Dielectric relaxation time spectroscopy for tissue characterisation

This thesis is concerned with Electrical Impedance Spectroscopy (EIS), a noninvasive technique for characterising biological tissue and distinguishing pathology. The thesis is focused on the development of an improved method for extracting physiologically related parameters from the measured impedance data in vivo and instrumentation for spectroscopic measurements. In EIS, the electrical properties of physiological tissues, defined by their composition and structure, are measured as functions of frequency. Experimental observations of the existence of dielectric dispersions caused by distributions of dielectric relaxation time (DRT) constants were made on different types of biological material. It is postulated that widely used approaches for modelling these electrical properties are fundamentally flawed. The research work concentrates on the reconstruction of DRT spectra directly from the measured frequency response. The reconstruction problem involves inversion of a linear operator and like many inverse problems, is complicated by the ill-posed nature of the problem. In this thesis an inversion algorithm - Galerkin Regularised Inverse Method (GRIM) - based on standard mathematical methods is developed. The DRT spectrum establishes a link between the raw impedance data and the physiological structure and function of biological tissues. The GRIM yields a large number of independent parameters each related to process on a different scale. Special care was taken in testing the method on simulated data and improving its resolution. The thesis is also concerned with the design and practical implementation of EIS systems. Two approaches are considered: systems based on commercially available Impedance Analysers and systems designed specially for studies in vivo. To evaluate the GRIM, an Impedance Analyser, benefitting from a higher accuracy and a wider frequency range, is used. To meet the more rigorous specification demanded for studies on living human tissues, an electrical impedance spectrometer is developed. The suitability of different current sources is investigated. This research work includes studies of animal tissue in vitro and in vivo. Optimal experiments are defined in terms of the measurement frequency range and the entire experimental protocol for dielectric spectroscopy is established. These biological data are used to evaluate the GRIM. A comparison between different tissue classes in vivo is made. From studying ischemic tissues, it is postulated and verified that physiological differences and changes can be measured using the technique of DRT spectroscopy

An Automated Bayesian Pipeline for Rapid Analysis of Single-Molecule Binding Data [preprint]

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. We benchmarked the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also used the pipeline to extend our understanding of TtAgo by measuring the protein\u27s binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites

Instabilité télomérique et progression tumorale : mécanismes épigénétiques de reprogrammation cellulaire

Le raccourcissement télomérique est la source majeure de l'instabilité chromosomique (CIN) au cours de la progression tumorale. Nous avons montré que les cellules humaines embryonnaires de rein (cellules HEK) ayant traversé une période de CIN subissent des vastes changements dans l'expression des microARNs, ce qui induit une transition épithélio-mésenchymateuse (TEM), un processus permettant aux cellules cancéreuses épithéliales migrer et envahir de nouveaux tissus et former des métastases. Notre travail a aussi suggéré que les cellules ayant subi une TEM étaient capables de former des tumeurs dans un microenvironnement sénescent. De surcroît, cette évolution dans la capacité tumorale était associée à une dérégulation supplémentaire des microARNs et à l'acquisition des propriétés des cellules souches. Afin d'étudier comment ce potentiel est mis en place au cours de l'instabilité chromosomique et au contact avec le microenvironnement sénescent, nous avons modulé les niveaux d'expression de miR-145 et avons démontré que la répression de miR-145 était nécessaire pour le développement des caractéristiques des cellules souches. Afin de mieux comprendre l'impact de CIN sur le programme génétique des cellules épithéliales, nous avons utilisé des approches de haut débit et avons caractérisé les changements des paysages chromatiniens et leur mise en place dans les cellules ayant traversé une période de CIN. Nos résultats révèlent pour la première fois que l'instabilité télomérique modifie profondément la distribution des marques d'histones en conduisant aux changements d'expression des gènes et au processus de transformation des cellules épithéliales pré-tumorales.Telomere shortening is a major source of chromosome instability (CIN) at early stages during carcinogenesis. However, the mechanisms through which telomere-driven CIN (T-CIN) contributes to the acquisition of tumor phenotypes remain uncharacterized. We have shown that human epithelial kidney (HEK) cells undergo massive microRNA deregulation upon CIN, in particular a miR-200-dependent epithelial-mesenchymal transition (EMT), which is thought to enable epithelial cancer cells to migrate and invade other tissues to form metastases. Our work also indicated that CIN+ cells that underwent EMT were able to form tumors in a senescent microenvironment. Notably, this progression in tumor capacity was associated with further microRNA deregulation and the manifestation of enhanced stem-like properties. To investigate how stem-like properties are acquired in CIN+ cells in the contact with senescent microenvironment we adapted knockdown and overexpression approaches to modulate miR-145 expression, and demonstrated that enhanced stem-like properties depended on miR-145 repression. To fully apprehend the impact of CIN on the genetic program of epithelial cells, we used an unbiased approach to characterize the chromatin state of HEK CIN+ cells and uncover genome wide redistributions that were in direct correlation with gene expression changes. Our results reveal for the first time that T-CIN profoundly modifies the chromatin landscape genome-wide thereby fueling the transformation process of pre-tumor epithelial cells

Psychological types of value orientations of personality in modern russian society

The article is dedicated to the most frequent types of value orientations in Russian society. Their connecting with psychological peculiarities is presented

Peculiarities of Trust and Distrust in Students of Higher Schools of Economics

The present study introduces the results of the empiric research of different types of trust. It also reveals some peculiarities of the manifestation of trust and distrust towards people in students at various stages of their professional growth

An automated Bayesian pipeline for rapid analysis of single-molecule binding data

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also use the pipeline to extend our understanding of TtAgo by measuring the protein\u27s binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites

Effects of Protein Unfolding on Aggregation and Gelation in Lysozyme Solutions

In this work, we investigate the role of folding/unfolding equilibrium in protein aggregation and formation of a gel network. Near the neutral pH and at a low buffer ionic strength, the formation of the gel network around unfolding conditions prevents investigations of protein aggregation. In this study, by deploying the fact that in lysozyme solutions the time of folding/unfolding is much shorter than the characteristic time of gelation, we have prevented gelation by rapidly heating the solution up to the unfolding temperature (~80 °C) for a short time (~30 min.) followed by fast cooling to the room temperature. Dynamic light scattering measurements show that if the gelation is prevented, nanosized irreversible aggregates (about 10–15 nm radius) form over a time scale of 10 days. These small aggregates persist and aggregate further into larger aggregates over several weeks. If gelation is not prevented, the nanosized aggregates become the building blocks for the gel network and define its mesh length scale. These results support our previously published conclusion on the nature of mesoscopic aggregates commonly observed in solutions of lysozyme, namely that aggregates do not form from lysozyme monomers in their native folded state. Only with the emergence of a small fraction of unfolded proteins molecules will the aggregates start to appear and grow.https://doi.org/10.3390/biom1009126