The study of crowding effects on particle diffusion is a large subject with implications in many scientific areas. The studies span from pure theoretical calculations to experiments actually measuring the movement of proteins diffusing in a cell. Even though the subject is important and has been studied heavily there are still aspects not fully understood.   This report describes a Monte Carlo simulation approach (Gillespie algorithm) to study the effects of crowding on particle diffusion in a quasi one-dimensional system. With quasi meaning that the particles diffuses on a one-dimensional lattice but has the possibility to disassociate from the lattice and then rebind at a latter stage. Different binding strategies are considered: rebinding to the same location and randomly choosing the binding location. The focus of the study is how these strategies affects the mobility (diffusion coefficient) of a tracer particle. The main result of this thesis is a graph showing the diffusion coefficient as a function of the binding rate for different binding strategies and particle densities. We provide analytical estimates for the diffusion coefficient in the unbinding rate limits which show good agreement with the simulations. / Hur "trängsel" (från engelskans "crowding" t ex molecular crowding) påverkar diffusionsprocesser är viktigt inom många olika vetenskapliga områden. Forskningen som för tillfället utförs sträcker sig från rent teoretiska beräkningar till experiments där man kan följa enskilda proteiners rörelse i en cell. Även fast ämnet är viktig och väl undersökt finns det fortfarande många aspekter som man inte förstår till fullo. I det här examensarbetet beskrivs en Monte Carlo metod (Gillespie algoritmen) för att studera hur trängsel påverkar en partikel som diffunderar i ett "nästan" en-dimensonellt system. Det är nästan en-dimensionellt i det avsedde att partiklarna diffunderar på ett gitter men kan binda av från gittret och binda tillbaka i ett senare skedde. Olika metoder för hur partiklarna binder till gittret undersöks: Återbinding till avbindingsplatsen och slumpmässigt vald återbindingsplats. Fokus ligger på att förklara hur dessa påverkar mobiliteten (diffusionskonstanten) av en spårningspartikel (tracer particle). Resultatet är en graf som visar diffusionskonstanten för spårningspartikeln som en funktion av avbindingsfrekvens för olika bindingstrategier och partikeldensiteter. Vi ger också analytiska resultat i gränsvärdet för höga och låga avbindingstakter vilka stämmer bra överens med simuleringar.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23333号 / 生博第451号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 永尾 雅哉, 教授 片山 高嶺, 教授 谷口 雄一 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

<p>Open questions remain on the influence of various conditions and ion behavior on the hydration-shell of oily molecules. My research uses Raman spectroscopy and Raman multivariate curve resolution to study the hydration-shell of oily molecules as tools to help answer some of these open questions.</p><p>More specifically, I present results on the effect of molecular crowding on the structure of water around various oily molecules, and report the effect of molecular crowding on hydrophobic crossover. These results are important, as crowding has the potential to influence several fields, such as biology and environmental sciences. This work shows that increasing molecular concentration results in oil-oil crowding, decreases the tetrahedrality of the water structure around the oily molecules, and subsequently, the crossover temperature.</p><p>In addition to studying the hydration-shell under crowded conditions, I also present work on ion affiliation for the hydration-shell of an oily molecule. Ion affiliation for oil/water interfaces has been an ongoing topic of research since the Hoffmeister experiments because of their effect on biological processes. This study focuses on hydroxide and its affiliation for tert-butyl alcohol in comparison to other electrolytes. These results show iodide is less repelled by the oil/water interface in comparison to hydroxide.</p><p>Finally, I present findings on the influence of hydrogen peroxide in comparison to other small molecules on the water structure of an oily molecule. Hydrogen peroxide has been shown to reach supercooled temperatures, which may be useful in future studies of liquid phase transitions or studies on solute behavior at supercooled conditions. It is found that hydrogen peroxide does not significantly influence the water structure around tert-butyl alcohol, while other small molecules display significant water structure changes.</p><p>All these projects aim to contribute results to heated debates, as well as share information for future experiments.</p>

The oncogene c-MYC has guanine-rich and complementary cytosine-rich sequences in its P1 promoter region. The P1 promoter is responsible for over 90% of the c-MYC expression. Downregulation of c-MYC expression represents a novel therapeutic approach to more than 50% of all cancers. A stable i-motif formed by the c-MYC C-rich sequence would be an attractive target for cancer treatment. We have previously shown that c-MYC promoter sequences can form stable i-motifs in acidic solution (pH 4.5-5.5). The question is whether c-MYC promoter sequence i-motif will be stable at physiological pH. In this work, we have investigated the stability of mutant c-MYC i-motif in solutions having pH values from 4 to 7 and containing co-solutes or molecular crowding agents. The crowded nuclear environment was modeled by the addition of polyethylene glycol (PEG, having molecular weights from 200 to 12000 g/mol) at concentrations of 10% to 40% w/w. Circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC) were used to establish the presence and stability of c-MYC i-motifs in buffer solutions having pH values of 4 to 7. The results of these studies are: 1) the addition of up to 20% w/w glycerol does not increase i-motif stability, 2) the addition of 30% PEG results in an increase in i-motif stability to pH values as high as 6.7, 3) i-motif stability is increased with increased PEG concentration and increased PEG molecular weight, and 4) the effects of PEG size and concentration are not linear, with larger PEGs forming DNA/PEG complexes, which destabilize the i-motif. In summary, we have shown that the c-MYC i-motif can exist as a stable structure at pH as high as 6.7 in a crowded environment. Molecular crowding, largely an excluded volume effect, drives the formation of the more compact i-motif, even at higher pH values where the cytosine imino-nitrogen is deprotonated and neutral C-C pairs can form only two H-bonds. Based on this research, it seems possible that a stable c-MYC promoter sequence i-motif could form at physiological pH and would be a reasonable drug target for new cancer therapies.

During the past decade the effects of macromolecular crowding on reaction pathways is gaining in prominence. The stress is to move out of the realms of ideal solution studies and make conceptual modifications that consider non-ideality as a variable in our calculations. In recent years it has been shown that molecular crowding exerts significant effects on all in vivo processes, from DNA conformational changes, protein folding to DNA-protein interactions, enzyme pathways and signalling pathways. Both thermodynamic as well as kinetic parameters vary by orders of magnitude in uncrowded buffer system as compared to those in the crowded cellular milieu. Ignoring these differences will restrict our knowledge of biology to a “model system” with few practical understandings. The recent expansion of the genome database has stimulated a study on numerous previously unknown proteins. This has whetted our thirst to model the cellular determinants in a more comprehensive manner. Intracellular extract would have been the ideal solution to re-create the cellular environment. However, studies conducted in this solution will be contaminated by interference with other biologically active molecule and relevant statistical data cannot be extracted out from it. Recent advances in methodologies to mimic the cellular crowding include use of inert macromolecules to reduce the volume occupancy of target molecules and the use of immobilization techniques to increase the surface density of molecules in a small volumetric region. The use of crowding agents often results in non-specific interaction and side-reactions like aggregation of the target molecules with the crowding agents themselves. Immobilization of one of the interacting partners reduces the probability of aggregation and precipitation of bio-macromolecules by restricting their degrees of freedom. Covalent linkage of molecules on solid support is used extensively in research for creating a homogeneous surface of bound molecules which can be interrogated for their reactivity. However, when it comes to biomolecules, direct immobilization on solid support or use of organic linkers often results in denaturation. The use of bio-affinity immobilization techniques can help us overcome this problem. Since mild conditions are needed to regenerate such a surface, it finds universal applicability as bio-memory chips. This thesis focuses on our attempts to design a physiologically viable immobilization technique for following rotein-protein/protein-DNA interactions. The work explores the mechanism for biological interactions related to transcription process in E. coli. Chapter 1 deals with the literary survey of the importance and effects of molecular crowding on biological reactions. It gives a brief history of the efforts been made so far by experimentalists, to mimic macromolecular crowding and the methods applied. The chapter tries to project an all-round perspective of the pros and cons of different immobilization techniques as a means to achieve a high surface density of molecules and the advancements so far. Chapter 2 deals with the detailed technicality and applicability of the Langmuir-Blodgett method. It discusses the rationale behind our developing this technique as an alternate means of bio-affinity immobilization, under physiologically compatible conditions. It then goes on to describe our efforts to follow the sequence-specific and sequential assembly process of a functional RNA polymerase enzyme with one immobilized partner and also explore the role of omega subunit of RNAP in the reconstitution pathway. This chapter uses the assembly process of a multi-subunit enzyme to evaluate the efficiency of the LB system as a universal two-dimensional scaffold to follow sequence-specific protein-ligand interaction. Chapter 3 discusses the application of LB technique to quantitatively evaluate the kinetics and thermodynamics of promoter-RNA polymerase interaction under conditions of reduced dimensionality. Here, we follow the interaction of T7A1 phage promoter with Escherichia coli RNA polymerase using our Langmuir-Blodgett technique. The changes in mechanistic pathway and trapping of kinetic intermediates are discussed in detail due to the imposed restriction in the degrees of freedom of the system. The sensitivity of this detection method is compared vis-a-vis conventional immobilization methods like SPR. This chapter firmly establishes the universal application of LB technique as a means to emulate molecular crowding and as a sensitive assay for studying the effects of such crowding on vital biological reaction pathway. Chapter 4 describes the mechanistic pathway for the physical binding of MsDps1 protein with long dsDNA in order to physically protect DNA during oxidative stress. The chapter describes in detail the mechanism of physical sequestering of non-specific DNA strands and compaction of the genome under conditions where a kinetic bottleneck has been applied. The data obtained is compared with results obtained in the previous chapter for the sequence-specific DNA-protein interaction in order to understand the difference in recognition process between regulatory and structural proteins binding to DNA. Chapter 5 deals with the evaluation of the σ-competition model in E. coli for three different sigma factors (all belonging to the σ-70 family). Here again, we have evaluated the kinetic and thermodynamic parameters governing the binding of core RNAP with its different sigma factors (σ70, σ32and σ38) and performed a comparative study for the binding of each sigma factor to its core using two different non-homogeneous immobilization techniques. The data has been analyzed globally to resolve the discrepancies associated with establishing the relative affinity of the different sigma factors for the same core RNA polymerase under physiological conditions. Chapter 6 summarizes the work presented in this thesis. In the Appendix section we have followed the unzipping of promoter DNA sequence using Optical Tweezers in an attempt to follow the temporal fluctuations occurring in biological reactions in real time and at a single molecule level.

L'objectif général de cette thèse est d'analyser les processus de diffusion et les processus de réaction-diffusion dans plusieurs types de conditions non-idéales, et d'identifier dans quelle mesure ces conditions non idéales influencent la mobilité des particules et les réactions entre les molécules. Dans la première partie de la thèse, nous nous concentrons sur les effets de l'encombrement macromoléculaire sur la mobilité, ainsi élaborant une description des processus de diffusion dans des milieux densément peuplés. Tous les processus sont analysés à partir de la description microscopique du mouvement des agents individuels sous forme de marche aléatoire, tenant compte de l'espace occupé par les particules voisines. La deuxième partie de la thèse vise à caractériser le rôle de la géométrie de l'environnement et de la réactivité des corps qui y sont contenus sur la réaction entre des molécules sélectionnées. La théorie classique de Smoluchowski, formulée pour les réactions contrôlées par la diffusion dans un milieu dilué, est ainsi adaptée à des domaines arbitrairement décorés par des obstacles, dont certains réactifs, et l'équation stationnaire de diffusion est résolue avec des techniques d’analyse harmonique. Finalement, le calcul explicit de la constante de réaction et la dérivation des formules approximées sont utilisés pour étudier des applications biologiques et nano-technologiques. / The overall purpose of this thesis is to analyze diffusion processes and diffusion-reaction processes in different types of non-ideal conditions, and to identify to which extent these non-ideal conditions influence the mobility of particles and the rate of the reactions occurring between molecules. In the first part of the thesis we concentrate on the effects of macromolecular crowding on the mobility of the agents, providing therefore a description of various diffusion processes in densely populated media. All the processes are analyzed by modeling the dynamics of the single agents as microscopic stochastic processes that keep track of the macromolecular crowding. The second part of the thesis aims at characterizing the role of the environment’s geometry (obstacles, compartmentalization) and distributed reactivity (competitive reactants, traps) on the reaction between selected molecules. The Smoluchowski theory for diffusion influenced reactions is thus adapted to domains arbitrarily decorated with obstacles and reactive boundaries, and the stationary diffusion equation is explicitly solved through harmonic-based techniques. The explicit calculation of the reaction rate constant and the derivation of simple approximated formulas are used for investigating nano-technological applications and naturally occurring reactions.

No description available.

Biopolymer networks play a major role as part of the cytoskeleton. They provide stable structures and act as a medium for signal transport. These features encourage the application of such networks as organic computation devices. While research on this topic is not advanced yet, previous results are very promising. The protein actin in particular appears advantageous. It can be arranged to various stable structures and transmit several signals. In this study aster shaped networks were self-assembled via entropic forces by the crowding agent methyl cellulose. These networks are characterised by a regular and uniquely thick bundle structure, but have so far only been accounted in droplets of 100 μm diameter. We report now regular asters in an area of a few mm2 that could be observed even after months. Such stability outside of an organism is striking and underlines the great potential actin aster networks display.

Un polypeptide néosynthétisé est capable de trouver rapidement le chemin vers sa structure tri-dimensionnelle finale, en passant par des intermédiaires partiellement structurés. L'acquisition d'information sur le rôle et l'importance de ces intermédiaires est rendue difficile parce qu'ils se forment très rapidement pendant la réaction de repliement et que cette période de temps n'est pas accessible aux appareils de mélange usuels.<br />L'objet de cette thèse était de caractériser les évènements cinétiques initiaux du repliement des protéines, notamment de l'apomyoglobine (apoMb), en utilisant des appareils de mélange ultra-rapide. Un appareil de type stopped-flow équipé d'une micro-cuve a permis de diminuer le temps mort de ce type de mélangeur. Une réaction bimoléculaire (NATA et NBS), à permis d'évaluer le temps mort à 400±10 µs, dans un mode d'utilisation permettant de suivre simultanément le signal de fluorescence et le signal de dichroïsme circulaire dans l'UV lointain. L'apoMb est une protéine particulièrement intéressante pour l'étude des évènements précoces du repliement des protéines. Le stopped-flow ultra rapide, a permis de suivre des cinétiques (k jusqu'à 1500 s-1) et montré que chaque étape précédemment identifiée, conduisant l'apoMb de sa forme dépliée à sa forme native (soit les réactions UIa, IaIb, et IbN), présente les caractéristiques typiques d'une réaction à deux états, hautement coopérative.<br />Nous avons étudié l'effet d'osmolytes sur les cinétiques et sur la stabilité à l'équilibre des formes U, I et N de l'apoMb. Des études cinétiques en présence de sucrose ont permis d'observer le comportement de la réactions UIa. Ces résultats indiquent que le sucrose déstabilise de manière relative la forme U et l'état de transition de la réaction UIa, par rapport à la forme Ia. L'étape limitante ne correspondrait donc pas à une compaction de la chaîne peptidique. Dans les mêmes conditions, l'étude de la transition IbN permet d'observer que l'état de transition présente des caractéristiques proches de Ib. Ces résultats, décrivant l'effet osmophobique sur l'intermédiaire I, ainsi que des résultats préliminaires de l'effet d'encombrement moléculaire sur le repliement du cytochrome C, sont discutés dans ce mémoire.

In this thesis we show how reactive processes can be affected by the presence of different types of spatial constraints, so much so that their nonlinear dynamics can be qualitatively altered or that new and unexpected behaviors can be produced. To understand how this interplay can occur in general terms, we theoretically investigate four very different examples of this situation. <p><p>The first system we study is a reversible trimolecular chemical reaction which is taking place in closed one-dimensional lattices. We show that the low dimensionality may or may not prevent the reaction from reaching its equilibrium state, depending on the microscopic properties of the molecular reactive mechanism. <p><p>The second reactive process we consider is a network of biological interactions between pigment cells on the skin of zebrafish. We show that the combination of short-range and long-range contact-mediated feedbacks can promote a Turing instability which gives rise to stationary patterns in space with intrinsic wavelength, without the need of any kind of motion.<p><p>Then we investigate the behavior of a typical chemical oscillator (the Brusselator) when it is constrained in a finite space. We show that molecular crowding can in such cases promote new nonlinear dynamical behaviors, affect the usual ones or even destroy them. <p><p>Finally we look at the situation where the constraint is given by the presence of a solid porous matrix that can react with a perfect gas in an exothermic way. We show on one hand that the interplay between reaction, heat flux and mass transport can give rise to the propagation of adsorption waves, and on the other hand that the coupling between the chemical reaction and the changes in the structural properties of the matrix can produce sustained chemomechanical oscillations. <p><p>These results show that spatial constraints can affect the kinetics of reactions, and are able to produce otherwise absent nonlinear dynamical behaviors. As a consequence of this, the usual understanding of the nonlinear dynamics of reactive systems can be put into question or even disproved. In order to have a better understanding of these systems we must acknowledge that mechanical and structural feedbacks can be important components of many reactive systems, and that they can be the very source of complex and fascinating phenomena.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished