Two-Dimensional Gel Electrophoresis Image Registration Using Block-Matching Techniques and Deformation Models

[Abstract] Block-matching techniques have been widely used in the task of estimating displacement in medical images, and they represent the best approach in scenes with deformable structures such as tissues, fluids, and gels. In this article, a new iterative block-matching technique—based on successive deformation, search, fitting, filtering, and interpolation stages—is proposed to measure elastic displacements in two-dimensional polyacrylamide gel electrophoresis (2D–PAGE) images. The proposed technique uses different deformation models in the task of correlating proteins in real 2D electrophoresis gel images, obtaining an accuracy of 96.6% and improving the results obtained with other techniques. This technique represents a general solution, being easy to adapt to different 2D deformable cases and providing an experimental reference for block-matching algorithms.Galicia. Consellería de Economía e Industria; 10MDS014CTGalicia. Consellería de Economía e Industria; 10SIN105004PRInstituto de Salud Carlos III; PI13/0028

Computational Methods on Study of Differentially Expressed Proteins in Maize Proteomes Associated with Resistance to Aflatoxin Accumulation

Plant breeders have focused on improving maize resistance to Aspergillus flavus infection and aflatoxin accumulation by breeding with genotypes having the desirable traits. Various maize inbred lines have been developed for the breeding of resistance. Identification of differentially expressed proteins among such maize inbred lines will facilitate the development of gene markers and expedite the breeding process. Computational biology and proteomics approaches on the investigation of differentially expressed proteins were explored in this research. The major research objectives included 1) application of computational methods in homology and comparative modeling to study 3D protein structures and identify single nucleotide polymorphisms (SNPs) involved in changes of protein structures and functions, which can in turn increase the efficiency of the development of DNA markers; 2) investigation of methods on total protein profiling including purification, separation, visualization, and computational analysis at the proteome level. Special research goals were set on the development of open source computational methods using Matlab image processing tools to quantify and compare protein expression levels visualized by 2D protein electrophoresis gel techniques

Registration and 3D reconstruction of histological sections: application to mammary gland development

Tesis doctoral inédita. Universidad Autónoma de Madrid, Escuela Politécnica Superior, mayo de 200

The Role of Endothelial Mechanosensing in Capillary Development and Organization.

Ischemic injury is a leading cause of morbidity and mortality with the most common causes being heart attack, stroke, and peripheral artery disease. Therapies attempt to improve healing, in part, by promoting angiogenesis in these ischemic sites. Angiogenic invasion and maturation into a new capillary network may be affected by the altered microstructure and the mechanical properties of the ischemic tissue, in particular, the extracellular matrix (ECM). It is known that endothelial cells (EC) are mechanosensitive and reorient in response to both shear and normal stresses in vessels. Further, they generate traction forces and displacements in 2D culture to coordinate motion. However, the question of whether EC use cell-generated ECM forces to communicate in 3D culture to direct capillary organization and anastomosis is currently unresolved. Hydrogels formed from natural extracellular matrix (ECM) proteins readily support the formation of vasculature in vitro. The ECM is a highly ordered meshwork of various macromolecules. This anisotropic microstructure produces non-linear viscoelastic mechanical properties which confound attempts towards modeling the mechanical environment around cells. To overcome these issues, we developed a biosynthetic hydrogel consisting of polyethylene glycol diacrylamide conjugated to macromolecular type-I collagen. Through acrylamide-based cross-links, these materials allow for independent control of physical properties and bulk ligand concentration. Photoencapsulation of EC and fibroblasts within this hydrogel material and their subsequent co-culture led to the formation of capillary vessel-like networks with well-defined hollow lumens. Patterned hydrogel constructs were produced to assess angiogenic invasion independently of other stages of EC organization. ECM displacements were observed over time and mechanical modeling was used to compute cell-generated stresses and strains. We found that regions of strain exceeding 9% and stress exceeding 1,500 pico-Newtons per square micron co-localized with regions of capillary invasion (r=0.44). Thus, capillaries were found to generate stresses which propagated though the ECM. Through these studies, we developed an engineered ECM which enabled the magnitudes of cell-generated stresses during a complex 3D morphogenetic process to be quantified for the first time. These findings could yield a better understanding of the physical principles guiding capillary morphogenesis and provide new strategies for treating ischemic disease.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111562/1/rahsingh_1.pd

Shear-promoted drug encapsulation into red blood cells: a CFD model and μ-PIV analysis

The present work focuses on the main parameters that influence shear-promoted encapsulation of drugs into erythrocytes. A CFD model was built to investigate the fluid dynamics of a suspension of particles flowing in a commercial micro channel. Micro Particle Image Velocimetry (μ-PIV) allowed to take into account for the real properties of the red blood cell (RBC), thus having a deeper understanding of the process. Coupling these results with an analytical diffusion model, suitable working conditions were defined for different values of haematocrit

A Methodology to Develop Computer Vision Systems in Civil Engineering: Applications in Material Testing and Fish Tracking

[Resumen] La Visión Artificial proporciona una nueva y prometedora aproximación al campo de la Ingeniería Civil, donde es extremadamente importante medir con precisión diferentes procesos. Sin embargo, la Visión Artificial es un campo muy amplio que abarca multitud de técnicas y objetivos, y definir una aproximación de desarrollo sistemática es problemático. En esta tesis se propone una nueva metodología para desarrollar estos sistemas considerando las características y requisitos de la Ingeniería Civil. Siguiendo esta metodología se han desarrollado dos sistemas: Un sistema para la medición de desplazamientos y deformaciones en imágenes de ensayos de resistencia de materiales. Solucionando las limitaciones de los actuales sensores físicos que interfieren con el ensayo y solo proporcionan mediciones en un punto y una dirección determinada. Un sistema para la medición de la trayectoria de peces en escalas de hendidura vertical, con el que se pretende solucionar las carencias en el diseño de escalas obteniendo información sobre el comportamiento de los peces. Estas aplicaciones representan contribuciones significativas en el área, y demuestran que la metodología definida e implementada proporciona un marco de trabajo sistemático y confiable para el desarrollo de sistemas de Visión Artificial en Ingeniería Civil.[Resumo] A Visión Artificial proporciona unha nova e prometedora aproximación ó campo da Enxeñería Civil, onde é extremadamente importante medir con precisión diferentes procesos. Sen embargo, a Visión Artificial é un campo moi amplo que abarca multitude de técnicas e obxectivos, e definir unha aproximación de desenvolvemento sistemática é problemático. En esta tese proponse unha nova metodoloxía para desenvolver estes sistemas considerando as características e requisitos da Enxeñería Civil. Seguindo esta metodoloxía desenvolvéronse dous sistemas: Un sistema para a medición de desprazamentos e deformacións en imaxes de ensaios de resistencia de materiais. Solucionando as limitacións dos actuais sensores físicos que interfiren co ensaio e só proporcionan medicións nun punto e nunha dirección determinada. Un sistema para a medición da traxectoria de peixes en escalas de fenda vertical, co que se pretende solucionar as carencias no deseño de escalas obtendo información sobre o comportamento dos peixes. Estas aplicacións representan contribucións significativas na área, e demostran que a metodoloxía definida e implementada proporciona un marco de traballo sistemático e confiable para o desenvolvemento de sistemas de Visión Artificial en Enxeñería Civil.[Abstract] Computer Vision provides a new and promising approach to Civil Engineering, where it is extremely important to measure with accuracy real world processes. However, Computer Vision is a broad field, involving several techniques and topics, and the task of defining a systematic development approach is problematic. In this thesis a new methodology is carried out to develop these systems attending to the special characteristics and requirements of Civil Engineering. Following this methodology, two systems were developed: A system to measure displacements from real images of material surfaces taken during strength tests. This technique solves the limitation of current physical sensors, which interfere with the assay and which are limited to obtaining measurements in a single point of the material and in a single direction of the movement. A system to measure the trajectory of fishes in vertical slot fishways, whose purpose is to solve current lacks in the design of fishways by providing information of fish behavior. These applications represent significant contributions to the field and show that the defined and implemented methodology provides a systematic and reliable framework to develop a Computer Vision system in Civil Engineering

Development of microstructured and protein patterned hydrogels to investigate the influence of the microenvironment on cancer cells.

Ovarian cancer is one of the most lethal gynaecological diseases. One of the reasons for its poor survival rates is its typically late diagnosis at an advanced stage of tumour progression. A better understanding of why and how cancer initiates and develops is crucial for early diagnosis and the development of improved treatment methods. To this point, investigations of mutations on genes of cells to reduce mortality of ovarian cancer have not lead to the desired outcome. Recently, increased attention has been paid to the influencing factors in cancer cell progression such as the cellular microenvironment. It is known that the natural microenvironment of tumours differs greatly from that of healthy tissues and that modification of the cell’s environment causes cellular responses such as changes in morphology, protein expressions, cell division and migration behaviour. In this work, an experimental set-up was developed to trap cells in defined 3D wells to investigate how physical properties of the microenvironment influence ovarian can- cer cells. Polyacrlyamide gels were simultaneously structured and protein patterned to create a platform for cell experiments, which allows the tuning of individual physical properties of the microenvironment of cells (such as stiffness, available volume and protein compositions for cell attachment) independently of each other. The designed and optimised fabrication process begins with optical lithography to transfer a pattern onto a Silicon (Si) substrate and a dry etching step to obtain an array of pillars while transforming the Si-substrate into a Si-mould. The Si-mould serves as a stamp during a µ-contact printing approach to transfer defined patterns of protein, and simultaneously as a mould during polyacrylamide polymerisation. The resulting microstructured and protein patterned polyacrylamide gels can then be used as cell culture substrates for cell experiments. Si-moulds and polyacrylamide gels were characterised with scanning electron microscopy, atomic force microscopy and confocal laser scanning microscopy. By investigating four differently sized circular patterns (with diameters of 20 µm, 30 µm, 40µm and 60 µm) and four different stiffnesses of the polyacrylamide gels (1 kPa, 8 kPa, 30 kPa and 100 kPa), it was shown that the fabrication process is robust and easy to adjust. Computational analysis protocols were developed and established for traction force microscopy and brightness fluctuation analysis of cells, including the corresponding bright- ness autocorrelation functions. It was shown that microstructured and protein patterned polyacrylamide gels can be used for investigations of protein expressions, cellular traction forces and brightness fluctuations of cells. While the analyses of protein expressions and autocorrelation functions of the brightness fluctuations need further improvement, the results of the traction force experiments allow a first hypothesis to be formulated: cellular traction forces increase with decreasing volume available to the cell, as indicated by analysis of cell experiments on ovarian cancer cells of the cell line SKOV3. The developed microstructured and protein patterned polyacrylamide gels are an important step to gain a better understanding on how mechanical properties of the microenvironment influence cellular responses. This experimental setup can be easily adapted and optimised for further investigations of cancer cells and can thus help in the development of new treatment approaches

Collagen 1 fibers and hypoxic tumor microenvironments in breast cancer and their effect on transport of molecules within the tumor matrix

The work presented in this thesis represents a culmination of four years of research work focused on understanding the role of collagen I (Col1) fibers, a major component of the tumor extracellular matrix in breast cancer. We have investigated the relationship of Col1 fibers with metastasis, hypoxia, macromolecular transport, water diffusion and fractional anisotropy using clinical breast cancer specimens and human breast cancer xenografts genetically engineered to fluoresce under hypoxia. The thesis describes in the detail of the rationale, study design, results and discussion for our studies, which have been either published or submitted as four separate papers. The work here also presents multimodal in vivo imaging techniques which could be used clinically for non-invasive diagnoses of breast cancer progression. These techniques will help provide better patient management

Electrochemical and Electrokinetic Tools for Surface Activity Characterization and Proteomics Analysis

Scanning electrochemical microscopy (SECM) has developed into an excellent and versatile technique to image heterogeneous chemical reactivities on a surface. Therefore SECM applications cover a wide range of different fields such as biology and forensic sciences. One interesting application that has been found in proteomics, and explored herein, is the use of SECM as a read-out tool for protein microseparations. Proteomic research is a long process where two main steps are contained: separation of one protein from a protein mixture and detection of the separated proteins with a high sensitivity and if required selectivity. Shortening the experimental time of each one of these steps while their quality is kept or improved, should be the strategy to win this exciting race. Although microelectrophoresis affords faster protein separation, the smaller amount of sample employed requires a more sensitive protein detection method. In the first part of the present thesis an approach to solve the latter milestone has been found based on the coupling of miniaturized electrophoresis and SECM. As a result, a complete miniaturized (i.e. 1 cm × 0.5 cm) isoelectric focusing (IEF) protein separation was scanned by SECM, providing protein detection with a high sensitivity and high resolution. Additionally, protein detection by SECM was performed by different strategies ranging from general to selective approaches based on the tagging of free cysteines and other nucleophiles in proteins and peptides by benzoquinone. The tagged proteins are detected by the mediated reduction of benzoquinone with a redox species produced electrochemically at the SECM tip. After careful optimization, a sensitivity in the low ng mm-2 range was reached for bovine serum albumin. One of the major advantages of the present technique is that the selectivity of the protein tagging can be tuned by changing the pH of the reaction media. Depending on the requirements, cysteine selective or general detection can therefore be achieved with a high sensitivity. Despite the time reduction achieved with microelectrophoresis and the successful coupling with SECM for sensitive protein detection, conventional SECM setups are limited to scan line by line the whole studied area carrying long experimental times. Additionally, since the response of the sensing microelectrode depends on the probe-substrate distance, discrimination between surface reactivity and topographic artifacts coming from the sample topology is not obvious. The outlined above SECM drawbacks motivate us in the second part of this thesis to address those points. Therefore, some recent developments that show how SECM can be used for reactivity analysis of large, corrugated, tilted and dry surfaces are presented. This extension of SECM is made possible by the use of specialized microelectrode probes fabricated in a soft polymer film integrating for instance, microfluidic systems for delivering microliter volumes of redox-active mediator on a dry sample. These soft structures are then scanned in a contact mode on the substrate, and the originated current from the redox cycling of a mediator is used to construct a reactivity image of the specimen. Due to the use of probes containing individually addressable multiplexed electrodes, it is possible to substantially decrease the recording time while a high quality image resolution is maintained. The electrochemical characterization of the proposed probes was performed by cyclic voltammetry, approach curves and lateral line scans over insulating and conductive substrates of different roughness