Fermeture de bulles de dénaturation de l'ADN couplées à l'élasticité de l'ADN

La compréhension physique des processus biologiques tels que la transcription nécessite de bien connaître la physique de l'ADN double brin. Une de ses propriétés thermodynamiques remarquable est sa dénaturation à une température particulière, lors de laquelle il se déroule et se sépare en deux brins après avoir formé des bulles (segments de paires de bases ouvertes consécutives). La dynamique de dénaturation jusqu'ici été étudiée l'échelle de la paire de base, ignorant ainsi les degrés de la chaîne. Ces études n'expliquent pas les temps de fermeture trés longs, de 20 $100\mu$s, mesurés par Alain-Bonnet et al.température ambiante pour des bulles de 18 paires de base. Dans cette thèse nous nous interessons la fermeture de grandes bulles de dénaturation thermalisées, l'aide de simulations de dynamique Brownienne d'un modèle simple "gros grains"de l'ADN. Nous montrons que la fermeture se fait en deux temps : d'abord, la bulle initiale se ferme rapidement jusqu'à ce qu'elle atteigne un état métastable, causé par les grandes énergies de courbure et de torsion emmagasinées dans la bulle. Ensuite, la fermeture de la bulle metastable se fait en fonction de la longueur de l'ADN et des parametres elastiques, soit apres la diffusion rotationnelle des "bras" rigides jusqu'à l'alignement de ceux-ci, soit lorsque la bulle a diffusée jusqu'un bout de la chaîne, ou soit localement lors d'une activation thermique. Nous montrons ainsi que le mécanisme physique associé des longs temps de fermeture est le couplage entre les degrés de liberté d'appariement et de conformations de l'ADN.The physical understainding of biological processes such as transcription requires the knowledge of double-stranded DNA (dsDNA) is its denaturation, at the melting temperature, in which it unwinds into two single-stranded DNAs via the formation of denaturation bubbles (segment of consecutive unpaired base-pairs). the dynamics of denaturation has beenstudies so far at the base-pair (bp) scale, ignoring conformational chaindegrees of freedom. These studies do not explain the very long closure times of 20 to 100µs, measured by atlan-Bonnet et al., of 18 bps long bubbles at room temperature. In this thesis, we study the closure of pre-equilibrated large bubbles, by using Brownian dynamics simulations of two simple DNA coarse-grained models. We show that the closure occurs via two steps : first, a fast zipping of the initial bubble occurs until a meta-stable state is reached, due to the large bending and twisting energies stored in the bubble. Then, the mete-stable bubble closes either via rotational diffusion of the stiff side arms until their alignment, or bubble diffusion until it reaches the chain end, or locally by thermal activation, depending on the DNA length and elastic moduli. We show that the physical mechanism behind these long timescales is therefore the dynamical coupling between base-pair and chain degrees of freedom

Acanthocyte Sedimentation Rate as a Diagnostic Biomarker for Neuroacanthocytosis Syndromes: Experimental Evidence and Physical Justification

(1) Background: Chorea-acanthocytosis and McLeod syndrome are the core diseases among the group of rare neurodegenerative disorders called neuroacanthocytosis syndromes (NASs). NAS patients have a variable number of irregularly spiky erythrocytes, so-called acanthocytes. Their detection is a crucial but error-prone parameter in the diagnosis of NASs, often leading to misdiagnoses. (2) Methods: We measured the standard Westergren erythrocyte sedimentation rate (ESR) of various blood samples from NAS patients and healthy controls. Furthermore, we manipulated the ESR by swapping the erythrocytes and plasma of different individuals, as well as replacing plasma with dextran. These measurements were complemented by clinical laboratory data and single-cell adhesion force measurements. Additionally, we followed theoretical modeling approaches. (3) Results: We show that the acanthocyte sedimentation rate (ASR) with a two-hour read-out is significantly prolonged in chorea-acanthocytosis and McLeod syndrome without overlap compared to the ESR of the controls. Mechanistically, through modern colloidal physics, we show that acanthocyte aggregation and plasma fibrinogen levels slow down the sedimentation. Moreover, the inverse of ASR correlates with the number of acanthocytes (R 2 = 0.61, p = 0.004). (4) Conclusions: The ASR/ESR is a clear, robust and easily obtainable diagnostic marker. Independently of NASs, we also regard this study as a hallmark of the physical view of erythrocyte sedimentation by describing anticoagulated blood in stasis as a percolating gel, allowing the application of colloidal physics theory

The Erythrocyte Sedimentation Rate and Its Relation to Cell Shape and Rigidity of Red Blood Cells from Chorea-Acanthocytosis Patients in an Off-Label Treatment with Dasatinib

Background: Chorea-acanthocytosis (ChAc) is a rare hereditary neurodegenerative disease with deformed red blood cells (RBCs), so-called acanthocytes, as a typical marker of the disease. Erythrocyte sedimentation rate (ESR) was recently proposed as a diagnostic biomarker. To date, there is no treatment option for affected patients, but promising therapy candidates, such as dasatinib, a Lyn-kinase inhibitor, have been identified. Methods: RBCs of two ChAc patients during and after dasatinib treatment were characterized by the ESR, clinical hematology parameters and the 3D shape classification in stasis based on an artificial neural network. Furthermore, mathematical modeling was performed to understand the contribution of cell morphology and cell rigidity to the ESR. Microfluidic measurements were used to compare the RBC rigidity between ChAc patients and healthy controls. Results: The mechano-morphological characterization of RBCs from two ChAc patients in an off-label treatment with dasatinib revealed differences in the ESR and the acanthocyte count during and after the treatment period, which could not directly be related to each other. Clinical hematology parameters were in the normal range. Mathematical modeling indicated that RBC rigidity is more important for delayed ESR than cell shape. Microfluidic experiments confirmed a higher rigidity in the normocytes of ChAc patients compared to healthy controls. Conclusions: The results increase our understanding of the role of acanthocytes and their associated properties in the ESR, but the data are too sparse to answer the question of whether the ESR is a suitable biomarker for treatment success, whereas a correlation between hematological and neuronal phenotype is still subject to verification

Multifractal analysis of HIV-1 genomes

Pathogens like HIV-1, which evolve into many closely related variants displaying differential infectivity and evolutionary dynamics in a short time scale, require fast and accurate classification. Conventional whole genome sequence alignment-based methods are computationally expensive and involve complex analysis. Alignment-free methodologies are increasingly being used to effectively differentiate genomic variations between viral species. Multifractal analysis, which explores the self-similar nature of genomes, is an alignment-free methodology that has been applied to study such variations. However, whether multifractal analysis can quantify variations between closely related genomes, such as the HIV-1 subtypes, is an open question. Here we address the above by implementing the multifractal analysis on four retroviral genomes (HIV-1, HIV-2, SIVcpz, and HTLV-1), and demonstrate that individual multifractal properties can differentiate between different retrovirus types easily. However, the individual multifractal measures do not resolve within-group variations for different known subtypes of HIV-1 M group. We show here that these known subtypes can instead be classified correctly using a combination of the crucial multifractal measures. This method is simple and computationally fast in comparison to the conventional alignment-based methods for whole genome phylogenetic analysis

Closure of DNA denaturation bubbles coupled to DNA elasticity

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Strand diffusion-limited closure of denaturation bubbles in DNA

The closure dynamics of a pre-equilibrated DNA denaturation bubble is studied using both Brownian dynamics simulations and an analytical approach. The numerical model consists of two semi-flexible interacting single strands (ssDNA) and a bending modulus which depends on the base-pair state, with double-strand DNA (dsDNA) segments being 50 times stiffer than ssDNA ones. For DNA lengths from N=20 to 100 base-pairs (bp) and initial bubble sizes of N−6 bp, long closure times of 0.1 to 4 μs are found, following a scaling law in N2.4. The bubble starts to close by a fast zipping which stops when the bubble reaches a highly bent metastable state of length around 10 bp. The limiting final step to complete closure is controlled by the dsDNA “arms” rotational diffusion, with closure occurring once they are nearly aligned. The central role of chain bending, which cannot be accounted for in one-dimensional models, is thus illuminated

Erythrocyte Sedimentation: Collapse of a High-Volume-Fraction Soft-Particle Gel

The erythrocyte sedimentation rate is one of the oldest medical diagnostic methods whose physical mechanisms remain debatable up to date. Using both light microscopy and mesoscale cell-level simulations, we show that erythrocytes form a soft-colloid gel. Furthermore, the high volume fraction of erythrocytes, their deformability, and weak attraction lead to unusual properties of this gel. A theoretical model for the gravitational collapse is developed, whose predictions are in agreement with detailed macroscopic measurements of the interface velocity

Erythrocyte sedimentation: Effect of aggregation energy on gel structure during collapse

The erythrocyte (or red blood cell) sedimentation rate (ESR) is commonly interpreted as a measure of cell aggregation and as a biomarker of inflammation. It is well known that an increase of fibrinogen concentration, an aggregation-inducing protein for erythrocytes, leads to an increase of the sedimentation rate of erythrocytes, which is generally explained through the formation and faster settling of large disjoint aggregates. However, many aspects of erythrocyte sedimentation conform well with the collapse of a colloidal gel rather than with the sedimentation of disjoint aggregates. Using experiments and cell-level numerical simulations, we systematically investigate the dependence of ESR on fibrinogen concentration and its relation to the microstructure of the gel-like erythrocyte suspension. We show that for physiological aggregation interactions, an increase in the attraction strength between cells results in a cell network with larger void spaces. This geometrical change in the network structure occurs due to anisotropic shape and deformability of erythrocytes and leads to an increased gel permeability and faster sedimentation. Our results provide a comprehensive relation between the ESR and the cell-level structure of erythrocyte suspensions and support the gel hypothesis in the interpretation of blood sedimentation