Targeted methylation profiling of single laser-capture microdissected post-mortem brain cells by adapted limiting dilution bisulfite pyrosequencing (LDBSP)

A reoccurring issue in neuroepigenomic studies, especially in the context of neurodegenerative disease, is the use of (heterogeneous) bulk tissue, which generates noise during epigenetic profiling. A workable solution to this issue is to quantify epigenetic patterns in individually isolated neuronal cells using laser capture microdissection (LCM). For this purpose, we established a novel approach for targeted DNA methylation profiling of individual genes that relies on a combination of LCM and limiting dilution bisulfite pyrosequencing (LDBSP). Using this approach, we determined cytosine-phosphate-guanine (CpG) methylation rates of single alleles derived from 50 neurons that were isolated from unfixed post-mortem brain tissue. In the present manuscript, we describe the general workflow and, as a showcase, demonstrate how targeted methylation analysis of various genes, in this case, RHBDF2, OXT, TNXB, DNAJB13, PGLYRP1, C3, and LMX1B, can be performed simultaneously. By doing so, we describe an adapted data analysis pipeline for LDBSP, allowing one to include and correct CpG methylation rates derived from multi-allele reactions. In addition, we show that the efficiency of LDBSP on DNA derived from LCM neurons is similar to the efficiency obtained in previously published studies using this technique on other cell types. Overall, the method described here provides the user with a more accurate estimation of the DNA methylation status of each target gene in the analyzed cell pools, thereby adding further validity to this approach

The utilisation of social and behavioural science through consulting.

The thesis is concerned with organisational consulting, and the 'theories' which guide social and behavioural consultants in what they do. To preserve the 'integrity of the phenomena' the research has utilised an interviewing methodology to obtain accounts which reveal 'personal theories'. The aim has been to achieve an adequate phenomenology of consultants' ideas, rooted in their personal lives and organisational role situations, and not just to treat consultancy as the disembodied application of skills and knowledge. Consultants' ideas and practices can thereby be viewed in relation to their role-contexts, and can be seen as adapted to specific operating situations, particularly in the comparison of internal,commercial and academic consultants. Thus far, the study makes a substantive contribution to the understanding of social consultancy by locating ideas and practices in role circumstances. But such consultants are also ah occupational group, sharing a common role-context. The role is the product of wider organisational and societal processes. Beyond the specific slant given by differences in their immediate work-role, therefore, there appear common features in their working models. Two paradigms, the negotiative and systems, are identified and analysed as projections of consultants' role experiences which were also functional for clients, insofar as they developed the cohesion of managers as a group and their capacity to cope with problems facing organisations in the period 1960-79. Ideas and practices are thus viewed, ideologically, in relation to an historical period and social formation. By considering consultants' ideas, as ideology, in relation to their market situation (expressed in role) we confront a central question in social theory - the relation between ideas and the material structures and processes of society. At this point the study therefore attempts to connect the sociology of knowledge directly with the theory of ideology, and to make a substantive contribution to each