarios with significant perspective warping. Methods are presented that allow the inverse perspective mapping traditionally applied to images to be applied instead to features computed from those images. For the special case of HOG-like features, which are used by many modern object detection systems, these methods are shown to provide substantial performance benefits over unadapted detectors while achieving real-time frame rates, orders of magnitude faster than comparable image warping methods.

The statistical tools and algorithms presented here are especially promising for mobile cameras, providing the ability to autocalibrate and adapt to the camera pose in real time. In addition, these methods have wide-ranging potential applications in diverse areas of computer vision, robotics, and imaging.

Molecular docking and geographical information systems as tools to assess the potential impact of veterinary medicines on non-target organisms and the environment

Veterinary medicines (VMs) from agricultural industry can enter the environment in a number of ways. This includes direct exposure through aquaculture, accidental spillage and disposal, and indirect entry by leaching from manure or runoff after treatment. Many compounds used in animal treatments have ecotoxic properties that may have chronic or sometimes lethal effects when they come into contact with non-target organisms. VMs enter the environment in mixtures, potentially having additive effects. Traditional ecotoxicology tests are used to determine the lethal and sometimes reproductive effects on freshwater and terrestrial organisms. However, organisms used in ecotoxicology tests can be unrepresentative of the populations that are likely to be exposed to the compound in the environment. Most often the tests are on single compound toxicity but mixture effects may be significant and should be included in ecotoxicology testing. This work investigates the use, measured environmental concentrations (MECs) and potential impact of sea lice treatments on salmon farms in Scotland. Alternative methods for ecotoxicology testing including mixture toxicity, and the use of in silico techniques to predict the chronic impact of VMs on different species of aquatic organisms were also investigated. The Scottish Environmental Protection Agency (SEPA) provided information on the use of five sea lice treatments from 2008-2011 on Scottish salmon farms. This information was combined with the recently available data on sediment MECs for the years 2009-2012 provided by SEPA using ArcGIS 10.1. In depth analysis of this data showed that from a total of 55 sites, 30 sites had a MEC higher than the maximum allowable concentration (MAC) as set out by SEPA for emamectin benzoate and 7 sites had a higher MEC than MAC for teflubenzuron. A number of sites that were up to 16 km away from the nearest salmon farm reported as using either emamectin benzoate or teflubenzuron measured positive for the two treatments. There was no relationship between current direction and the distribution of the sea lice treatments, nor was there any evidence for alternative sources of the compounds e.g. land treatments. The sites that had MECs higher than the MAC could pose a risk to non-target organisms and disrupt the species dynamics of the area. There was evidence that some marine protected sites might be at risk of exposure to these compounds. To complement this work, effects on acute mixture toxicity of the 5 sea lice treatments, plus one major metabolite 3-phenoxybenzoic acid (3PBA), were measured using an assay using the bioluminescent bacteria Aliivibrio fischeri. When exposed to the 5 sea lice treatments and 3PBA A. fischeri showed a response to 3PBA, emamectin benzoate and azamethiphos as well as combinations of the three. In order to establish any additive effect of the sea lice treatments, the efficacy of two mixture prediction equations, concentration addition (CA) and independent action ii(IA) were tested using the results from single compound dose response curves. In this instance IA was the more effective prediction method with a linear regression confidence interval of 82.6% compared with 22.6% of CA. In silico molecular docking was carried out to predict the chronic effects of 15 VMs (including the five used as sea lice control). Molecular docking has been proposed as an alternative screening method for the chronic effects of large animal treatments on non-target organisms. Oestrogen receptor alpha (ERα) of 7 non-target bony fish and the African clawed frog Xenopus laevis were modelled using SwissModel. These models were then ‘docked’ to oestradiol, the synthetic oestrogen ethinylestradiol, two known xenoestrogens dichlorodiphenyltrichloroethane (DDT) and bisphenol A (BPA), the antioestrogen breast cancer treatment tamoxifen and 15 VMs using Auto Dock 4. Based on the results of this work, four VMs were identified as being possible xenoestrogens or anti-oestrogens; these were cypermethrin, deltamethrin, fenbendazole and teflubenzuron. Further investigation, using in vitro assays, into these four VMs has been suggested as future work. A modified recombinant yeast oestrogen screen (YES) was attempted using the cDNA of the ERα of the zebrafish Danio rerio and the rainbow trout Oncorhynchus mykiss. Due to time and difficulties in cloning protocols this work was unable to be completed. Use of such in vitro assays would allow for further investigation of the highlighted VMs into their oestrogenic potential. In conclusion, VMs used as sea lice treatments, such as teflubenzuron and emamectin benzoate may be more persistent and have a wider range in the environment than previously thought. Mixtures of sea lice treatments have been found to persist together in the environment, and effects of these mixtures on the bacteria A. fischeri can be predicted using the IA equation. Finally, molecular docking may be a suitable tool to predict chronic endocrine disrupting effects and identify varying degrees of impact on the ERα of nine species of aquatic organisms.

Survival or death: a dual role of autophagy in stress-induced pericyte loss in diabetic retinopathy

Aims/hypothesis
Intra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects.
Methods
Immunohistochemistry was used to detect autophagy marker LC3B in human and murine diabetic and non-diabetic retinas. Cultured human retinal capillary pericytes (HRCPs) were treated with in vitro-modified heavily-oxidised glycated LDL (HOG-LDL) vs native LDL (N-LDL) with or without autophagy modulators: green fluorescent protein–LC3 transfection; small interfering RNAs against Beclin-1, c-Jun NH(2)-terminal kinase (JNK) and C/EBP-homologous protein (CHOP); autophagy inhibitor 3-MA (5 mmol/l) and/or caspase inhibitor Z-VAD-fmk (100 μmol/l). Autophagy, cell viability, oxidative stress, endoplasmic reticulum stress, JNK activation, apoptosis and CHOP expression were assessed by western blots, CCK-8 assay and TUNEL assay. Finally, HOG-LDL vs N-LDL were injected intravitreally to STZ-induced diabetic vs control rats (yielding 50 and 200 mg protein/l intravitreal concentration) and, after 7 days, retinas were analysed for ER stress, autophagy and apoptosis.
Results
Intra-retinal autophagy (LC3B staining) was increased in diabetic vs non-diabetic humans and mice. In HRCPs, 50 mg/l HOG-LDL elicited autophagy without altering cell viability, and inhibition of autophagy decreased survival. At 100–200 mg/l, HOG-LDL caused significant cell death, and inhibition of either autophagy or apoptosis improved survival. Further, 25–200 mg/l HOG-LDL dose-dependently induced oxidative and ER stress. JNK activation was implicated in autophagy but not in apoptosis. In diabetic rat retina, 50 mg/l intravitreal HOG-LDL elicited autophagy and ER stress but not apoptosis; 200 mg/l elicited greater ER stress and apoptosis.
Conclusions
Autophagy has a dual role in diabetic retinopathy: under mild stress (50 mg/l HOG-LDL) it is protective; under more severe stress (200 mg/l HOG-LDL) it promotes cell death.

Video anomaly detection in real-time on a Power-Aware Heterogeneous Platform

FPGAs and GPUs are often used when real-time performance in video processing is required. An accelerated processor is chosen based on task-specific priorities (power consumption, processing time and detection accuracy), and this decision is normally made once at design time. All three characteristics are important, particularly in battery-powered systems. Here we propose a method for moving selection of processing platform from a single design-time choice to a continuous run time one.We implement Histogram of Oriented Gradients (HOG) detectors for cars and people and Mixture of Gaussians (MoG) motion detectors running across FPGA, GPU and CPU in a heterogeneous system. We use this to detect illegally parked vehicles in urban scenes. Power, time and accuracy information for each detector is characterised. An anomaly measure is assigned to each detected object based on its trajectory and location, when compared to learned contextual movement patterns. This drives processor and implementation selection, so that scenes with high behavioural anomalies are processed with faster but more power hungry implementations, but routine or static time periods are processed with power-optimised, less accurate, slower versions. Real-time performance is evaluated on video datasets including i-LIDS. Compared to power-optimised static selection, automatic dynamic implementation mapping is 10% more accurate but draws 12W extra power in our testbed desktop system.

Seasonal changes in broodstock spawning performance and egg quality in ballan wrasse (Labrus bergylta)

Sea lice continue to be one of the largest issues for the salmon farming industry and the use of ballan wrasse (Labrus bergylta) as a biological control is considered to be one of the most sustainable solutions in development. Broodstock management has proved challenging in the initial phases due to the significant lack of understanding of basic reproductive physiology and behaviour in the species. The aim of the study was to monitor captive breeding populations throughout a spawning season to examine timing and duration of spawning,quantify egg production, and look at seasonal changes in egg quality parameters as well as investigate the parental contribution to spawning events. A clear spawning rhythm was shown with 3-5 spawning periods inclusive of spawning windows lasting 1-9 days followed by inter spawning intervals of 8-12 days. Fertilization rate remained consistently high (> 87.5%) over the spawning season and did not differ significantly between spawning populations. Hatch rate was variable (0-97.5 %), but peaked in the middle of the spawning season. Meanoocyte diameter and gum layer thickness decreased slightly over the spawning season with no significant differences between spawning populations. Fatty acid (FA) profile of eggs remained consistent throughout the season and with the exception of high levels of ARA (3.8 ± 0.5 % of total FA) the FA profile was similar to that observed in other marine fish species. Parental contribution analysis showed 3 out of 6 spawning events to be single paired mating while the remaining 3 had contributions from multiple parents. Furthermore, the proposed multiple batch spawning nature of this species was confirmed with proof of a single femalecontributing to two separate spawning events. Overall this work represents the first comprehensive data set of spawning activity of captive ballan wrasse, and as such and will be helpful in formulating sustainable broodstock management plans for the species.

Navegação de robô móvel em ambiente com humanos

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Mecânica

Pediculose

A relação parasita-hospedeiro entre o piolho e o Homem representa um dos maiores casos de sucesso de ectoparasitoses. Apesar de clinicamente a infestação por piolhos não provocar grandes danos á saúde o impacto social mental e económico é substancial. Não há programas que priorizem o controle de ectoparasitas na saúde. Uma crise emergente no controlo da pediculose, devido à falta de opções de novos pediculicidas, abriu caminho à eventualidade de resistências. A ocorrência de mutações, deve-se em grande parte ao facto do abuso indiscriminado e utilização de produtos que estavam a ser comercializados em ampla escala como Over-the-counter drugs (OTC), particularmente a permetrina e piretroides.

Aproveitamento de subprodutos da indústria de carne suína: caracterização físico-química do queijo de porco

Headcheese is a meat sausage originated from Europe made from hog slaughter by-products. It is a much appreciated product in the South of Brazil which is increasingly established in the market, however it does not have official regulations yet. This study aimed to present the physicochemical characterization of headcheese in a western Santa Catarina industry supervised by Companhia Integrada de Desenvolvimento Agrícola de Santa Catarina and assess 10 different brands to find the relationship between chemical composition and texture profile analysis (TPA). Thus, the chemical composition, energy value, total nitrite, lipid oxidation and physical parameters (color and texture) were evaluated. The product exhibited great variability in moisture content, lipid and protein because the different formulations, processing and intrinsic and extrinsic characteristics of raw material. The utilization of offal provided higher cholesterol and iron levels, and the high content of collagen was accountable for the shear force responses (7.84 ± 1.68 N). The product showed higher amount of sodium, due to the use of additives, but calcium levels were compatible with other sausages. There was a predominance of polyunsaturated fatty acids and polyunsaturated fatty acids/saturated fatty acids ratio was more favorable than other sausage in the same category. Nitrite assured preservation effects and thus lower product levels of oxidation were observed. The high Water Activity and pH 6.5 showed that the product is susceptible to growth of pathogens and requires cooling for preservation. Its brownish occurred due to cooking and production of metmyoglobin. There was a strong positive correlation between collagen and attributes of TPA, especially for chewiness (r = 0.855). The use of Hierarchical Cluster Analysis and Principal Component Analysis were able to separate three groups based on the amount of collagen and texture attributes, especially hardness, gumminess and chewiness.

Lindano vs. cipermetrina en el tratamiento de la pediculosis capitis

Se presentan los resultados de un estudio comparativo cuasi experimental de tipo clínico y a doble ciego, entre Lindano y Cipermetrina, en el tratamiento de la pediculosis cápitis, realizado en 150 niños de la Parroquia rural de Tarqui, de la escuela Alfonso Moreno Mora, en el que intervinieron 75 pacientes que corresponden al 50 por ciento, tratados con Lindano, y 75 pacientes que corresponden al 50 por ciento, tratados con Cipermetrina. En la muestra estuvieron presentes los siguiente diagnósticos: Pacientes con Liendres 81 que corresponden al 54 por ciento, pacientes con piojos 1 el 0.7 por ciento y pacientes con piojos y Liendres 68 que representan el 45.3 por ciento. Además se valoraron varios aspectos entre los cuales destacan: 1] El número de aplicaciones que necesitaron los niños para curar con cipermetrina y Lindano: Primera aplicación con Cipermetrina curaron 65, que representa el 43.33 por ciento, con Lindano 40 el 26.66 por ciento, segunda aplicación con Cipermetrina 10 el 6.67 por ciento, y con Lindano 34 el 22.67 por ciento, y tercera aplicación con Lindano 1 niño el 0.67 por ciento. 2) Efectos colaterales tales como: Lindano: a] Conjuntivitis: 2 niños 1.34 por ciento, b] Dermatitis: 5 niños 3.33 por ciento; Cipermetrina: a] Dermatitis: 2 niños el 1.33 por ciento

Literatura catalana infantil: continguts, metodologia i avaluació

En aquest article presentem els resultats de la reflexió, l’estudi i la discussió que s’ha produït sobre la metodologia docent de l’assignatura Literatura catalana infantil (17557), a partir dels nous enfocaments relacionats amb les TIC i les possibilitats que ofereix la xarxa, i de la recerca sobre altres experiències docents vinculades a l’ensenyament de la literatura, amb especial interès per l’Aprenentatge Basat en Projectes (sobre el qual hem organitzat, amb el suport de l’ICE, un curs de formació per al professorat del Departament de Filologia Catalana). La investigació sobre metodologies ha propiciat una revisió i modificació de l’avaluació de l’assignatura així com també d’alguns continguts específics, com ara la narrativa audiovisual, que s’incorporarà en forma de discussió als continguts relacionats amb el gèneres de la literatura infantil i juvenil. Al capdavall, el treball realitzat contribueix a generar un discurs propi sobre l’ensenyament de la literatura, especialment dirigit a l’educació primària i, per tant, incideix directament en la formació de l’alumnat de la Facultat d’Educació en la qual s’imparteix aquesta assignatura.