Optimisation de la cryo microscopie électronique pour les études des minicercles d'ADN

RESUME : Bien que les propriétés physiques de la structure de l'ADN aient été intensivement étudiées pendant plus de 50 ans il y a encore beaucoup de questions importantes qui attendent des réponses. Par exemple, qu'arrive-t-il à la structure de la double hélice d'ADN nue (sans protéines liées) lorsqu'elle est fortement courbée, de la même manière que dans les nucléosomes? Cet ADN nu est-il facilement plié (il reste dans le régime élastique) ou réduit-il la contrainte de flexion en formant des sites hyperflexibles «kinks» (il sort du régime élastique en cassant l'empilement des paires de bases à certains endroits) ? La microscopie électronique peut fournir une réponse à cette question par visualisation directe des minicercles d'ADN de la longueur d'un tour de nucléosome (environ 90 paires de bases). Pour que la réponse soit scientifiquement valide, on doit observer les molécules d'ADN lorsqu'elles sont en suspension dans la solution d'intérêt et sans que des colorations, produits chimiques ou fixatifs n'aient été ajoutés, étant donné que ceux-ci peuvent changer les propriétés de l'ADN. La technique de la cryo-microscopie électronique (cryo-EM) développée par le groupe de Jacques Dubochet au début des années 80, permet la visualisation directe des molécules d'ADN suspendues dans des couche minces vitrifiées de solutions aqueuses. Toutefois, le faible contraste qui caractérise la cryo-EM combinée avec la très petite taille des minicercles d'ADN rendent nécessaire l'optimisation de plusieurs étapes, aussi bien dans la préparation des échantillons que dans le processus d'acquisition d'images afin d'obtenir deux clichés stéréo qui permettent la reconstruction 3-D des minicercles d'ADN. Dans la première partie de ma thèse, je décris l'optimisation de certains paramètres pour la cryoEM et des processus d'acquisition d'image utilisant comme objets de test des plasmides et d'autres molécules d'ADN. Dans la deuxième partie, je .décris comment j'ai construit les minicercles d'ADN de 94 bp et comment j'ai introduit des modifications structurelles comme des coupures ou des lacunes. Dans la troisième partie, je décris l'analyse des reconstructions des rninicercles d'ADN. Cette analyse, appuyée par des tests biochimiques, indique fortement que des molécules d'ADN sont capables de former de petites molécules circulaires de 94 bp sans dépasser les limites d'élasticité, indiquant que les minicercles adoptent une forme circulaire régulière où la flexion est redistribuée le long la molécule. ABSTRACT : Although physical properties of DNA structure have been intensively studied for over 50 years there are still many important questions that need to be answered. For example, what happens to protein-free double-stranded DNA when it is strongly bent, as in DNA forming nucleosomes? Is such protein-free DNA smoothly bent (i.e. it remains within elastic limits of DNA rigidity) or does it release its bending stress by forming sharp kinks (i.e. it exits the elastic regime and breaks the stacking between neighbouring base-pairs in localized regions)? Electron microscopy can provide an answer to this question by directly visualizing DNA minicircles that have the size of nucleosome gyres (ca 90 bp). For the answer to be scientifically valid, one needs to observe DNA molecules while they are still suspended in the solution of interest and no staining chemicals or fixatives have been added since these can change the properties of the DNA. CryoEM techniques developed by Jacques Dubochet's group beginning in the 1980's permit direct visualization of DNA molecules suspended in cryo-vitrified layers of aqueous solutions. However, a relatively weak contrast of cryo-EM preparations combined with the very small size of the DNA minicircles made it necessary to optimize many of the steps and parameters of the cryo-EM specimen preparation and image acquisition processes in order to obtain stereo-pairs of images that permit the 3-D reconstruction of the observed DNA minicircles. In the first part of my thesis I describe the optimization of the cryo-EM preparation and the image acquisition processes using plasmid size DNA molecules as a test object. In the second part, I describe how I formed the 94 by DNA minicircles and how I introduced structural modifications like nicks or gaps. In the third part, I describe the cryo-EM analysis of the constructed DNA minicircles. That analysis, supported by biochemical tests, strongly indicates that DNA minicircles as small as 94 by remain within the elastic limits of DNA structure, i.e. the minicircles adopt a regular circular shape where bending is redistributed along the molecules.

Standards for the Assessment of Need process under Part 2 of the Disability Act 2005Consultation Process

Standards for the Assessment of Need process under Part 2 of the Disability Act 2005 In 2004, the Irish Government launched the National Disability Strategy as a framework of positive action measures to support the participation of people with disabilities in Irish society. Two new pieces of legislation â?" the Education for Persons with Special Education Needs Act, 2004 (EPSEN Act 2004 hereafter) and the Disability Act, 2005 â?" form an integral part of this strategy and deal with the special education and/or health needs of persons. Click here to download PDF 279kb The Report on the Consultation Process on Standards for the Assessment of Need process as referred to on page 6 of the Standards document above. Click here to download PDF 369kb

In Our Own Words: Report of the Consultation Process on the National Positive Ageing Strategy

In Our Own Words: Report of the Consultation Process on the National Positive Ageing Strategy If you wish to receive this document in an alternative format, please email positiveageing@health.gov.ie or telephone 01-635 3184

Facilitation Process Concerning the Difficulties in Implementing A Vision For Change in the South Tipperary and Carlow Kilkenny Catchment Area Mental Health Service

Facilitation Process Concerning the Difficulties in Implementing A Vision For Change in the South Tipperary and Carlow Kilkenny Catchment Area Mental Health Service Click here to download PDF 4.27MB

National Strategy on Dementia Summary of Consultation Process

The Programme for Government gives a commitment to develop a National Strategy on Dementia by 2013 which will increase awareness, ensure early diagnosis and intervention, and enhance community based services for people living with this condition. During 2012, following the completion of the Research Review in preparation for the National Strategy, the Department carried out a public consultation to inform its development. This report is a summary of the responses and submissions received. Click here to download PDF 271KB  

Framework Agreement between the Minister for Health and the Irish Medical Organisation in respect of a process for engagement concerning the GMS/GP Contract

Minister of State with responsibility for Primary Care, Alex White TD, today (4 June 2014) concluded a series of meetings with the Irish Medical Organisation (IMO) with the signing of the Framework Agreement between the Minister of Health, the HSE and the Irish Medical Organisation (IMO) setting out a process for engagement concerning the GMS/GP contract and other publicly funded contracts involving General Practitioners (GPs). Download document here

Report of the Consultation Process on new legislation to replace the Dentists Act, 1985

  This is a report of a public consultation which the Department of Health undertook in June 2013 on new legislation to replace the Dentists Act 1985. The report outlines the views and opinions of those who completed questionnaires or made submissions to the Department on the proposed new legislation. One hundred and twenty five questionnaires/submissions were received. Of these, 98 were submissions made by personal respondents and 27 were made by corporate respondents. Download this document as a PDF The breakdown of these respondents is as follows: This report will inform the development of policy on the key issues, which will be the next stage in the process towards developing new dental legislation.

Modelling the mechanical aspects of the curing process of magneto-sensitive elastomeric materials

In this paper, a phenomenologically motivated magneto-mechanically coupled finite strain elastic framework for simulating the curing process of polymers in the presence of a magnetic load is proposed. This approach is in line with previous works by Hossain and co-workers on finite strain curing modelling framework for the purely mechanical polymer curing (Hossain et al., 2009b). The proposed thermodynamically consistent approach is independent of any particular free energy function that may be used for the fully-cured magneto-sensitive polymer modelling, i.e. any phenomenological or micromechanical-inspired free energy can be inserted into the main modelling framework. For the fabrication of magneto-sensitive polymers, micron-size ferromagnetic particles are mixed with the liquid matrix material in the uncured stage. The particles align in a preferred direction with the application of a magnetic field during the curing process. The polymer curing process is a complex (visco) elastic process that transforms a fluid to a solid with time. Such transformation process is modelled by an appropriate constitutive relation which takes into account the temporal evolution of the material parameters appearing in a particular energy function. For demonstration in this work, a frequently used energy function is chosen, i.e. the classical Mooney-Rivlin free energy enhanced by coupling terms. Several representative numerical examples are demonstrated that prove the capability of our approach to correctly capture common features in polymers undergoing curing processes in the presence of a magneto-mechanical coupled load.

Construcció d'una aplicació web utilitzant el mètode Rational Unified Process

Construcció d'una aplicació web a partir de les especificacions d'un client imaginari. Estudi i utilització del mètode Rational Unified Process, el més habitual actualment en la construcció de software. Disseny d'una base de dades i implementació del model lògic mitjançant un SGBD punter al mercat com Oracle.

A Grounded Theory Model of the Counselling Referral Process in Primary Care Methadone Treatment in Ireland.

In terms of the treatment of illicit drug abuse, methadone maintenance is a well researched and widely applied systematic response. The approach to primary care methadone treatment in Ireland is based on the methadone protocol. Primary care plays a central role in the delivery of methadone treatment. Beginning with a view that a system evolves within the constraints and influencing factors of its context, the aim of this thesis is to model the process that has developed by which patients on primary care methadone treatment are referred to counselling. It investigates the role primary care practitioners perceive they have in relation to managing the psychosocial aspects of the methadone patient's treatment regime. It analyzes individual medical practitioner counselling referral mechanisms to determine what common processes operate across different practitioners. It identifies the factors that influence the use of counselling on primary care methadone programmes and structures these in a cause/effect model. This research used interviews and documentary analysis to acquire grounded data. The sample consisted primarily of medical practitioners involved in the delivery of methadone programmes. Others closely involved in the implementation of drug treatment in the primary care context made up the balance of interviewees. The study used a grounded theory methodology to induce the process that was latent in the grounded data. Concepts emerging were grouped under the headings of referral factors, decision making factors and factors related to the unique positioning of primary care at the interface between medicine and society. The core finding was that, in primary care in Ireland, there is no psychological model to complement the pharmacological intervention of methadone substitution. The findings from this study offer insight into the factors at work and their impacts, in the context of the use of counselling in primary care methadone treatment. The study suggests a possible direction for further evolution of opiate abuse treatment in Ireland which would transform it from a harm reduction to a holistic patient centric paradigm.This resource was contributed by The National Documentation Centre on Drug Use.

A Process Analysis of Males and Females who Attended a Community Based Addiction Treatment Service: Towards an Expanded Paradigm

A process analysis was conducted in a community - based treatment programme for alcohol abuse. The aims of the study were: to evaluate assessment instruments and measures; to measure change following treatment; to monitor gender differences; to assess the importance of early and current relationships; and to evaluate the effects of therapists. Subjects (n=145, males 83/females 62) completed a semi-structured interview schedule, Severity of Alcohol Dependency Questionnaire (SADQ), Short Alcohol Dependence Data Questionnaire (SADD); General Health Questionnaire (GHQ 12), and Alcohol Problems Questionnaire (APQ). A further three non-standardised self-rated measures were devised by the author. Included was the opportunity to obtain qualitative data. Follow up data was collected at 3, 9 and 15 months following first assessment. The SADD, APQ and consumption measures using detailed drink diaries proved the most relevant assessment measures. Following treatment, there was significant reduction in clients' dependency levels at 3 months, maintained through 9 and 15 months. Key client-rated changes were progress in reducing consumption and alcohol problems leading to a better quality of life and health. Qualitative data augmented these quantitative results. Psychological and acquired cognitive behavioural skills emerged as the main reasons for positive change and the treatment programme was found to have played a significant role in their acquisition. It appears that addressing marital problems can lead to a reduction in alcohol dependency levels. Gender analysis showed that males and females were similar in demographic characteristics, alcohol history details and dependence levels. It was concluded that the differences found did not necessitate different treatment programmes for women. Early family relationships were more problematic for females. Therapist performance varied and that variance was reflected in their clients' outcomes.This resource was contributed by The National Documentation Centre on Drug Use.

A Study of a Staff Alcohol Policy in a Large Public Health Organisation, Looking at the Policy Content, the Policy Development and the Implementation Process, and the Policy Impact

There are different approaches to dealing with alcohol related problems in the workplace. A literature review indicates that two of the models that underpin programmes to deal with alcohol related problems in the workplace are the disease model and the health promotion model. The disease model considers alcoholism as an illness and uses curative techniques to restore the individual to sobriety. The health promotion model looks at the determinants of health and promotes changes in the environment and structures, which would support healthy behaviour in relation to alcohol. Employee Assistance Programmes (EAPs) may have elements of both theses models. Dealing with alcohol problems at work involves a captive audience and the workplace as a setting can be used to influence healthier lifestyles. A workplace alcohol policy is a mechanism through which alcohol related issues might be dealt with, and the necessary resources and commitment of managers and staff channelled to this end. The policy aims should be clear and unambiguous, and specific plans put in place for implementing all aspects of the policy. In the case of the alcohol policy in the organisation under study, the policy was underpinned by a health promotion ethos and the policy document reflects broad aims and objectives to support this. The steering group that oversaw the development of the policy had particular needs of their own which they brought to the development process. The common theme in their needs was how to identify and support employees with alcohol related problems within an equitable staff welfare system. The role of the supervisor was recognised as crucial and training was provided to introduce the skills needed for an early intervention and constructive confrontation with employees who had alcohol related problems. Opportunities provided by this policy initiative to deal with broader issues around alcohol and to consider the determinants of health in relation to alcohol were not fully utilised. The policy formalised the procedures for dealing with people who have alcohol related problems in an equitable and supportive manner. The wider aspect of the health promotion approach does not appear to have been a priority in the development and implementation of the policy.This resource was contributed by The National Documentation Centre on Drug Use.