Young children's understanding of disabilities: the influence of development, context and cognition

Throughout Europe, educational support for children with disabilities has moved towards a model of inclusive education. Such policy changes mean that for all children there will be an increased likelihood of working with and encountering children with differing disabilities and difficulties. Previous research had indicated that children had poorly differentiated views of developmental differences. The present study investigated children?s representations of different disabilities. Seventy-nine 8-9 and 10-11 year old Greek children from an urban school and a rural school completed an attitudes toward school inclusion rating scale and a semi-structured interview. Responses to the attitude scale provided generally positive views of educational inclusion. However, children were less positive about activities that might directly reflect upon themselves. Children?s responses in the interviews indicated that they were developing rich representations of differences and diversities. Children had the greatest understanding of sensory and physical disabilities, followed by learning disabilities. There was limited knowledge of dyslexia and hyperactivity and no child was familiar with the term autism. Both groups of children identified a range of developmental difficulties, with older children being more aware of specific learning disabilities, their origin and impact. Results are discussed in terms of children?s developing knowledge systems and the implications for educational practices

<i>Fxr1</i>-depletion does not impair myoblasts viability but specifically induces accumulation in G0/G1 phase to the detriment of mitosis.

<p>(A) PI incorporation in living siFxr1- or siControl-transfected cultures and subsequent FACS analysis was performed to show that viability of the culture is not affected by <i>Fxr1</i>-depletion. (B) MTT colorimetric assay show that the proliferation abilities of C2C12 cells are significantly impaired by <i>Fxr1</i>-depletion. (C) FACS analysis of the Propidium Iodure-stained DNA content of C2C12 cells transfected with siControl or siFxr1. Cells were analysed in asynchronous conditions or following synchronisation treatment for 8 hrs with the cell cycle blocker mimosine (late G1) followed by 16 hrs release in normal growth medium (D). In asynchronous conditions, cell cycle distribution is similar in siControl or siFxr1 transfected cells. Synchronisation of cells allows detecting significant differences in the distribution of the cells in the various cell cycle absence of FXR1P: increase in the G0/G1 proportion and decrease in the G2/M. Data are presented as means ± SEM of n = 4 experiments, with FACS analysis of a minimal cell population of 15,000 for each condition and each experiment. The asterisk (*) indicates p<0.05 of a Mann & Whitney test.</p

Knockdown of FXR1P induces premature cell cycle exit of myoblasts.

<p>(A) Immunofluorescence analysis of C2C12 cells transfected with siControl or siFxr1. Nuclei are stained with DAPI (blue) and cells expressing the proliferation marker Ki67 are labelled with FITC antibody (green). Scale bar: 75 µm. (B) Quantification of the number of DAPI-stained nuclei. (C) Quantification of the number of Ki67-positive cells over total number of nuclei quantified in (B). Quantification was performed using a macro developed with the ImageJ software. Data presented are mean of n = 4 experiments with analysis of 10 optical fields for each condition and each experiment. The asterisk (*) indicates p<0.05 for the Student T-test.</p

The γ portion of <i>p21</i> mRNA 3′UTR modulates the stability of the mRNA that is potentiated by FXR1P depletion.

<p>(A) Scheme of the constructs bearing various portions of <i>p21</i> mRNA 3′UTR (α, β and γ) used for luciferase assays. (B) Effect of <i>p21</i> 3′UTR−α, −β and −γ fragments on <i>Renilla luciferase</i> (<i>Ren</i>) mRNA levels in C2C12 cells transfected with control siRNAs (siControl) or siRNAs targeting <i>Fxr1</i> (siFxr1). Quantitative RT-PCR analysis of the levels of <i>Ren</i> mRNA normalised to <i>Firefly</i> (<i>Luc</i>) mRNA relative to the empty construct are presented. In siControl cells, only the γ fragment significantly increased <i>Ren</i> mRNA levels, this effect is potentiated by <i>Fxr1</i> depletion with siFxr1. In contrast, the α and β fragment have no effect on <i>Ren</i> mRNA levels, in the presence or absence of FXR1P. The results are presented as the means ±SEM of 4 experiments. (C) Effect of <i>p21</i> 3′UTR and its α, β and γ fragments on Renilla Luciferase activity in C2C12 cells transfected with control siRNAs (siControl) or siRNAs targeting <i>Fxr1</i> (siFxr1). Results presented here represent the mean of the ratio of Luc-FL, Luc-α, Luc-β and Luc-γ to Luc-empty signal. In siControl cells, only the γ fragment significantly increased luciferase activity. In siFxr1 transfected cells compared to controls, the β and γ fragments increased luciferase activity, while the α fragment has no effect. However, the amplitude of variation is greater with the γ fragment and this effect is potentiated by <i>Fxr1</i> depletion. Six independent experiments in triplicate for each transfection were quantified. For each transfection, Renilla was normalized to Firefly luciferase activity. RLU, relative luciferase units. (D) <i>Fxr1</i>-depletion increases the stability of endogenous <i>p21</i> mRNA. C2C12 transfected with siControl (empty squares) or siFxr1 (black squares) were treated with the transcription inhibitor actinomycin D for 8 hrs. <i>p21</i> mRNA levels were determined by quantitative RT-PCR at several time points and normalised to levels before treatment (t0). Percentage of remaining mRNA is plotted using a semi-log scale. Data presented represent the mean of n = 3 experiments. The asterisks * indicate p<0.05 of the Mann & Whitney test, while # and ## indicate respectively p<0.05 and p<0.01 of the Wilcoxon test.</p

FXR1P selectively binds <i>in vitro</i> to the distal portion of <i>p21</i> mRNA 3′ UTR and associates <i>in vivo</i> with <i>p21</i> mRNA.

<p>(A) Scheme of the various portions of <i>p21</i> mRNA 3′ UTR (α, β and γ) used for <i>in vitro</i> binding assays. Note that the α fragment contains a characterized ARE motif. (B) Nitrocellulose filter binding assays to determine the portion of <i>p21</i> mRNA bound by FXR1P. Radiolabeled mRNA probes were incubated with increasing concentrations of recombinant FXR1P Isoe protein, the amount of radioactive probes recovered on filters after binding reaction is then plotted against the concentration of proteins. The portion of <i>FMR1</i> mRNA called N19 (known to be bound by FXR1P) and its truncated version (N19Δ35) were used as controls. This reveals that the distal portion of <i>p21</i> 3′UTR (γ fragment) and N19 are selectively bound by FXR1P. Both the α and β fragments from the 3′UTR of <i>p21</i> remain at background levels comparable to N19Δ35 binding to FXR1P. (C) Western-blot analysis of UV-crosslinking and immunoprecipitation (CLIP) assay performed on C2C12 lysates using polyclonal antibodies raised against the C-terminus of FXR1P (#830) and control rabbit IgG (R). Input lysates (lanes 1 & 2, Input, 1/50^th), immunoprecipitates (lanes 3 & 4, IP, 1/5^th) and post-immunoprecipitation supernatants (lanes 5 & 6, post, 1/50^th) were probed for FXR1P using the 3FX antibody. A selective enrichment in FXR1P medium and long isoforms is observed in #830 immunoprecipitate (lane 3), concomitant with a depletion in these isoforms in the post-immunoprecipitation supernatant (lane 4) as compared to corresponding controls (lane 3 & 5). (D) RT-PCR analysis of mRNAs associated with FXR1P complexes. RNA was extracted from input and immunoprecipitate fractions described in (C), and used as template for RT-PCR. RT-PCR products obtained from inputs and immunoprecipitations respectively from control with rabbit IgG (Lanes 1, 3) and immunoprecipitation of FXR1P using #830 (Lanes 2, 4) were separated and visualized by agarose gel electrophoresis. This reveals that <i>p21</i> mRNA is selectively enriched in the #830 immunoprecipitates, while the mRNAs encoding the myogenic determination factors Myogenin and MyoD or the unrelated mRNA encoding β-tubulin are not recovered in any immunoprecipitates. The symbol # indicates aspecific PCR products corresponding to <i>β-tub</i> primers dimers. DNA molecular weight markers presented on the gels are respectively 100, 200, 300, 400, 500, 600, 800 and 1000 bp.</p

The γ portion of <i>p21</i> mRNA 3′UTR contains an evolutionary conserved G-quadruplex structure with mRNA stabilization properties.

<p>(A) Cation-dependent termination of reverse transcription in the 3′-UTR full-length (FL) or γ fragment of <i>p21</i> mRNA. Strong and weak pauses of reverse transcriptase (RT) are, respectively, indicated by large and thin arrows. Numbers correspond to positions of RT pauses, position +1 being the first nucleotide following the stop codon. (B) Localization and conservation of the G-quadruplex structure detected in (A) on the sequences of <i>p21</i> 3′UTR from <i>Mus musculus</i> (Mmu) and <i>Homo sapiens</i> (Hsa). (C) Scheme of the constructs used for luciferase assays bearing the conserved G-quadruplex of γ <i>p21</i> mRNA (boxed) and two versions where the G-quadruplex has been deleted partially (Δ9) and fully (Δ38). (D) Effect of <i>p21</i> 3′UTR G-quadruplex and its deletions on <i>Renilla luciferase</i> (<i>Ren</i>) mRNA stability in C2C12 cells. Quantitative RT-PCR analysis of the levels of <i>Ren</i> mRNA normalised to <i>Firefly</i> (Luc) mRNA relative to the levels of the empty construct. The γ fragment bearing the G-quadruplex significantly increases <i>Ren</i> mRNA levels relative to empty vector. Partial or full deletion of the G-quadruplex sequence strongly increases <i>Ren</i> mRNA levels, both relative to empty vector and to the G-quadruplex bearing fragment. The results are presented as the mean of 4 experiments (±SEM). The asterisks * and # indicate p<0.05 respectively of the Wilcoxon test or of the Mann & Whitney test.</p

FXR1P overexpression in <i>Fxr1</i>-depleted C2C12 cells restores <i>p21</i> mRNA levels to normal.

<p>(A) Western-blot analysis (upper panel) of C2C12 cells transfected with empty pTL1 vector or pTL1.FXR1 Isoe (pTL1.Isoe) construct indicate a strong expression of FXR1P long isoform Isoe in transfected myoblasts. Quantitative RT-PCR (lower panel) reveals a significant decrease of <i>p21</i> mRNA levels in C2C12 myoblasts overexpressing FXR1 Isoe, as compared to control. Data are presented as means ± SEM of n = 3 independent experiments. (B) Western-blot analysis (upper panel) of C2C12 cells transfected with control siRNA (siC) or siFxr1 (siFx) and empty pTL1 vector or a mutated version of pTL1.FXR1 Isoe (pTL1.Isoe*) bearing 4 mismatches in siFxr1 recognition sequence indicate a reexpression of FXR1P long isoform Isoe in <i>Fxr1</i>-depleted transfected myoblasts. In the western blot FXR2P is indicated by (*) Quantitative RT-PCR (lower panel) reveals a significant increase of <i>p21</i> mRNA levels in C2C12 myoblasts transfected with siFxr1 (siFx) and the empty vector (pTL1), as compared to control. This increase is restored to normal levels when FXR1P Isoe expression is rescued by transfection of pTL1.FXR1 Isoe. Data are presented as means ± SEM of n = 3 independent experiments. The asterisks * indicate p<0.05 of the Wilcoxon paired test, ns indicates non significance.</p

FXR1P depletion in C2C12 cells and in myoblasts derived from FSHD myopathic patients biopsies contributes to a consistent increase in <i>p21</i> mRNA that translates into enhanced p21 protein levels.

<p>(A) Quantitative RT-PCR reveals a significant increase of <i>P21</i> mRNA in FSHD myoblats relative to control individuals. Data are presented as means ± SEM of n = 3 individuals/group. (B) Representative western-blot of p21 protein levels in siControl (siC) or siFxr1 (siFx)-transfected C2C12 cells. Densitometric quantification of western-blots reveal that depletion of FXR1P by siRNA transfection (siFxr1) leads to a significant increase of p21 protein levels relative to siControl-transfected cells. Data are presented as means ± SEM of n = 4 experiments. (C) Representative western-blot of P21 protein levels in FSHD patients and control individuals. Densitometric quantification of western-blots reveals that muscle biopsies of FSHD patients display a significant increase of P21 protein relative to controls. Data are presented as means ± SEM of n = 3 individuals/group. The asterisks * and ** indicate respectively p<0.05 and p<0.01 of the Mann & Whitney test.</p

^99mTcO₄⁻-, Auger-Mediated Thyroid Stunning: Dosimetric Requirements and Associated Molecular Events

<div><p>Low-energy Auger and conversion electrons deposit their energy in a very small volume (a few nm³) around the site of emission. From a radiotoxicological point of view the effects of low-energy electrons on normal tissues are largely unknown, understudied, and generally assumed to be negligible. In this context, the discovery that the low-energy electron emitter, ^99mTc, can induce stunning on primary thyrocytes <i>in vitro</i>, at low absorbed doses, is intriguing. Extrapolated <i>in vivo</i>, this observation suggests that a radioisotope as commonly used in nuclear medicine as ^99mTc may significantly influence thyroid physiology. The aims of this study were to determine whether ^99mTc pertechnetate (^99mTcO₄⁻) is capable of inducing thyroid stunning <i>in vivo</i>, to evaluate the absorbed dose of ^99mTcO₄⁻ required to induce this stunning, and to analyze the biological events associated/concomitant with this effect. Our results show that ^99mTcO₄⁻–mediated thyroid stunning can be observed <i>in vivo</i> in mouse thyroid. The threshold of the absorbed dose in the thyroid required to obtain a significant stunning effect is in the range of 20 Gy. This effect is associated with a reduced level of functional Na/I symporter (NIS) protein, with no significant cell death. It is reversible within a few days. At the cellular and molecular levels, a decrease in NIS mRNA, the generation of double-strand DNA breaks, and the activation of the p53 pathway are observed. Low-energy electrons emitted by ^99mTc can, therefore, induce thyroid stunning <i>in vivo</i> in mice, if it is exposed to an absorbed dose of at least 20 Gy, a level unlikely to be encountered in clinical practice. Nevertheless this report presents an unexpected effect of low-energy electrons on a normal tissue <i>in vivo</i>, and provides a unique experimental setup to understand the fine molecular mechanisms involved in their biological effects.</p></div

Longitudinal follow up of ^99mTcO₄⁻-mediated thyroid stunning by SPECT/CT.

<p>^99mTcO₄⁻ uptake in the thyroid was measured by SPECT/CT in 12 mice at day 0 (D0) and again at day 1 (D1, n = 3), day 2 (D2, n = 3), day 4 (D4, n = 3), or day 8 (D8, n = 3). The second scan was performed by injecting a dose of 50 MBq ^99mTcO₄⁻. (A) Representative SPECT/CT images of ^99mTcO₄⁻ uptake by the thyroid. The images are normalized (100%) to the voxel with the highest activity in the series. (B) Thyroid uptake presented as percentage of the value at day 0± SD. (*: p<0.05; ***: p<0.001).</p