Authority and ethics: A case for estrangement in educational research and research education

This article focuses attention onto an underexamined issue in the literature on educational research ethics: how ethical authority is established in educational research. We address this from a perspective that disrupts naturalised approaches to ethics, arguing that rather than seeking ‘rights’ or ‘wrongs’ researchers are always tasked with constructing ethical stances. Attention can then be placed onto the array of embodied and objectified resources that might be recruited in establishing these. Through an engagement with published academic accounts of ethical reflection and decision-making, the article explores the ways that educational researchers achieve or sometimes question their ethical security in respect of their research activity. The analysis we present draws out the referential strategies that constitute ethical subjectivity and maps the diversity of anchoring points that might be recruited in this action. We also draw attention to the process of recontextualisation that is inevitable when one activity (or aspect of an activity) regards another, introducing necessary incoherence into ethical practice. The case we present celebrates rather than seeking to conceal or repair such disruption.</p

Poly(A) tail length regulation plays a major role in the OPMD <i>Drosophila</i> model.

<p>A) Genetic rescue of wing position phenotypes with genes involved in poly(A) tail regulation. Percentage of wing posture defects in the presence or absence of heterozygous mutants, scored at day 6. OPMD flies were <i>w</i>^<i>1118</i><i>; Act88F-PABPN1-17ala/+</i> raised at 25°C. Note that OPMD phenotypes (affected wing posture) are not visible at day 2 with this transgene. **** <i>p</i>-value <0.0001, using the χ² test. B) Genetic screen with heterozygous mutants. Wing posture defects were scored at day 6. OPMD flies were <i>w</i>^<i>1118</i><i>; Act88F-PABPN1-17ala/+</i> raised at 25°C. <i>wispy</i> encodes a cytoplasmic poly(A) polymerase expressed in the female germline [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005092#pgen.1005092.ref042" target="_blank">42</a>]. **** <i>p</i>-value <0.0001, *** <i>p</i>-value <0.001, ** <i>p</i>-value <0.01, ns: not significant, using the χ² test. C-D) PAT assays in control (<i>w</i>^<i>1118</i><i>; Mhc-Gal4/+</i>) and OPMD (<i>w</i>^<i>1118</i><i>; UAS-PABPN1-17ala/+; Mhc-Gal4/+</i>) adult thoraxes from flies raised at 18°C, at days 2 and 6. C) mRNAs encoding subunits of mitochondrial respiratory chain complexes. D) mRNAs encoding muscle sarcomeric proteins. Arrows indicate poly(A) tails of 12A. Profiles of PAT assays using the ImageJ software are shown.</p

Deregulation of the mitochondrial pathway in the OPMD <i>Drosophila</i> model.

<p>A) Number of deregulated genes in the transcriptome of PABPN1-17ala-expressing muscles using microarrays. B) Venn’s diagram of overlapping down-regulated genes at days 2, 6 and 11. C) GO term enrichment in down-regulated genes in PABPN1-17ala-expressing muscles. D) Number of nuclear genes encoding mitochondrial respiratory chain complex subunits down-regulated in PABPN1-17ala-expressing muscles. E) Quantification of levels of mRNAs encoding mitochondrial respiratory chain subunits of complexes I and II in control and PABPN1-17ala-expressing muscles at day 2, using RT-qPCR. mRNA levels were normalized to <i>sop</i> mRNA. Similar results were obtained when normalization was with <i>Cpr100A</i> mRNA which is expressed in thorax cuticle. Means are from three biological replicates, error bars represent standard deviation. * <i>p</i>-value <0.05, *** <i>p</i>-value <0.001, ns: not significant, using the Student’s t-Test.</p

Decrease of mitochondrial protein levels in human OPMD muscle biopsies.

<p>A) Distribution of differentially expressed proteins from OPMD and control human muscle samples, following a quantitative label-free LC-MS profiling proteomic analysis. Proteins are sorted according to their GO terms in "cellular component" using DAVID. Each square represents a protein. The value of the fold change is color-coded. Yellow to red, over-expressed in OPMD samples (maximum 20-fold); blue, under-expressed in OPMD samples (minimum -20-fold). B) Number of subunits of the mitochondrial respiratory chain complexes down-regulated in OPMD patient muscle biopsies. C) Quantification of levels of mRNAs encoding mitochondrial respiratory chain subunits in control and OPMD patient muscle biopsies, using RT-qPCR. Normalization was with B2M (Beta-2 microglobulin) mRNA. Box plots are from 4–7 muscle biopsies, the median is indicated as an horizontal line within the box. * <i>p</i>-value <0.05 using the Student’s t-Test. For ATP5B and ATP5F1, the median is lower in OPMD than in control biopsies, although the difference is not recorded as significant. D) Quantification of mitochondrial DNA on muscle biopsies, using qPCR. The MT-RNR1 mitochondrial gene was quantified. Normalization was with B2M nuclear DNA. Means are from four biological replicates, error bars represent standard deviation. ns: not significant, using the Student’s t-Test.</p

Deregulation of the mitochondrial pathway in the OPMD mouse model.

<p>A) Number of deregulated genes in A17.1 mouse skeletal muscles using microarrays. T1, 6 weeks; T2, 18 weeks; T3, 26 weeks. B) Venn diagram of overlapping down-regulated genes at T1, T2 and T3. C) Number of nuclear genes encoding mitochondrial respiratory chain complex subunits down-regulated in A17.1 mouse muscles. D) Quantification of levels of mRNAs encoding mitochondrial respiratory chain subunits in control (WT) and A17.1 quadriceps skeletal muscle at T1, using RT-qPCR. Normalization was with <i>Rplp0</i> mRNA. Means are from three biological replicates, error bars represent standard deviation. * <i>p</i>-value <0.05, ** <i>p</i>-value <0.01, *** <i>p</i>-value <0.001, using the Student’s t-Test. E) ePAT assays of mRNAs encoding mitochondrial proteins in control (WT) and A17.1 quadriceps skeletal muscles. Arrows indicate poly(A) tails of 12A. Accumulation of 12A poly(A) tails was visible in A17.1 muscles at T2 and/or T3. <i>RpL32</i> is a negative control mRNA encoding a ribosomal protein. Profiles of ePAT assays using the ImageJ software are shown.</p

Smg binds to mRNAs encoding mitochondrial proteins and is involved in mRNA down-regulation due to PABPN1-17ala expression.

<p>A) Quantification of mRNA levels in control and PABPN1-17ala-expressing thoraxes in the presence or absence of heterozygous <i>twin</i> or <i>smg</i> mutations at day 6, using RT-qPCR. mRNA levels were normalized to <i>Cpr100A</i> mRNA. Means of two biological replicates quantified three times. Error bars represent standard deviation. * <i>p</i>-value <0. 05, ** <i>p</i>-value <0.01, *** <i>p</i>-value <0.001, ns: not significant, using the Student’s t-Test. B) Confocal images of immunostaining of indirect flight muscles from wild type and <i>smg</i> mutant (<i>smg</i>^{<i>PL00423</i>}<i>/Df(3L)scf-R6</i>) adult flies with anti-Smg (green). DNA was visualized with DAPI (blue). Smg protein levels were strongly reduced in <i>smg</i>^{<i>PL00423</i>}<i>/Df(3L)scf-R6</i> muscles compared to wild-type muscles. Scale bars: 5 μm. C) Western blots of protein extracts from wild-type and <i>smg</i> mutant thoraxes revealed with guinea pig (gp) and rabbit (rb) anti-Smg, showing the presence of Smg and its lower level in <i>smg</i> mutant. α-Tubulin was used as a loading control. D) Smg immunoprecipitations (IP) in <i>UASp-CCR4-HA/Mhc-Gal4</i> adult thoraxes, either in the presence or the absence of RNase A. Mock IP was with rabbit IgG. Input is the protein extract prior to immunoprecipitation. Western blots revealed with anti-Smg, anti-HA and anti-PABP2, showing CCR4-HA co-precipitation and the lack of PABP2 co-precipitation. E) Quantification of mRNA enrichment in Smg IP using RT-qPCR. The ratio of mRNA/control mRNA was set to 1 in the mock IP (black line). Normalization was with <i>sop</i> mRNA. <i>RpL32</i> mRNA is a negative control, which is not enriched in Smg IP. Means are from two independent IP quantified three times. Error bars represent standard error to the mean. F) Means of SRE scores of mRNAs down-regulated in PABPN1-17ala-expressing muscles and annotated with the term "mitochondrion" compared to those of control mRNAs, in <i>Drosophila</i> (left panel) and mouse (right panel). Twenty times 98 <i>Drosophila</i> and 10 times 407 mouse control genes were used. Error bars represent standard deviation.</p

Nuclear PABPN1 aggregates in the OPMD <i>Drosophila</i> model.

<p>A) Genetic rescue of wing position phenotypes with <i>twin</i>^{<i>KG00877</i>} hererozygous mutant. Percentage of wing posture defects in the presence or absence of <i>twin</i>^{<i>KG00877</i>}/+, scored at day 6. OPMD flies were <i>w</i>^<i>1118</i><i>; UAS-PABPN1-17ala/+; Mhc-Gal4/+</i> raised at 18°C. *** <i>p</i>-value <0.0001, using the χ² test. B) Immunostaining of indirect flight muscles from OPMD (<i>w</i>^<i>1118</i><i>; UAS-PABPN1-17ala/+; Mhc-Gal4/+)</i> and OPMD; <i>twin</i>^{<i>KG00877</i>}<i>/+</i> (<i>w</i>^<i>1118</i><i>; UAS-PABPN1-17ala/+; Mhc-Gal4/twin</i>^{<i>KG00877</i>}) adult flies at day 6 with anti-PABPN1 (green), showing nuclear aggregates. DNA was visualized with DAPI (blue). Nuclei are outlined with a dotted line. C) Quantification of PABPN1 nuclear aggregates. (Top) Percentages of nuclei with a nuclear PABPN1 aggregate in OPMD and OPMD; <i>twin</i>^{<i>KG00877</i>}<i>/+</i> indirect flight muscles (genotypes as in B) *** <i>p</i>-value <0.0001, using the χ² test. (Bottom) Quantification of nuclear aggregate areas. Each nuclear aggregate was delimited in a focal plan and the surface was calculated using ImageJ. Mean values of the areas with standard errors of the mean are indicated in arbitrary units Distribution of cross-sectional areas is shown as box plots (right), the median is indicated as an horizontal line within the box. ns: not significant, using the Student’s t-Test. Thoracic muscles were stained with anti-PABPN1 and DAPI and nuclear aggregates were visualized and scored using both staining. Quantification was from three independent experiments.</p

Molecular model of OPMD.

<p>The first molecular defect in OPMD would be a general decrease in the cleavage/polyadenylation reaction resulting from affected PABPN1 function. This would not lead to a reduction of mRNA levels at steady-state for most mRNAs, but would lead to such a decrease for mRNAs actively deadenylated by Smg/CCR4-NOT, among which mRNAs involved in mitochondrial function. This would result in mitochondrial dysfunction and in turn affected muscle function. Additional mechanisms of mRNA regulation occurring downstream of the first defect in cleavage/polyadenylation are also expected to be involved. CPSF, Cleavage and polyadenylation specificity factor; CstF, Cleavage stimulation factor; PAP, poly(A) polymerase.</p

Defective cleavage at poly(A) sites in muscles expressing PABPN1-17ala and in the <i>Pabp2</i> mutant.

<p>A) Schematic representation of primers (arrows) used to quantify uncleaved pre-mRNA. B) Quantification of uncleaved pre-mRNAs in control and PABPN1-17ala-expressing thoraxes at day 2, using RT-qPCR. Uncleaved RNAs were normalized to <i>Cpr100A</i> uncleaved RNA. <i>Cpr100A</i> is expressed in the cuticle and its expression remains unaffected by expression of PABPN1-17ala in muscles. Means of three biological replicates quantified three times. For (B) and (C), error bars represent standard deviation. * <i>p</i>-value <0.05, ** <i>p</i>-value <0.01, *** <i>p</i>-value <0.001, ns: not significant, using the Student’s t-Test. C) Quantification of uncleaved pre-mRNAs in control (<i>w</i>^<i>1118</i>) and <i>Pabp2</i> (<i>Pabp2</i>^<i>55</i><i>/Df(2R)CA53</i>) mutant first instar larvae, using RT-qPCR. Uncleaved RNAs were normalized to <i>RpS6</i> uncleaved RNA. The levels of <i>RpS6</i> uncleaved RNA normalized to <i>sop</i> mRNA were unaffected in <i>Pabp2</i> mutant larvae (right panel). Means of three biological replicates quantified three times.</p

Reduced mitochondrial activity in muscles expressing PABPN1-17ala.

<p>A) Quantification of mitochondrial DNA (mtDNA) content in control and PABPN1-17ala- expressing thoraxes at day 2, using qPCR. Three mitochondrial genes (<i>mt</i>:<i>CoI</i>, <i>mt</i>:<i>CoII</i> and <i>mt</i>:<i>cyt-b</i>) were analysed. Mitochondrial DNA levels were normalized to <i>RpL32</i> DNA. Means are from three biological replicates, error bars represent standard deviation. ns: not significant, using the Student’s t-Test. B) Activities of mitochondrial respiratory chain complexes were analysed by spectrophotometry from control and PABPN1-17ala-expressing thoraxes (genotypes as in A). Means are from five biological replicates, error bars represent standard deviation. * <i>p</i>-value <0.05, *** <i>p</i>-value <0.001, ns: not significant, using the Student’s t-Test. C) Quantification of mRNA levels of transcription factors regulating mitochondrial function in control and PABPN1-17ala-expressing thoraxes at day 2, using RT-qPCR (genotypes as in A). mRNA levels were normalized to <i>sop</i> mRNA. Means are from two biological replicates quantified three times, error bars represent standard deviation. *** <i>p</i>-value <0.001, ns: not significant, using the Student’s t-Test. D) Overexpression of <i>ewg</i> and <i>dERR</i> genes reduces the wing posture phenotypes of flies expressing PABPN1-17ala. Wing posture phenotypes were scored at day 6, at 18°C from OPMD (<i>w</i>^<i>1118</i><i>; UAS-PABPN1-17ala/+; Mhc-Gal4/+</i>), OPMD; <i>OE-spargel</i> (<i>w</i>^<i>1118</i><i>; UAS-PABPN1-17ala/+; Mhc-Gal4/</i>s<i>pargel</i>^{<i>EY05931</i>}), OPMD; <i>OE-delg</i> (<i>w</i>^<i>1118</i><i>; UAS-PABPN1-17ala</i>, <i>UAS-delg-HA/+; Mhc-Gal4/+</i>), OPMD; <i>OE-ewg</i> (<i>w</i>, <i>ewg</i>^{<i>EY05137</i>}<i>/Y; UAS-PABPN1-17ala/+; Mhc-Gal4/+</i>) and OPMD; <i>OE-dERR</i> (<i>w</i>^<i>1118</i><i>; UAS-PABPN1-17ala/+; Mhc-Gal4/dERR</i>^<i>G4389</i>) flies (n > 130). *** <i>p</i>-value <0.001, using the χ² test.</p