Establishing a proteomics-based monocyte assay to assess differential innate immune responses

The innate immune system represents the first line of organism defense. Monocytes and their derivatives, the macrophages, play a central role in this response. The monocyte response to various stimuli is complex and includes simultaneous activation in a variety of ways. The common methods currently used to estimate this response usually focus on one of the modes of activation. The aim of this thesis was to assess the possibility of designing an assay based on unbiased proteome analysis that would be capable of predicting the monocyte response to various challenges. The underlying hypothesis of the project was that the cell proteome response to different activating molecules would reflect the immunogenicity of these molecules. The assay is based on comparison between the proteomes of human monocytic THP-1 cells treated with a stimulus of interest and the ‘reference’ proteomes obtained from THP-1 cells treated with positive and negative controls. The study described in Paper I (Project 1) revealed two main types of monocyte responses and features of monocyte separation into adherent and non-adherent subpopulations. The first subpopulation differentiated into macrophages, while the second one was activated by cytokines according to the changes in their proteomes compared to the control cells. The Pam3CSK4 and PMA stimuli were concluded to be the best positive controls for pro-inflammatory activation and differentiation, respectively, among the tested nine treatments (Paper II, Project 2). Anti- cancer drugs with known mechanisms of action were chosen as positive controls for cell death (Paper III). The workflow of the assay was optimized and validated in Paper II (Project 2). The established assay was applied to identify the response of monocytes to peptide containing isoaspartate (Paper II, Project 3) and nanoparticles (Paper III, Project 3). The epidermal growth factor (EGF) peptide containing isoaspartate primed monocytes for differentiation but not for pro-inflammatory activation. The three studied nanoparticles induced distinct proteome signatures despite equivalent overall toxicity effect (induction of 50±10% cell death). The CdTe-NH2 nanoparticles induced the strongest response characterized by down-regulation of topoisomerases and similar to the effect of Camptothecin. The response to CuO nanoparticles included up-regulation of proteins involved in heat response. The gold nanoparticles showed the weakest response among the studied three nanoparticles and induced up-regulation of NF-κB and a number of proteins related to energy metabolism consistent with pro-inflammatory activation. The established and validated assay has several possibilities for extension as well as optimization for particular aims of the experiment. For instance, the identification of induced post- translational modifications upon stimulation can be performed (Paper IV, Project 4). Because of its versatility, robustness and specificity, this new assay is likely to find a niche among the more established immunological methods

THREE-DIMENSIONAL NUMERICAL SIMULATION OF TSUNAMI WAVES BASED ON THE NAVIER-STOKES EQUATIONS

A numerical algorithm of solving the three-dimensional system of Navier-Stokes equations to simulate free surface waves and flows with gravity is presented. The main problem here is to ensure that the gravity force is properly accounted in the presence of discontinuities in the medium density. The task is made more complicated due the use of unstructured computational grids with collocated placement of unknown quantities and splitting algorithms based on SIMPLE-type methods. To obtain correctly the hydrostatic pressure, it is suggested that the contribution of the gravitational force in the equation for pressure should be distinguished explicitly; the latter being calculated by using the solution of the two-phase medium gravitational balance problem. To ensure the balance of the gravity force and the pressure gradient in the case of rest an algorithm in which the pressure gradient in the equation of motion is replaced by a modification considering the gravitational force action is suggested. This method is demonstrated by the example of tsunami wave propagation in the real water area of the World Ocean

Simulation of Turbulent Convection at High Rayleigh Numbers

The paper considers the possibility of using different approaches to modeling turbulence under conditions of highly developed convection at high Rayleigh numbers. A number of industrially oriented problems with experimental data have been chosen for the study. It is shown that, at Rayleigh numbers from 109 to 1017, the application of the eddy-resolving LES model makes it possible to substantially increase the accuracy of modeling natural convection in comparison with the linear vortex viscosity model SST. This advantage is most pronounced for cases of a vertical temperature difference with the formation of a large zone of convection of strong intensity. The use of the Reynolds stress model EARSM is shown for cases of natural convective flow in domains with dihedral angles in the simulated region and the predominance of secondary currents. When simulating a less intense convective flow, when the temperature difference is reached at one boundary, the differences in the approaches used to model turbulence are less significant. It is shown that, with increasing values of Rayleigh numbers, errors in the determination of thermohydraulic characteristics increase and, for more accurate determination of them, it is expedient to use eddy-resolving approaches to the modeling of turbulence

PECULARITIES OF FUNCTIONING OF THE BIBLICAL TEXT IN A NOVEL’S PLOT: ON THE PROBLEM OF INTERPRETATION OF BIBLICAL QUOTATIONS AND ALLUSIONS IN DOSTOEVSKY’S “CRIME AND PUNISHMENT”

This article analyzes the interpretations of  biblical texts in  Dostoevsky’s novel “Crime and Punishment”. It reveals the cases of ambiguity in  determining the  sources and the character of  functioning of  biblical allusions and quotations in  the  novel’s plot, examines the reasons for the textural discrepancies, and determines criteria for studying the biblical text in  Dostoevsky’s works. For a  more complete and accurate understanding of intertextual relationships, it is important to study biblical intertexts not only on a textual but on plot and fi gurative levels too, as far as the semantics of  certain quotations and allusions is  ultimately determined by  the characteristics of their insertion into the author’s text and their interaction with the cultural tradition that is represented not only in the biblical text, but in spiritual literature and the folk perception of biblical stories as well

An integrated assessment of the quality of teaching Mathematics in school

This article presents a diagnostic criteria-based methodology for a systematic and complex assessment of the quality of teaching Mathematics in school. This methodology may help assess the prospects of applied approaches to teaching Mathematics as well as monitor it and describe in terms of ensuring the high quality of general education in Mathematics. We used Kolmogorov’s “convolution of qualities” as a basis for developing this methodology for the systematic and complex diagnostics of mathematical education in school. This methodology is used to assess the quality and optimization of complex objects in mechanics, chemical industry, economics, and higher education. The suggested integral assessment (systematic and complex diagnostics) was successfully applied to evaluate the quality of teaching Mathematics in schools of the Volgograd region. We defined three levels of mathematical education quality: discrete (minimal and restricted level), fragmentary (average functional level), and integrated and comprehensive (a rather high level)

ANALYSIS OF THE MANAGEMENT SYSTEM IN THE FIELD OF ENVIRONMENTAL PROTECTION OF RUSSIAN CHEMICAL COMPANIES

Since 2007, many chemical industrial companies in the Russian Federation have been actively involved in the Responsible Care® international voluntary program. To implement this program, vast bodies of data on environmental impact assessments needs to be collected. This allows us to analyse the environment-oriented trends in economic and social activities, and to record the achievements and problems in this field. The collected large bodies of data are in many cases heterogeneous, since the report has been a voluntary initiative. To analyse the existing trends in business processes, authors applied the methodology for system analysis of large bodies of data and used their own heuristic approximation algorithm for the treatment of accumulated data. This algorithm gives us the unique possibility of evaluating the performance of both individual chemical companies in the framework of the Responsible Care® program and the Russian chemical industry as a whole

Proteomics Reveals a Role for Attachment in Monocyte Differentiation into Efficient Proinflammatory Macrophages

Monocytes are blood-borne cells of the innate immune system. They can be differentiated and activated into proinflammatory macrophages that might be employed in tumor immune therapy. Monocyte exposure to lipopolysaccharide (LPS) is a standard method to induce a pro­inflammatory macrophage state, with the resultant population comprising both adherent and nonadherent cells. In the current study, we aimed to identify the differences in proteomes of these monocyte subpopulations, which addresses a more general question about the role of attachment in monocyte differentiation. Label-free proteomics of a model of human monocytes (THP-1 cell line) revealed that the cells remaining in suspension upon LPS treatment were activated by cytokines and primed for rapid responsiveness to pathogens. In terms of proteome change, the adhesion process was orthogonal to activation. Adherent cells exhibited signs of differentiation and enhanced innate immune responsivity, being closer to macrophages. These findings indicate that adherent, LPS-treated cells would be more appropriate for use in tumor therapeutic applications

Establishing a Proteomics-Based Monocyte Assay To Assess Differential Innate Immune Activation Responses

Innate immune cells are complex systems that can be simultaneously activated in a variety of ways. Common methods currently used to estimate the response of innate immune cells to stimuli are usually biased toward a single mode of activation. The aim of this study was to assess the possibility of designing an assay based on unbiased proteome analysis that would be capable of predicting the complex response of the innate immune system to various challenges. Monocytes were used as representative cells of the innate immune system. The underlying hypothesis was that their proteome response to different activating molecules would reflect the immunogenicity of these molecules. To identify the main modes of response, we treated the human monocytic THP-1 cell line with nine different stimuli. Differentiation and activation were determined to be the two major modes of monocyte response, with PMA causing the strongest differentiation and Pam3CSK4 causing the strongest proinflammatory activation. The established assay was applied to characterize the monocyte response to epidermal growth factor peptide containing isoaspartate, which induced differentiation but not proinflammatory activation. Because of its versatility, robustness, and specificity, this new assay is likely to find a niche among the more established immunological methods

Proteomics Reveals a Role for Attachment in Monocyte Differentiation into Efficient Proinflammatory Macrophages

Monocytes are blood-borne cells of the innate immune system. They can be differentiated and activated into proinflammatory macrophages that might be employed in tumor immune therapy. Monocyte exposure to lipopolysaccharide (LPS) is a standard method to induce a pro­inflammatory macrophage state, with the resultant population comprising both adherent and nonadherent cells. In the current study, we aimed to identify the differences in proteomes of these monocyte subpopulations, which addresses a more general question about the role of attachment in monocyte differentiation. Label-free proteomics of a model of human monocytes (THP-1 cell line) revealed that the cells remaining in suspension upon LPS treatment were activated by cytokines and primed for rapid responsiveness to pathogens. In terms of proteome change, the adhesion process was orthogonal to activation. Adherent cells exhibited signs of differentiation and enhanced innate immune responsivity, being closer to macrophages. These findings indicate that adherent, LPS-treated cells would be more appropriate for use in tumor therapeutic applications