Building counter culture: the radical praxis of social movement milieux

This thesis falls into two parts. The first (chapters one to three) states the problematic of the research, develops a critique of the dominant “social movements” literature as unhelpful for understanding the counter culture and argues that the latter can more effectively be theorised in terms of the implicit theory of social movement found within agency-oriented Western Marxism and socialist feminism. This latter theory is developed as an understanding of movement as direction, developing from the local rationalities of everyday life through articulated but partial campaigns to a “movement project” which attempts to deploy such local rationalities to restructure the social whole. Within these terms, it argues for an understanding of counter culture as a movement project from below within disorganised capitalism. This mode of analysis is seen as that of a historical sociology geared to the production of open concepts which can be used by participants to theorise the context of their own choices. The second part (chapters four to eight) theorises the issues involved in researching social movements within this perspective, entailing the need to engage with tacit knowledge, to thematise conflicts and collusion between researcher and participants. The findings chapters use qualitative interviews from a Dublin movement milieu to develop an analysis, grounded in participation, of the local rationalities of the counter culture. In this section the key findings are a rationality of autonomy as self-development, which is shown to underlie processes of distancing and problems of commitment, and a rationality of radicalised reflexivity, which resolves the problem of institutionalisation through the deployment of a wide range of “techniques of the self”. The analysis attempts to locate this reading within the life-histories of participants but also within the historical development of the counter culture, examplifying the ability of the concepts developed in this thesis to engage with the problems facing participants

Flanking Restriction Enhanced Pulldown (FREP).

<p>(a) An 82 bp biotinylated DNA fragment is conjugated to streptavidin-coated magnetic Dynabeads (Invitrogen). This fragment is engineered to include a 31bp gene specific (“bait”) sequence (black box), flanked by restriction enzyme cleavage sites for BamH I proximally (blue box) and EcoR I distally (red box), as well as 20bp DNA fragments to allow PCR amplification of the whole unit. (b) DNA-beads are mixed with nuclear extract. A free 82 bp non-biotinylated DNA fragment can be included in the control reaction at this stage as a specific competitor. (c) Magnetic separation and wash to remove non-DNA binding proteins. (d) EcoR I digestion to release 3’ DNA end-binding proteins, yielding the EcoR I fraction. (e) BamH I digestion to separate the sequence specific DNA binding proteins, BamH I fraction, from proteins that bind 5’ DNA and Dynabeads. (f) Mass spectrometry (MS) to identify proteins remaining within the BamH I fraction.</p

Mass spectrometry analysis identifies PARP-1 as the CCR6DNP binding protein.

<p>Mass spectrometry analysis identifies PARP-1 as the CCR6DNP binding protein.</p

The association of PARP-1 with CCR6DNP is sequence dependent and allele specific.

<p>(a) FREP+Western blot for PARP-1 with BamH I fractions. Lane 1: nuclear extract (NE) input, lane 2: NE with CCR6DNP/TG-beads, lane 3: NE with CCR6DNP/TG-beads with cold competitor (Free TG), and lane 4: NE with a non-specific DNA sequence (NS-beads). (b) To detect allele specificity, FREP was modified to cut directly with BamH I only, yielding a combined BamH I+EcoR I fraction, and SDS-PAGE was probed with anti-PARP-1. Anti-Ku86 antibody was used as the internal loading control to ensure comparable amounts of competitor DNA. CCR6DNP/TG-beads were incubated with NE and competed with equal amount of free competitors. Lane 1: no competitor; lane 2: free CCR6DNP/TG; lane 3: CCR6DNP/CG; lane 4: free CCR6DNP/CA; and lane 5: free non-specific DNA. Upper: Western blot; Lower: relative density from the Western blot. (c) ChIP assay with an anti-PARP-1 antibody on WT HCT116 cells and 5 mutants as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006292#pgen.1006292.g001" target="_blank">Fig 1</a> showing impaired binding of PARP-1 to the CCR6DNP. (d) ChIP assay with an anti-PARP-1 antibody on WT Jurkat T cells showing significant enrichment of the CCR6DNP fragments comparing to anti-IgG antibody. (e) Sequencing of the CCR6DNP fragments from the ChIP with WT HCT116 cells showing the significant enrichment of the T allele over the C allele by an anti-PARP-1 antibody. Results from (a), (b), (c), and (d) representative of 3 experiments. Data are shown as mean±s.d. (<i>n</i> = 3).</p

Characterization of mutated HCT116 clones generated by TALENs targeting the CCR6DNP.

<p>(a) Partial genomic arrangement of <i>CCR6</i> showing alternative transcripts CCR6-a and CCR6-b and the location of CCR6DNP. Only CCR-b could be detected in HCT116 and Jurkat T cells by real time PCR (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006292#pgen.1006292.s003" target="_blank">S3 Fig</a>). (b) Sequence of the three alleles of CCR6DNP in 7 representative targeted HCT116 clones. The CCR6DNP is underlined. (c) and (d) Expression of CCR6 in the 7 targeted clones by qPCR and Western blot. For qPCR, data are shown as mean±s.d. (<i>n</i> = 3). Sequence, sequence trace and expression data for all 17 mutated HCT116 clones are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006292#pgen.1006292.s001" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006292#pgen.1006292.s002" target="_blank">S2</a> Figs. Statistical significance in 1c reflects comparison to WT.</p

CCR6 expression in human cells treated with 3-aminobenzamide.

<p>(a) qPCR and (b) Western blot to show expression of CCR6 and PARP-1 in Jurkat T cells, HCT116 and Hela cells. Cells were treated with 0 mM (lane 1), 5 mM (lane 2) and 10 mM (lane 3) 3-AB for 72 hrs. For qPCR, data are shown as mean±s.d. (<i>n</i> = 3).</p

ST2 deficiency attenuates K/BxN arthritis.

<p>Arthritis was initiated in ST2^−/− mice and their WT littermates via intraperitoneal administration of K/BxN mouse serum on days 0 and 2 (n = 5/group). (A) Clinical score on a 0–12 scale, <i>P</i><0.0001, WT versus ST2^−/−. (B) Change in ankle thickness, <i>P</i><0.0001, WT versus ST2^−/−. (C) Histomorphometric quantification of arthritic tissue (5 ankles/group). (D) Cytokine mRNA in ankle lysates (10 ankles/group from two separate experiments) at day 8 or 10 arthritis. (E) Acute change in wrist and ankle thickness (“flare”) measured 30 minutes after initial serum administration (n = 5/group). Results shown are the mean ± SEM. Panels A–C&E reflect 1 of 2 experiments with similar results. *<i>P</i><0.05, **<i>P</i><0.01, WT versus ST2^−/−.</p

FREP to identify the binding of PARP-1 with CCR6DNP.

<p>(a) Silver stain and (b) densitometry to show proteins pulled down from CCR6DNP/TG-beads mixed with nuclear extract from THP-1 cells. Lane 1, BamH I fraction; Lane 2, BamH I fraction where pulldown was performed in the presence of a 40x excess of competitor (free CCR6DNP/TG). Arrow indicates the specific band sent for mass spectrometry analysis.</p

CCR6 expression in PARP-1 knockdown human cells.

<p>HCT116 cells (a) and Jurkat T cells (b) by Western blot (left), and qPCR analysis on PARP-1 (middle) and on CCR6 (right). For Western blot, whole cell extract was isolated and separated on SDS-PAGE gel. Western blot was detected with mouse anti-human PARP-1, CCR6 and α-tubulin antibodies simultaneously. Lane 1: negative control for shRNA. Lane 2: shRNA treatment for HCT116 cells or siRNA treatment for Jurkat T cells. For qPCR, data are shown as mean±s.d. (<i>n</i> = 3).</p

IL-33-mediated priming of MCs for immune complex-dependent arthritis.

<p>In the model proposed, synovial fibroblasts release IL-33 in a constitutive or induced manner. IL-33 causes phenotypic changes in neighboring MCs, including accumulation of cytokine mRNA and alteration in granule content, depicted as color change in “primed” MC. Upon exposure to immune complexes, primed MCs exhibit release pro-inflammatory mediators that further activate fibroblasts, promote neutrophil recruitment, and contribute to arthritis severity. Reciprocal signals from MCs stimulated via ST2 enhance IL-33 production by fibroblasts, constituting a MC-fibroblast amplification loop.</p