Emergence of collective modes and tri-dimensional structures from epithelial confinement.

Many in vivo processes, including morphogenesis or tumour maturation, involve small populations of cells within a spatially restricted region. However, the basic mechanisms underlying the dynamics of confined cell assemblies remain largely to be deciphered and would greatly benefit from well-controlled in vitro experiments. Here we show that confluent epithelial cells cultured on finite population-sized domains, exhibit collective low-frequency radial displacement modes as well as stochastic global rotation reversals. A simple mathematical model, in which cells are described as persistent random walkers that adapt their motion to that of their neighbours, captures the essential characteristics of these breathing oscillations. As these epithelia mature, a tri-dimensional peripheral cell cord develops at the domain edge by differential extrusion, as a result of the additional degrees of freedom of the border cells. These results demonstrate that epithelial confinement alone can induce morphogenesis-like processes including spontaneous collective pulsations and transition from 2D to 3D.

Padronizaçäo da técnica antigenemia para a detecçäo do citomegalovirus / Padronization of antigenemia metodology for detection of citomegalovirus

Antigenemia para citomegalovirus (CMV) é um importante marcador de evoluçäo de doença e eficácia de tratamento em pacientes imunocomprometidos. O objetivo desse estudo foi comparar diferentes técnicas de processamentos e de imunomarcaçäo para a detecçäo da proteína da matrix do CMV pp65 em leucócitos do sangue periférico. Amostras de sangue coletadas de pacientes submetidos ao transplante de medula óssea (TMO) foram processadas e imunomarcadas por diferentes metodologias. Separou-se leucócitos de sangue periférico, utilizando-se duas técnicas, sedimentaçäo espontânea a 37§C (Processamento 1) e a sedimentaçäo com Dextran (Processamento 2), após a lise eritrocitária procedeu-se a contagem dos leucócitos, ajuste da densidade celular e o preparo das lâminas que continham (2x10 a quinta potência) células, por citocentrifugaçäo. As lâminas, obtidas através das diferentes técnicas de processamentos, foram coradas, utilizando-se a metodologia de Imunoperoxidase (IP) e os resultados obtidos foram analisados de acordo com parâmetros qualitativos e quantitativos. Também avaliou-se duas diferentes técnicas de imunomarcaçäo: IFI Imunofluorescência Indireta) e IP onde comparou-se o número de células positivas. Obteve-se lâminas de melhor qualidade pelo processamento 1 e um maior número de célu;as positivas com técnica de IP

Linear stability of scroll waves.

A full linear stability of a straight scroll wave in an excitable medium is presented. The five eigenmode branches which correspond to deformation in the third dimension of the five main modes of two-dimensional (2D) spiral dynamics are found to play a dominant role. For untwisted scroll waves, modulations in the third dimension have stabilizing or destabilizing effects on the different modes depending on the parameter regimes, in partial agreement with previous predictions. The influence of twist on the different branches is investigated. In particular, the sproing instability is seen to arise from the twist-induced deformation of the translation branches above a threshold twist.

Fast global oscillations in networks of integrate-and-fire neurons with low firing rates.

We study analytically the dynamics of a network of sparsely connected inhibitory integrate-and-fire neurons in a regime where individual neurons emit spikes irregularly and at a low rate. In the limit when the number of neurons --> infinity, the network exhibits a sharp transition between a stationary and an oscillatory global activity regime where neurons are weakly synchronized. The activity becomes oscillatory when the inhibitory feedback is strong enough. The period of the global oscillation is found to be mainly controlled by synaptic times but depends also on the characteristics of the external input. In large but finite networks, the analysis shows that global oscillations of finite coherence time generically exist both above and below the critical inhibition threshold. Their characteristics are determined as functions of systems parameters in these two different regions. The results are found to be in good agreement with numerical simulations.

Theory of spiral wave dynamics in weakly excitable media: asymptotic reduction to a kinematic model and applications.

In a weakly excitable medium, characterized by a large threshold stimulus, the free end of an isolated broken plane wave (wave tip) can either rotate (steadily or unsteadily) around a large excitable core, thereby producing a spiral pattern, or retract, causing the wave to vanish at boundaries. An asymptotic analysis of spiral motion and retraction is carried out in this weakly excitable large core regime starting from the free-boundary limit of the reaction-diffusion models, valid when the excited region is delimited by a thin interface. The wave description is shown to naturally split between the tip region and a far region that are smoothly matched on an intermediate scale. This separation allows us to rigorously derive an equation of motion for the wave tip, with the large scale motion of the spiral wave front slaved to the tip. This kinematic description provides both a physical picture and exact predictions for a wide range of wave behavior, including (i) steady rotation (frequency and core radius), (ii) exact treatment of the meandering instability in the free-boundary limit with the prediction that the frequency of unstable motion is half the primary steady frequency, (iii) drift under external actions (external field with application to axisymmetric scroll ring motion in three dimensions, and spatial- or/and time-dependent variation of excitability), and (iv) the dynamics of multiarmed spiral waves with the prediction that steadily rotating waves with two or more arms are linearly unstable. Numerical simulations of FitzHugh-Nagumo kinetics are used to test several aspects of our results. In addition, we discuss the semiquantitative extension of this theory to finite cores and pinpoint mathematical subtleties related to the thin interface limit of singly diffusive reaction-diffusion models.

Regulation of the multiple promoters of the human aldolase A gene: response of its two ubiquitous promoters to agents promoting cell proliferation.

The human aldolase A gene is transcribed from three distinct promoters, the two ubiquitous promoters PN and PH and the muscle specific promoter PM. In the present study, we investigate further aldolase A mRNA structure and expression. We demonstrate that the upstream N-type exon is, in fact, extremely heterogeneous. RNAse H mapping experiments permit quantification of relative abundance of N, M, and H type mRNAs and show that the level of transcripts containing the downstream H-type exon is at least 30 times higher than that of those containing N exon, in all tissues tested. Aldolase A level is up-regulated in proliferating cells. Here we show that both N and H type mRNAs, although barely detectable in normal liver, are highly expressed in human hepatomas biopsies. Furthermore, in human lymphocytes, N-type mRNA level is enhanced by serum treatment, while in cultured Hep G2 cells, both N-type and H-type mRNA levels are increased by serum and by the tumor promoting agent PMA. Using CAT constructs in transfection experiments, we demonstrate that the H exon plus its upstream region can function autonomously: the 420 base pairs upstream of the H exon are sufficient to confer to promoter PH an efficiency comparable that of the complete SV40 early promoter and enhancer in two cell lines.

Characterization of three optional promoters in the 5' region of the human aldolase A gene.

We undertook cloning and sequencing of the 5' portion of the human aldolase A gene to elucidate the mechanisms that govern synthesis of its different mRNAs. The sequenced gene is the only active gene in human-rodent fibroblastic somatic hybrids, while the other aldolase A-related sequences are inactive. S1 mapping and primer extension analysis enabled us to demonstrate that three promoter regions were implicated in the initiation of different aldolase A mRNAs, differing only in their 5' non-coding extremities. A distal promoter, N (non-specific), governs the synthesis of a 5' non-coding region of 142 bases composed of two exons, N1 and N2, which are found in a variety of tissues. A median promoter, M (muscle), is only active in skeletal muscle, and initiates the transcription by a 5' non-coding exon of 45 bases. Finally, a proximal promoter, H (housekeeping), contained in a "G + C-rich island", permits transcription of three colinear mRNAs containing 172, 126 or 112 bases of 5' non-coding sequence; their expression seems ubiquitous. These three promoters are arranged in 1.5 X 10(3) base-pairs of DNA. Homologies between rat and human genomic sequences and the absence of homology between promoters or 5' non-coding exons of the same species exclude a recent duplication of the promoter regions.