Material manifestations of memory: genocide and commemoration in Rwanda

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Introduction

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Education for sustainability as activity: Towards explaining sustainability curriculum and policy enactment in rural and regional Australia

<p>Paper presented at AARE 2015</p

<i>ND42</i> or <i>sicily</i> overexpression can rescue <i>pink1</i> mutant phenotypes in flies.

<p>Overexpression of two <i>ND42</i> transgenes, <i>ND42</i> and <i>ND42-HA</i>, in a wild type background has no effect on climbing (A) or flight behavior (B). In <i>pink1^B9</i> mutants, climbing (C) and flight ability (D), normalized to control, is significantly rescued by overexpression <i>ND42</i> or <i>sicily</i>. Histograms indicate mean ± s.e.m. (E) Transmission electron microscopy of flight muscle shows partial rescue of mitochondrial disruption. Scale bar  = 1 µm. Overexpression was driven by the ubiquitous driver <i>da-GAL4</i>. Control genotype is <i>da-GAL4</i>/+. Number of animals tested, n>50. * <i>P</i><0.05, *** <i>P</i><0.001, **** <i>P</i><0.0001, One-way ANOVA with Bonferroni correction. Comparisons are with control (A, B) or <i>pink1^B9</i> mutants (C, D).</p

Analysis of ND42 Ser-250 phospho-variant rescue of <i>pink1</i> mutant climbing defect and complex I deficiency.

<p>Transgenes from different sources, labeled ‘HB’ <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004815#pgen.1004815-Zhang1" target="_blank">[29]</a> and ‘BDS’ <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004815#pgen.1004815-Morais2" target="_blank">[25]</a>, expressing wild type <i>ND42</i> (WT), non-phosphorylatable (SA) or phospho-mimetic (SD) variants of Ser-250 were tested for rescue of climbing (A), flight (B) and complex I (C) deficiencies in <i>pink1^B9</i> mutants. For comparison, transgenic expression of the yeast complex I equivalent, <i>NDI1</i>, was also tested. Overexpression was driven by the ubiquitous driver <i>da-GAL4</i>. Control genotype is <i>da-GAL4</i>/+. * <i>P<</i>0.05, ** <i>P<</i>0.01, **** <i>P</i><0.0001, One-way ANOVA with Bonferroni correction. Comparisons are with <i>pink1^B9</i> mutants unless otherwise shown.</p

List of genes that suppress <i>pink1</i> dsRNA induced mitochondrial fusion.

<p>List of genes that suppress <i>pink1</i> dsRNA induced mitochondrial fusion.</p

<i>ND42</i> or <i>sicily</i> overexpression does not rescue <i>parkin</i> mutant phenotypes in flies.

<p>In <i>park²⁵</i> mutants, climbing (A) and flight ability (B), normalized to control, is not rescued by <i>ND42</i> or <i>sicily</i> overexpression. Histograms indicate mean ± s.e.m. (C) Transmission electron microscopy of flight muscle shows widespread disruption of mitochondrial integrity. Scale bar  = 1 µm. Overexpression was driven by the ubiquitous driver <i>da-GAL4</i>. Control genotype is <i>da-GAL4</i>/+. Number of animals tested, n>50. * <i>P</i><0.05, *** <i>P</i><0.001, **** <i>P</i><0.0001, One-way ANOVA with Bonferroni correction. Comparisons are with <i>pink1^B9</i> mutants.</p

<i>ND42</i> RNAi but not other complex I subunits phenocopies <i>pink1</i> RNAi.

<p>(A) <i>ND42</i> RNAi in <i>Drosophila</i> S2R+ cells stained with MitoTracker Red causes tubulation of the mitochondrial network, similar to <i>pink1</i> RNAi. <i>ND42</i> RNAi does not further perturb morphology in conjunction with <i>pink1</i> RNAi. (B) Quantification of mitochondrial morphology as in A, scored in triplicate experiments. (C) RNAi of selected subunits of complex I or rotenone treatment do not phenocopy <i>pink1</i> RNAi. (D) Quantification of morphology scored in triplicate experiments as in C. Histograms indicate mean ± s.d. of triplicate experiments. Inverted, binary images are shown below each fluorescence image to aid clarity of mitochondrial morphology. n>30 cells per experiment. Scale bar  = 10 µm.</p

List of genes that phenocopy <i>pink1</i> dsRNA induced mitochondrial fusion.

<p>List of genes that phenocopy <i>pink1</i> dsRNA induced mitochondrial fusion.</p

<i>NDUFA10</i> knockdown slightly reduces CCCP-induced Parkin translocation and mitophagy.

<p>(A) In HeLa cells stably transfected to express YFP-Parkin, before CCCP toxification (0 h) YFP-Parkin (green) has a diffuse cytoplasmic distribution in control (ctrl) siRNA treated cells. Following 4 h CCCP, YFP-Parkin co-localizes with mitochondria labeled with ATP5A immunostaining (red). <i>PINK1</i> siRNA treatment almost completely abolishes YFP-Parkin translocation. (B) Quantification of YFP-Parkin translocation as in A. (C) Stably transfected HeLa cells expressing YFP-Parkin, before CCCP treatment (0 h, ctrl) have a normal (“High”) mitochondrial content. Following 24 h treatment with CCCP, a high proportion of control cells (ctrl) show complete degradation (“none”) or perinuclear aggregated (“low”) mitochondria, visualized by ATP5A immunostaining (red). <i>PINK1</i> siRNA treatment almost completely abolishes mitophagy. (D) Quantification of mitochondrial content as in C. Histograms indicate mean ± s.d. of triplicate experiments. n>30 cells per experiment. Scale bar  = 20 µm. * <i>P<</i>0.05, ** <i>P<</i>0.01, *** <i>P<</i>0.001, Student's <i>t</i>-test compared with respective conditions in control siRNA.</p