Automated analysis of quantitative image data using isomorphic functional mixed models, with application to proteomics data

Image data are increasingly encountered and are of growing importance in many areas of science. Much of these data are quantitative image data, which are characterized by intensities that represent some measurement of interest in the scanned images. The data typically consist of multiple images on the same domain and the goal of the research is to combine the quantitative information across images to make inference about populations or interventions. In this paper we present a unified analysis framework for the analysis of quantitative image data using a Bayesian functional mixed model approach. This framework is flexible enough to handle complex, irregular images with many local features, and can model the simultaneous effects of multiple factors on the image intensities and account for the correlation between images induced by the design. We introduce a general isomorphic modeling approach to fitting the functional mixed model, of which the wavelet-based functional mixed model is one special case. With suitable modeling choices, this approach leads to efficient calculations and can result in flexible modeling and adaptive smoothing of the salient features in the data. The proposed method has the following advantages: it can be run automatically, it produces inferential plots indicating which regions of the image are associated with each factor, it simultaneously considers the practical and statistical significance of findings, and it controls the false discovery rate.Comment: Published in at http://dx.doi.org/10.1214/10-AOAS407 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

A robust high-sensitivity algorithm for automated detection of proteins in two-dimensional electrophoresis gels

The automated interpretation of two-dimensional gel electrophoresis images used in protein separation and analysis presents a formidable problem in the detection and characterization of ill-defined spatial objects. We describe in this paper a hierarchical algorithm that provides a robust, high-sensitivity solution to this problem, which can be easily adapted to a variety of experimental situations. The software implementation of this algorithm functions as part of a complete package designed for general protein gel analysis applications

Investigation of the effects of weight loss on Ovis aries muscle: a proteomic study on three breeds with different levels of adaptation to nutritional stress

Tese de mestrado. Biologia (Biologia Celular e Biotecnologia). Universidade de Lisboa, Faculdade de Ciências, 2011Nos trópicos, a produção animal em regime extensivo enfrenta grandes obstáculos, talvez o maior dos quais é a perda de peso sazonal, devida à época seca existente nos países tropicais com uma pluviosidade muito baixa ou até mesmo inexistente. Em muitos países tropicais, a produção animal foca-se sobretudo na ovelha e, principalmente nos sistemas tradicionais, recorre ao uso de espécies nativas pois estas, duma forma geral, apresentam maior resistência às condições ambientais comparativamente com as espécies seleccionadas. Factores importantes para a escolha das espécies nativas sobre as seleccionadas são a tolerância a longos períodos sem alimento nem água, às altas temperaturas e a insectos causadores de doenças, como por exemplo a miíase – designação geral para doença parasitária causada pela larva de moscas ou outros dípteros. A escassez de alimento leva a que o animal perca peso, podendo atingir uma diminuição de peso na ordem de 40% em ovelhas. É evidente a necessidade para desenvolvimentos nas áreas da produção animal para evitar as quebras na produção e rendimento. Na Austrália, uma medida para reduzir essa diminuição de produção passa pelo progressivo abandono das raças e sistemas de produção tradicionais e pela adopção de raças ‘alternativas’. Um típico exemplo é a passagem de sistemas de produção de lã para sistemas de produção de carne. Os sistemas de produção de lã, normalmente com apenas a raça Merino, estão numa tendência de abandono, devido principalmente aos custos elevados de manutenção e mão-de-obra bem como à diminuição do valor da lã nos mercados internacionais. Sistemas de produção de lã baseados em Merino passam a ser sistemas de produção de carne com raças de pêlo, como por exemplo as raças Dorper e Damara, as raças ‘alternativas’ mais importantes na Austrália. Estas três raças apresentam diferenças ao nível da tolerância à escassez de alimento: a Merino, sendo uma raça oriunda de selecção, apresenta o nível mais baixo de tolerância; a Dorper apresenta o nível de tolerância intermédio; a Damara é a raça mais tolerante, das três, à escassez de alimento. Dado que a escassez de alimento conduzirá eventualmente à perda de peso, a perda de peso deve-se essencialmente pela utilização das reservas energéticas do corpo do animal: primeiro há consumo do tecido adiposo com reduzido consumo proteico, seguindo-se um aumento da degradação das proteínas, é de esperar que haja diferenças na expressão das proteínas entre um animal bem alimentado e outro em perda de peso (subalimentado). Assim, este trabalho pretende identificar essas proteínas cuja expressão varia entre condições de favoráveis e desfavoráveis ao ganho de peso. Para tal recorreu-se à técnica de Electroforese Bidimensional usando 6 grupos experimentais, grupo de controlo e grupo com subnutrição para cada uma das três raças, com quatro réplicas biológicas, amostras de quatro animais diferentes por grupo experimental. Obtiveram-se assim 24 géis, com um total de 1228 spots, dos quais apenas 22 eram estatisticamente significativos (p<0.05) e tinham um poder de pelo menos 0,8. Todos os 22 spots foram seleccionados para identificação, em que 16 foram identificados com sucesso. Estas 16 proteínas distribuem-se, de acordo com a sua função, por proteínas do metabolismo (23%), proteínas estruturais (18%) e proteínas do aparelho contráctil (32%).In tropical countries, extensive ruminant production faces several constraints, of which the most important is seasonal weight loss, which is caused by a dry season with very low rain levels or no rain at all. In the majority of these countries, the animal most used for production is sheep, namely indigenous breeds, as these breeds can, generally speaking, endure harsher environment conditions than selected breeds. Being able to better tolerate lack of food and water is a very important feature an animal should have regarding extensive production systems. In Australia, currently, the sheep industry is mainly focused on the Merino breed. This breed, introduced in Australia by the Europeans and a derivative from the European breed, i.e. a selected breed, is wool breed and as such is mainly used for wool production, with very few animals being used for meat. This, however, creates a problem for these industries: with the decrease in price of wool and the increase of labour costs, it is becoming less and less economically viable to produce Merino for wool. Because of this, increasing number of producers are looking for alternative breeds that can better withstand the lack of food and tolerate some diseases that affect greatly Merino sheep. Two breeds are standing out due to their ability to withstand prolonged undernutrition: the Damara and the Dorper. These two breeds have high and intermediate tolerance to undernutrition, respectively, while the Merino have very low tolerance to undernutrition. If the undernutrition, and the associated weight loss, is long enough, there will be protein break down in response to the need for energy to maintain the animal’s metabolism. In regard to the interested in selecting traits like weight loss resistance for increased animal production rates, this work aims at providing information about protein markers which may be used to access tolerance to undernutrition and be used as a tool to better select the desired traits in the animals. A proteomic approach of Two-Dimensional Gel Electrophoresis was used to compare gastrocnemius muscle protein expression patterns between six experimental groups: three sheep breeds (Damara, Dorper and Australian Merino) grouped in a control (not feed restricted) and a feed restricted group; samples from four animals for each group were used. A total of 24 gels was obtained. After staining and analysis, 22 spots were detected as significantly (p<0.05) different and with a power of at least 0.8. All spots were selected for identification, 16 were successfully identified, with the identified proteins ranging from structural, metabolic and contractile apparatus roles

Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models.

To-date, most invasion or migration assays use a modified Boyden chamber-like design to assess migration as single-cell or scratch assays on coated or uncoated planar plastic surfaces. Here, we describe a 96-well microplate-based, high-content, three-dimensional cell culture assay capable of assessing invasion dynamics and molecular signatures thereof. On applying our invasion assay, we were able to demonstrate significant effects on the invasion capacity of fibroblast cell lines, as well as primary lung fibroblasts. Administration of epidermal growth factor resulted in a substantial increase of cellular invasion, thus making this technique suitable for high-throughput pharmacological screening of novel compounds regulating invasive and migratory pathways of primary cells. Our assay also correlates cellular invasiveness to molecular events. Thus, we argue of having developed a powerful and versatile toolbox for an extensive profiling of invasive cells in a 96-well format. This will have a major impact on research in disease areas like fibrosis, metastatic cancers, or chronic inflammatory states

Quantitative transcription factor binding kinetics at the single-molecule level

We have investigated the binding interaction between the bacteriophage lambda repressor CI and its target DNA using total internal reflection fluorescence microscopy. Large, step-wise changes in the intensity of the red fluorescent protein fused to CI were observed as it associated and dissociated from individually labeled single molecule DNA targets. The stochastic association and dissociation were characterized by Poisson statistics. Dark and bright intervals were measured for thousands of individual events. The exponential distribution of the intervals allowed direct determination of the association and dissociation rate constants, ka and kd respectively. We resolved in detail how ka and kd varied as a function of 3 control parameters, the DNA length L, the CI dimer concentration, and the binding affinity. Our results show that although interaction with non-operator DNA sequences are observable, CI binding to the operator site is not dependent on the length of flanking non-operator DNA.Comment: 34 pages, 10 figures, accepted by Biophysical Journa

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time

We present a single-molecule tool called the CoPro (Concentration of Proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrates its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a 4-fold shift towards higher values in concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, which is almost exclusively localized in the nucleus under high and..

The influence of microgravity on invasive growth in Saccharomyces cerevisiae

This study investigates the effects of microgravity on colony growth and the morphological transition from single cells to short invasive filaments in the model eukaryotic organism Saccharomyces cerevisiae. Two-dimensional spreading of the yeast colonies grown on semi-solid agar medium was reduced under microgravity in the Sigma 1278b laboratory strain but not in the CMBSESA1 industrial strain. This was supported by the Sigma 1278b proteome map under microgravity conditions, which revealed upregulation of proteins linked to anaerobic conditions. The Sigma 1278b strain showed a reduced invasive growth in the center of the yeast colony. Bud scar distribution was slightly affected, with a switch toward more random budding. Together, microgravity conditions disturb spatially programmed budding patterns and generate strain-dependent growth differences in yeast colonies on semi-solid medium