Mapping three dimensional interactions between biomolecules and electric fields.

Electroporation is a technique that induces the formation of open pores in cell membranes by the application of an electric field. Electroporation is widely practiced in research and clinical work for transfection of genetic sequences and drug molecule transport through the membrane barrier. However, a full theoretical explanation of the molecular mechanisms and thermodynamics responsible for pore formation, structure, and longevity does not yet exist. Advances in molecular dynamics simulations have enabled theoretical studies of electroporation with previously unobtainable fidelity spanning biologically relevant timescales. All-atom simulations utilizing the recently developed method of computational electrophysiology demonstrate that pore size correlates to the magnitude of the applied electric flux. This insight suggests improvements to electroporation protocols and instrument design to increase treatment efficacy while simultaneously decreasing cell mortality. Data processing, that scales and centers each simulation frame, generates a pore-centric matrix of voxels representing the time-averaged charge density of the simulation volume. This processing enabled the calculation of the first high resolution, three-dimensional maps of the electric fields that act to create and stabilize the pore. Applying this capability to individual moieties gives additional insight to how electrostatic forces between biomolecules and membrane structures give cell membranes their remarkable properties. Complementary processing of atom types, instead of partial charges, produces a similarly scaled, stabilized, and time averaged matrix of moiety number densities. Plotted in three dimensions, these density data reveal additional membrane structure detail that have not previously been reported

A practical guide to computer simulations

Here practical aspects of conducting research via computer simulations are discussed. The following issues are addressed: software engineering, object-oriented software development, programming style, macros, make files, scripts, libraries, random numbers, testing, debugging, data plotting, curve fitting, finite-size scaling, information retrieval, and preparing presentations. Because of the limited space, usually only short introductions to the specific areas are given and references to more extensive literature are cited. All examples of code are in C/C++.Comment: 69 pages, with permission of Wiley-VCH, see http://www.wiley-vch.de (some screenshots with poor quality due to arXiv size restrictions) A comprehensively extended version will appear in spring 2009 as book at Word-Scientific, see http://www.worldscibooks.com/physics/6988.htm

A parallel computing-visualization framework for polycrystalline minerals

In this report, we have reported some preliminary results in the development of a parallel computing-visualization framework for large-scale molecular dynamics simulations of polycrystals of minerals, which are geophysically relevant for Earth’s mantle. First, we have generated the input configurations of atoms belonging to various grains distributed in the space in a way, which resembles the polycrystalline structure of the minerals. The Input configuration is developed using Voronoi geometry. Thus generated polycrystalline system is simulated using the PolyCrystal Molecular Dynamics algorithm. Performance tests conducted using up to 256 processors and a couple of millions of atoms have shown that the computation time per MD step remains under 20 seconds. The other important part is the development of an efficient visualization system to interactively explore the massive three dimensional and time-dependent datasets produced by MD simulations. Some results are presented for the simulation of two-grain structure. The proposed framework is expected to be useful in simulations of more realistic and complex rheological (mechanical) properties of important Earth forming mineral phases under different conditions of stresses and temperatures

Three-dimensional Structure of the Central Mitotic Spindle of Diatoma vulgare

Central mitotic spindles in Diatoma vulgare have been investigated using serial sections and electron microscopy. Spindles at both early stages (before metaphase) and later stages of mitosis (metaphase to telophase) have been analyzed. We have used computer graphics technology to facilitate the analysis and to produce stereo images of the central spindle reconstructed in three dimensions. We find that at prometaphase, when the nuclear envelope is dissassembling, the spindle is constructed from two sets of polar microtubules (MTs) that interdigitate to form a zone of overlap. As the chromosomes become organized into the metaphase configuration, the polar MTs, the spindle, and the zone of overlap all elongate, while the number of MTs in the central spindle decreases from greater than 700 to approximately 250. Most of the tubules lost are short ones that reside near the spindle poles. The previously described decrease in the length of the zone of overlap during anaphase central spindle elongation is clearly demonstrated in stereo images. In addition, we have used our three-dimensional data to determine the lengths of the spindle MTs at various times during mitotis. The distribution of lengths is bimodal during prometaphase, but the short tubules disappear and the long tubules elongate as mitosis proceeds. The distributions of MT lengths are compared to the length distributions of MTs polymerized in vitro, and a model is presented to account for our findings about both MT length changes and microtubule movements

Hydrates in supersaturated binary sulfuric acid‐water vapor

Portions of the free energy surface giving the reversible work W required to form a droplet containing n1 molecules of H2O and n2 molecules of H2SO4 from an initially homogeneous vapor have been calculated for a variety of relative humidities and H2SO4 vapor concentrations. These surfaces are displayed as three dimensional perspective plots. The surfaces predict the existence of stable H2SO4 hydrates in the vapor phase, and the number of hydrates, Nh, with h molecules of H2O per hydrate has been calculated for three different values of the relative humidity. The results of these calculations indicate that virtually all the H2SO4 present, especially for relative humidities greater than 100%, exists in hydrate form. As the relative humidity is increased (i.e., greater than 200%), the distribution of hydrate sized agrees well with that found by Giauque et al. for solid H2O–H2SO4 mixtures. Hydrate formation can exert an appreciable effect on the process of vapor phase nucleation in H2O–H2SO4 mixtures, and must be considered in any theory of nucleation rate

A Farewell to Flat Biology. Three-dimensional Cell Culture Models in Cancer Drug Target Identification and Validation

Cells of epithelial origin, e.g. from breast and prostate cancers, effectively differentiate into complex multicellular structures when cultured in three-dimensions (3D) instead of conventional two-dimensional (2D) adherent surfaces. The spectrum of different organotypic morphologies is highly dependent on the culture environment that can be either non-adherent or scaffold-based. When embedded in physiological extracellular matrices (ECMs), such as laminin-rich basement membrane extracts, normal epithelial cells differentiate into acinar spheroids reminiscent of glandular ductal structures. Transformed cancer cells, in contrast, typically fail to undergo acinar morphogenic patterns, forming poorly differentiated or invasive multicellular structures. The 3D cancer spheroids are widely accepted to better recapitulate various tumorigenic processes and drug responses. So far, however, 3D models have been employed predominantly in the Academia, whereas the pharmaceutical industry has yet to adopt a more widely and routine use. This is mainly due to poor characterisation of cell models, lack of standardised workflows and high throughput cell culture platforms, and the availability of proper readout and quantification tools. In this thesis, a complete workflow has been established entailing well-characterised 3D cell culture models for prostate cancer, a standardised 3D cell culture routine based on high-throughput-ready platform, automated image acquisition with concomitant morphometric image analysis, and data visualisation, in order to enable large-scale high-content screens. Our integrated suite of software and statistical analysis tools were optimised and validated using a comprehensive panel of prostate cancer cell lines and 3D models. The tools quantify multiple key cancer-relevant morphological features, ranging from cancer cell invasion through multicellular differentiation to growth, and detect dynamic changes both in morphology and function, such as cell death and apoptosis, in response to experimental perturbations including RNA interference and small molecule inhibitors. Our panel of cell lines included many non-transformed and most currently available classic prostate cancer cell lines, which were characterised for their morphogenetic properties in 3D laminin-rich ECM. The phenotypes and gene expression profiles were evaluated concerning their relevance for pre-clinical drug discovery, disease modelling and basic research. In addition, a spontaneous model for invasive transformation was discovered, displaying a highdegree of epithelial plasticity. This plasticity is mediated by an abundant bioactive serum lipid, lysophosphatidic acid (LPA), and its receptor LPAR1. The invasive transformation was caused by abrupt cytoskeletal rearrangement through impaired G protein alpha 12/13 and RhoA/ROCK, and mediated by upregulated adenylyl cyclase/cyclic AMP (cAMP)/protein kinase A, and Rac/ PAK pathways. The spontaneous invasion model tangibly exemplifies the biological relevance of organotypic cell culture models. Overall, this thesis work underlines the power of novel morphometric screening tools in drug discovery.Siirretty Doriast

Comparing stenotic blood flow in three- and two-dimensional arterial renderings using computational fluid dynamics and multiphase mean age theory.

Over one million invasive coronary angiography procedures are performed annually in patients who experience chest pain or are known to have coronary artery disease. The procedure is carried out to ascertain the degree of arterial blockage (stenosis) that hinders blood flow to the heart. A cardiologist performing the procedure determines the physiological degree of a stenosis by either visual estimation, which is routine practice, or by invasively measuring fractional flow reserve (FFR), which is the current gold standard that has been demonstrated to improve patient outcomes and temper the cost of healthcare. Nevertheless, FFR is performed in only 10–20% of patients because it is invasive, expensive, and requires more radiation exposure. New computational methods utilizing three-dimensional renderings processed from coronary angiograms can provide an accurate, highly sensitive, non-invasive method to assess stenotic significance without using FFR. While beneficial, this technique requires intensive computer processing power and calculation runtimes on the order of several hours. An approach to reduce computational time involves alike computing of two-dimensional arterial slices cut from the three-dimensional source renderings. The main objective was to determine if two-dimensional processing can also provide an accurate and highly sensitive method to assess stenotic significance at a fraction of the computational expense. Blood flow was analyzed in five patient cases below and five patient cases above the commonly accepted FFR threshold value for intervention of 0.80. Following the generation of two orthogonal slices from DICOM-derived three-dimensional renderings, pulsing blood flow was simulated with CFD, and multiphase mean age theory was applied to calculate the mean age of red blood cells as a diagnostic metric. Two-dimensional processing typically exhibited a correlation with FFR only in the geometries of vertically-oriented slices. This was ascribed to the possibility of uncaptured stenotic blood flow characteristics in the limited testing of only two angles of a full arterial segment.Mean ages for the three-dimensional cases were many orders of magnitude higher than those of the corresponding two-dimensional cases. This was attributed to red blood cell collisions and distal recirculatory eddies near a stenosisbeing less expressed in the simplicity of the two-dimensional slices when compared to the complexity of the three-dimensional source renderings. A mean age threshold for determining stent intervention was estimated for the two-dimensional cases since limited sample size disallowed rigorous statistical analysis. The data suggested an arbitrary value equal to ~2.5. Nine out of ten cases correlated with FFR, with just one false negative diagnosis. In published virtual FFR techniques, false diagnosis typically occurs in 10–13% of the cases. Computational runtime for two-dimensional cases was less than 2% of the runtime for corresponding three-dimensional cases. Preliminary results indicate two-dimensional processing may efficiently detect and assess stenoses non-invasively, provided that it holds up to rigorous statistical analysis following testing of at least 80–100 more cases, plus several additional slice angles

Engineered three-dimensional microenvironments as functional in vitro models of stromal tissues

Currently, the majority of current cultures is still carried out with long-established techniques like the exploitation of 2D supports, the use of tissue-derived immortalized cell lines, and the administration of un-physiological doses of soluble factors to induce a biological response. However, the lack of structural and physical cues often leads to biological artifacts, from the total loss of cellular function to the lack of correlation between the predicted and actual results when the experimental model shifts from in vitro to in vivo. Hence, in this work I test the hypothesis that recapitulating in vitro crucial chemo-physical components of the native cell environment can uniquely maintain the original function and the phenotype of cultured cells. Therefore, the critical aspects are (i) the choice of a suitable source of cells, and (ii) the engineering of the culture conditions. In first instance, it is proposed that freshly isolated adult cells, as opposed to cell lines, are needed to mimic physiological and pathological processes occurring in animal tissues and organs. Secondly, in vitro culture conditions need to be adapted to support cell viability, function, and growth. In particular, the proposed approach relies on the combination of the cells with a suitable biomaterial able to provide a 3D environment for cell adhesion and suitable to allow complex spatial interactions with neighboring cells. The concept of the third dimension as a critical parameter able to influence cell physiology is challenged in different contexts. The complexity of the proposed culture systems, due to the high number of variables among 2D and 3D experimental groups, is such that the precise dissection of the single contributions is not obvious. However, we propose that the combination of a physiological 3D architecture with a suitable biomaterial provide technological and biological advantages able to trigger further investigations. Notably, the material itself can be chosen so to mimic the native organ, e.g. the mineralized matrix of bone substituted in vitro by a ceramic material. Additionally, we suggest that the use of bioreactors as supportive technologies can exploit the full potential of 3D cell cultures. Despite implying an increase in the complexity of the procedures required to execute experiments based on 3D cell cultures, it is proposed that the relevance of the results surpasses the efforts required to implement new culture models. In the first chapter of my thesis, I focused on the validation of a platform for the expansion of bone-marrow derived stromal cells (MSC). As a result, the bioreactor-based platform was validated not only as a streamlined approach to expand MSC that maintain at a higher extent progenitor features, but also as a valuable tool to recreate in vitro an engineered stromal niche. In the second chapter of the thesis the focus was moved to exploit the unique features of 3D cultures on the recapitulation of the thymic stroma in vitro. This chapter describes the evolution of a culture system able to manufacture in vitro a thymic organoid constituted by TEC that can suits as a model to investigate thymus physiology. Finally, in the third chapter of the thesis, the concept of 3D stromal tissue engineering is applied to the hematopoietic niche, a specialized microenvironment devoted to regulate hematopoietic stem cells (HSC) quiescence and activity through a wide array of chemo-physical cues. Starting from previous reports in which freshly harvested bone marrow- or adipose tissue-derived cells can be cultured within porous scaffolds, allowing the formation of an organized 3D stromal tissue, we propose that cellularized constructs can be cultured in perfusion bioreactors to reconstruct the HSC niche through the controlled modulation of several parameters. Taken together, these results highlight that an increase in the complexity of the traditional culture systems is crucial to better recapitulate the functional microenvironment of stromal and stroma-dependent cells or stem cells. Growing and handling cells in a 3D structure combined with a compliant biomaterial and bioengineering tools can dramatically increase the relevance of scientific data, enable unpredecented modalities to control the artificial microenvironment, and decrease the need of costly, time consuming, and ethically debated in vivo experiments

Micromagnetic Simulation of Three-dimensional Nanoarchitectures

The thesis discusses micromagnetic simulation studies on high-frequency magnetic dynamics in three-dimensional ferromagnetic nanoarchitectures made of interconnected magnetic nanowire networks. Such artificial magnetic materials with nanoscale features have recently emerged as a vivid topic of research, as their geometry has a decisive impact on their magnetic properties. By studying their static magnetization structure, we find that these systems display a behavior analogous to that of 3D artificial spin ice lattices, with frustrated interactions and the emergence of monopole-like defect structures at the wires' intersection points. Our simulations reveal a high activity of these defect sites in the magnonic high-frequency spectrum. We study various 3D nanoarchitectures and show that their geometry and magnetization state results in characteristic high-frequency signatures. Controlling these features could open new pathways for magnonics research and reprogrammable magnetic metamaterials