Régine Teulier
email: teulier@shs.polytechnique.fr

Strategizing a journal with PRP. How individual work becomes collective judgement

Keywords: Strategizing, routines, micro-practices, peer review process, Knowledge engineering

The paper focuses on micro-practices in use that permit simultaneously two strategizing processes. First that individual work becomes a collective and institutional position through processes participating to RIO in the PRP. Second that doing strategy day after day and making the core process evolve through the evolving set of micro-practices involve in the RIO. To argue about these two transformations, we study the micro-practices, processes and routines underlying the Peer Review Process and its practices over time while the issues are built, following general strategic aims. We adopt a practice-oriented perspective, which allows us to understand how routines are acted in social and individual activities. We use descriptive method but also knowledge engineering methods. These last ones are useful to describe knowledge processes; it focuses on task, a notion that may be compared to that one of routine. An empirical study allows us to light on day to day strategizing practices in the PRP, through a longitudinal study of an academic journal.

Introduction

Strategy is something people do [START_REF] Jarzabkowski | Strategy as Practices: Recursiveness, adaptation and practice in use[END_REF]. To observe and analyse strategizing is quite difficult, one promising way is to have interest for routines [START_REF] Feldman | Organizational routines as a source of continuous change[END_REF] and micro-practices [START_REF] Rouleau | Micro-practices of strategic sensemaking and sensegiving: how middle managers interpret ands sell change every day[END_REF], [START_REF] Jarzabkowski | Strategic Practices: an activity theory perspective on continuity and change[END_REF].

The « micro level » [START_REF] Feldman | Resources in emerging structures and processes of change[END_REF] shows interest for processes which are not concerned by the whole organization, but that can be observed about a small part of its activity and some of the actors, in their day-to-day activity. This approach allows a synthesis view between « organizing » perspectives and « practice-based »ones. It uses the notions of routine and procedure that became well known in management science, for example, linked to the core competencies of the firm [START_REF] Hamel | Competing for the future[END_REF]. They are consistent with some theoretical foundations in economy through the evolutionary theory of economic change (Nelson et Winter, 1982).

We propose to use Feldman definitions (2000): « Routines are temporal structures that are often used as a way of accomplishing organizational work. » or more precisely « repeated patterns of behaviour that are bound by rules and customs and that do not change very much from one iteration to another » or (Feldman, 1995, p 6) « repeated actions carried out by two or more interdependent actors ». Thus, routines are defined as repeated procedures among the procedures that are the « standard operating procedures » allowing doing a task in the meaning of Cyert and March (1963).

The distinction between routines and procedures is then essentially due for Feldman to this feature of repeated action. Routines are that particular procedure: those, which are, repeated ones.

That implies several consequences: there are the procedures which are applied without thinking to it, those which are call into question only border line and when problems occur, those which concretise the « path dependency »of the firm (Nelson et Winter, 1982). Evolutions and adaptations are nevertheless frequent as Feldman shows it, we shall discuss that point.

Routines can also be considered as the way the actors translate the procedure into activity, they put into acts what is organizationally planned to do. Procedures are explicit, often encoded and written, well known of the hierarchy. Thus, routines should be instantiations of procedures, they should be more « variables » than the procedures that may differ progressively from routines and micro-practices. However that distinction seems not stabilised among these different works and we have to notice that the two concepts are indifferently used.

More clearly, micro-practices are referring directly to the notions of routines and procedures in the frame of activity of the actors. For some authors, as Feldman they seems equivalent to routines and if the term of practice is used, even at a « micro » level, the particular qualification of micropractices is not used.

For other authors [START_REF] Rouleau | Micro-practices of strategic sensemaking and sensegiving: how middle managers interpret ands sell change every day[END_REF], the term of micro-practices is used in a particular meaning. [START_REF] Rouleau | Micro-practices of strategic sensemaking and sensegiving: how middle managers interpret ands sell change every day[END_REF] situates the micro-practices at the individual level: « All individuals put micropractices of translation into action every day… », routines at the organizational level and conversations at the social level [START_REF] Rouleau | Micro-practices of strategic sensemaking and sensegiving: how middle managers interpret ands sell change every day[END_REF](Rouleau, , p. 1425)). The routines that are an organizational feature are lived every day by people in a personal way, and that produce micro-practices. [(22], p. 1431: « It appears that middle managers, through their tacit knowledge, strategize by enacting a set of micro-practices that are produced in each routine and conversation surrounding the change. » or by the same author [START_REF] Rouleau | Micro-practices of strategic sensemaking and sensegiving: how middle managers interpret ands sell change every day[END_REF](Rouleau, , p. 1432): «all the routines and conversations examined combined synergistically the four micro-practices into arrangements of verbal and symbolic signs». This author refers particularly to Giddens [START_REF] Giddens | The constitution of society[END_REF]: «In other words, these micro-practices were produced through routines and conversations as the result of mundane human competence in action » [START_REF] Jarzabkowski | Strategic Practices: an activity theory perspective on continuity and change[END_REF] use also the concept of micro-practices. This author defines practices referring to activity theory (Vygotski, 1997) and the concrete activity context as an activity system (Engestrom, 1993). This view is consistent with that one that proposes to consider the organization as a set of activity systems [START_REF] Blackler | Knowledge and the theory of organization : organizations as activity systems and the reframing of management[END_REF]. In that perspective, this author considers that practices are mainly a link between actors, according the social and historic dimension of Vygotski. In that view, micro-practices are the way the actors « habit » organizational routines and made them evolve.

The greatest interest of considering routines is to allow linking practices and strategizing. The point is to enlighten precisely how the firm orientations are putting in day-to-day acts and « how people are invested in the doing the real world», as [START_REF] Cook | Bridging epistemologies : the generative dance between organizational knowledge and organizational knowing[END_REF] invited us to observe. Doing so, the analyst focuses on an organizational processes granularity level, which include day-to-day actors' practices [START_REF] Charlet | Teulier L'ingénierie des connaissances[END_REF]. The routines granularity can be high: so that they include the description of several actors' activity during several months. But they can also be con-stituted of actors' activity during some minutes. Thus, the routines granularity is highly variable.

Routines including routines, «Within each of these routines there are multiples routines, and there is some variance in what is included in each of routines depending on who is describing them. » [START_REF] Feldman | Organizational routines as a source of continuous change[END_REF], several granularity levels can be implied in the same case.

Knowledge engineering methods are useful to describe tasks and knowledge intensive processes.

Knowledge engineering does not focus on routines, but the notion of task may be compared to that one of routine. Knowledge engineering aims are to build concepts, tools and methods to model knowledge and develop software using knowledge and supporting user' work [START_REF] Charlet | Teulier L'ingénierie des connaissances[END_REF].

Most of the time, the starting point is a synthetic activity description that is preliminary step for modelling.

KADS methodology concentrates a lot of works and debates in knowledge engineering in

Europe during the ninety. This methodology is based on problem solving methods [START_REF] Newell | Human problem solving[END_REF]. It argues that it is built and not existing before modelling, and that models have to be constructed to be guidelines to the knowledge acquisition process. It argues that problem solving methods are various but not infinite and that we can recognize in very various using fields some generic problem solving methods that can be modelled and re-used in these different fields.

Therefore, the aim of methodology is to build library of models.

The description of practice by a discourse organised with concepts may be compared to the Kads models of organization, of the task, of the agent, of knowledge and of organization.

The task model is composed in several views. Schreiber et al define it as following (Schreiber et al, 2000, p 45): « the notion of task, although important, has different connotations. As a commonsense concept, it is a human activity to achieve some purpose. In the above organizational study it has been viewed in the (not incompatible) sense of a well-defined subpart of a business process.

The notion of task has also emerged as a crucial one in the theory and methodology of knowledge systems and of knowledge sharing and reuse. » Schreiber et al define the task so that it may make sense in the two fields (Fig. 2), in the one of modelling actors and in the one of practitioners. To do that, they define the task as a subpart of a business model presenting the following characteristics: it represents a goal-oriented activity adding value to the organization, it handles inputs and delivers desired outputs in a structured and controlled way, it consumes resources it requires and provides knowledge and competences, it is carried out according to given quality and performance criteria, it is performed by responsible and accountable agents.

Observation of routines

To pay attention to practices, to identify and to give a title to the tasks or to the routines is an important starting point? It needs to distinguish among different actors' practices and among different knowledge. It's a first step which « distinguishes » an activity frame from a complicated organizational background, full of activity, alternate exchanges that implies several set of actors which are not all concerned with the studied routine. It. constitutes for itself a step of analysis and design, which need observations, methods and interpretation. This step is common to an identification of routines and to the modelling of problem solving methods as it is done in knowledge engineering. We call that step characterisation.

Methods

Describing routines in the practice of management of a students residence halls: budgeting for maintenance, hiring, training the student staff, moving students at the beginning of the year and closing the residence halls, Feldman use for identifying routines the formulation proposed by the concerned actors. (Feldman, 2000, p. 614) « Organizational members identified five routines for me ». That way of routines identification is close to the actors sense making, it is consistent with the ethnomethodologic approach which found this author method (among four meta-theories) (Feldman 1995, p.4) « Ethnomethodologists look for processes by which people make sense of their interactions and the institutions through which they live ». We will not discuss that theoretical point in this paper.

Routines description is done through a short story about actors' activity and some characteristics as incomes, outcomes, and used resources. By the routine process, actors transform incomes in outcomes. Among different resources for the routine: actors, activities, knowledge, knowing and others routines. We propose here, to extend this approach by discussing the qualification of knowledge. In fact, knowledge seems to us a main resource that can be extended by artefacts, that can permit to determine routines types, and that permit to link routines and competencies.

Data gathering

We have observed the editorial board during three years, about thirteen day meetings, and forty individuals interviews of two hours. The meetings and interview were recorded with audio or video.

Organizational setting

This scientific journal is a french language journal, it is publishing papers of a various and distributed scientific community, and animate that community. The main processes that we have distinguished in the editing board practices and that we suggest, as routines are the following: selecting scientific papers by peers review process, management of a texts set, issue composition. Each of these three main activities is made of a set of plus or less coordinated and well-arranged processes.

These three processes are massively overlapped and interacting together. The main process is the Peer review process" as, described by [START_REF] Rowland | The peer review process[END_REF] according a lot of authors "…the four main functions of the scholarly literature are dissemination of current knowledge, archiving of the canonical knowledge base, quality control of published information and assignment of priority and credit for authors."

Description of routines

We shall describe the global routine of PRP, then focus on a sub-routine "assignment of reviewers to a paper", and analyse in details how the individual work is transformed in collective work in the process "first reviews back".

The global schema of peer review process.

In the peer review process, we can distinguish the following routines in the editing board practices that we have observed:

• Assign a reporter (a redactor who is specifically in charge of a paper review process)

• Assign three reviewers

• Received review notes, evaluate them, synthesize them, prepare the collective decision by a synthesized reporter note • Made an editing board decision about the paper • Change notifications to author

• Evaluate the Vn+1 / Vn evolution and the remarks integration or not, in a short loop managed by the reporter or in a long loop with back review by one or two reviewers.

To characterize rapidly this process with few words, we can say that it is an activity of selecting papers for publishing. The process goes on several steps: a reporter manage the individual paper curs us, several reviewers are selected among a population and an assignment paper/reviewers is done. Reviewer's notifications are collectively validated and legitimated by the editing board. The peer review process is a core activity in scientific activity. Beyond the notion of selection, it's a complex way to influence scientific production and partly a co-construction of results, because the notifications addressed to the authors largely contribute to the direct elaboration of the paper.

Synthesize Synthesis and Decision proposal

FIG 1 -Organizing the peer review process, as a routine and as set of tasks.

Analyse

Review As we have seen in fig. 1, individual and collective phases are interred related. We shall analyse a part of the PRP: the reviewer's assignment to a paper, to enlighten more concretely how this interrelation between individual and collective work occurs. Among a population of reviewers, a sub-set is determined that can be assigned to the paper. It is affected to the paper. Then a resource constituted of a sub-set of three reviewers is selected and allocated to this paper. In order to produce a synthetic representation, at the same time in terms of task and in terms of routine, we propose the following schema. That schema represents incomes, outcomes and processes, at a higher granularity than in a task model.

We shall represent the process of reviewer's assignment in terms of tasks. At the beginning of this process, the assignment of a reporter. The process of reviewer's assignment to a paper is the task of assignment in the Kads library. It consists in pairing two objects belonging at two different sets. The goal of the task is to create the relation between a paper and three reviewers. Two main notions of that model are the notion of resource and the notion of assignment. The resource of the task is a set of reviewers, they are a resource fro the editing board. The assignment process allocates three reviewers to a paper. Inputs are the set of papers presented by the editor in chief, the set of possible reviewers as known by the editing board, the sub-set of reviewers proposed by the reporter. Outputs are the set of pairs reviewers / paper. The figure 3 The frame of this transformation.

The transformation of individual work in a collective result in an editing board in academic journals is not a simple validation; it needs a real collective work of elaboration, so that we can really talk of transformation. The machinery transforming individual work requires several processes implying a mutation in the kind of judgement. This transformation is not a mechanic one: the result is not easy to foresee. To perform and to act this transformation, a representative collective group is required: in our case the editing board.

The individual position requires becoming a collective position to take a recognized value in the social world. Through this transformation, it's become the judgement of a journal and through this organization; it becomes the judgement of a scientific community. So, as the schema 2 illustrates it, a small collective group, which represents more than itself, formulates the judgement: a largest human group or network as a scientific community. So, the small collective group (here an editing board) stands instead of the largest group, it is representing it, and that is making sense. This relation of representation may be institutional one (in the case of a court for example) or a trust, reputation, and cultural relation (in the case of an academic journal) we shall not discuss that point here.

FIG. 2 -The collective work of a common position is an elaborative process.

The process of change from an individual position to a collective position in an editing board is a fascinating process. The reporter is the only one present person who had read the paper; the three reviewers are not present. One or two of the chief editor had read the paper at the previous step (one or two months before), but it was very often a quick reading. Among this kind of processes, we had chosen to analyse the process of the V1 evaluation or first reviews back.

A collective position elaboration process : the first reviews back.

We shall describe in detail this collective elaboration process. We can distinguish three subprocesses (cf. fig. 5), which are, first of all the deliverable of the individual work of the reporter, which is to say an arguments set made of evaluation and proposal, then a debate among all the redactors, and finally the stabilisation by consensus on a collective position.

Presentation, argumentation of the reporter

In the initial step of PRP (cf. fig. 1), the redactors in chief had read and presented quickly the paper, then in the following step the redactor had made a preliminary report to propose reviewers. So the redactors eared about the paper for the third time on a few months time, but they never read it. Now the editing board begin the deep analyse of the paper.

The redactor had analysed the paper, analysed the reviews. In the meeting of editing board, the reporter describes the paper, but he essentially presents the reviews and enlightens them of his own appreciation, or read some specific parts of the paper if necessary. Then, he presents his own synthesis. He has to present, to argue and to convince (cf. fig. 4.), but essentially he has to inform his peers so that each of them could construct his own opinion.

FIG 4 -Modelling the reviewer's advertising to the board

The analysis of our audio data shows that the reporters' discourses are always very structured, the arguments are those, in use in the profession and they rely on the support of the connivance with the redactors. Some expressions that we can noted are the following: The paper focuses on that kind of problem in that kind of situation, the author tries to demonstrate that …To that, he uses such and such well-known results …its originality consists in … The demonstration lacks rigour in that point and that point … the paper is well written, easy to read … The paper does not add any progress from the work of X or Y (or it completes)… That kind of ritual language in the presentation routine offers guarantee that the other redactors could take over the advanced elements to make their own opinions. Analogies, shared references, categorisations are used, they reinforce the tacit connivance between peers. To conclude the basic well known points of such a Through this report, the reporter part is to orchestrate a global and equilibrated and collective vision about the paper by using reviews. It is not easy, as write Agarwal and al (2006) "an inevitable consequence of the review process is the emergence of differences among the philosophies, perspectives and interpretations of reviewers and authors". The redactor had to take in account all of them. It may become that the reporter use the reviews as a layer use testimonies, amplifying them or putting them into perspective. The reporter may have some doubt (The journal is a pluridisciplinary one) and that he tries to separate what is well known for him and about what he needs another appreciation about one review or another: is it too hard, or too biased, etc. The reporter presentation (as well as the reviewers reviews) is itself implicitly evaluated about its coherence, its ability to apply the implicit rules of the profession.

That sub-process « Presentation and argumentation » is strongly individual because it consists in presenting alone an individual work, but it is a « situated » presentation in a board meeting, so it is strongly oriented by this organisational situation and by the group phenomenon, that's why we represent it by the fig. 5, part in the individual work universe, part in collective work universe.

Debate, collective elaboration

As we have said, the process « first reviews back » is not a simple validation, a choice between yes or no, (for example, the decision is not made by voting), it is, in fact a consensus research.

And it is mainly located in that second sub process. Some part objections, shades of meaning, or doubts arisen and relativizent the first expressed opinion: that one of redactor. The redactors, depending of its coherence, perceive the redactor presentation. If the presentation lacks of coherence, the redactors react and oppose to. As well if the presentation presents a failure, but this case is quite rare.

Two points essentially made debate in the collective group and they are a way of strategizing.

Point 1: Has the journal interest to publish that paper? What kind of interest? The question is particularly of some evidence for papers which obtained R and R with limits and which theme is of a limited interest for the journal. Point 2: As we know that the V1 is rarely satisfying, what is the author ability to modify their paper? Accord the R and R, behind the strict actual state paper eva-luation is a project on what can become the paper, its potential and a gamble about the authors' capacity to make it evolved. The collective board consensus has to be built on a projection in the future, about what authors may really do, even they are not present and that it is difficult to evaluate their competencies, disponibility, and willingness to modify the paper. If "a reviewer is, first, a gatekeeper who makes sure that a manuscript has certain basic required features…" [START_REF] Seibert | Anatomy of an Rnad R (Or reviewers are an author's best friends…[END_REF] the editor (in the journal we had observed, he is a collective one) is a gatekeeper of the journal scope and of the journal strategy. Evaluate the paper adequacy to the journal scope is mostly done in the first part of PRP (cf. fig. 1), rests here to do through every R and R attributed the day to day journal strategy.

Consensus about a position

Thus, during the debate, the collective position is converging on the consensus. The consensual position is formulated several times, finally formulated into acts and validate by the group. The process takes end; it had taken about 15 minutes. Frequently, if a light objection or a doubt subsists, then another redactor, or redactor in chief propose to read the paper about an aspect that made discussion, and about what he is competent. Then the evolving constraints put on the paper or the redactor arbitration between two reviews positions are modified in the letter to author. The resulting consensus may be a redactor synthesis inflexion, or not, the point is that it is now a collective position: that one of the journal. And it may be very different from the reviewers' opinion as write [START_REF] Bergh | Editing the 2004 AMJ best article award winner[END_REF] "Remember, the editor is the ultimate decision maker and will be reading both the reviewer and author perspectives".

FIG. 5 -Relationship between individual and collective work. Articulated sub-processes.

Discussion : Strategizing day after day

Strategizing through PRP

The strategy axe of the journal is to make the global scientific debate progress. The way, the journal practice the PRP build day to day the strategy of the journal. As we had previously shown in the PRP description, according an R and R advertising to paper at the frontier of theme, or at the frontier of validated level.

Maintain the journal scope, but positioning this scope on new themes is also a challenge, in dayto-day practices, accepting papers that are hardly discussed in the editing board do it.

One of the strategic difficulties of a journal is to progress in quality and to change of niche among the concurrent journals, because of the fascia of academic evaluation which plays a conservative part [START_REF] Starbuck | How much better are the most-pretigious Journals ? The statistics of academic publication[END_REF].

Retroactive loops

The imbrication of individual and organisational in the PRP implies retroactive loops and of recursive processes that modifies each other's. It retroactive loop who allows sharing opinions and which allows also controlling it. These various loops, all together play a part of awareness in the organisation. For example, if individual work plays an important part in the process, the actor who plays it had been collectively chosen (as well as reviewer or as editor) and he is controlled by collective institutions at various steps of the academic life phases (Starbuck, Rynes). It is because the PRP is well established in a routine, because the reputation of each journal is based on practices around this routine PRP, and because these retroactive loops plays a part of control in the core process that the scientific community may build it trust.

An efficacity lying on usual and long processes intra et inter organizations.

The PRP process is organization specific, in the sense that it is embodied in the routines, but

"practice theory emphasizes the extra-organizational too-the practices deriving from the larger social fields or systems…" (Whittington, 2006). In fact, the PRP is largely and deeply shared through academic journals, the ways the different boards are practicing it can vary by a little dif-ference, due to the identity of the journal. We may further and consider with S. [START_REF] Rynes | Academy of management journal eidtors' forum on the review process. Making the most of the review process: lessons from award-winning authors[END_REF] that "each review cycle is also somewhat unique: an interaction of multiple parties, each with his or her own competencies, skills, personality traits and taken-for-granted assumptions and beliefs".

Exchanges between redactors are lying on shared and common large and deep knowledge, on individuals and collectives knowing which are consistent and very well experimented. They are codified, even if they are formulated, if they are for a large part implicit. How to proceed is well known in the academic universe, it may be qualified of "habitus" as «a set of practices » and behaviours that characterise a group [START_REF] Bourdieu | Le sens pratique[END_REF]. The PRP, is a at the same time, a process codified in a routine but also practices lying on individuals and collectives craft knowing, with tacit rules very adaptable with long learning. The dance between individual and organization include at the same time imposed figures and improvisation. Imposed figures supported by routines and which are useful in the field of professional practices intra and inter organization. In that dance between individual and organization, the peers group plays an essential function. In the academic journal that we have observed, it is the functioning of a stable group, where persons know each other, which plays the part of organisational control.

The organization we have observed, is a patricians community, which may be analyzed, as Whittington (2006) suggests it, by taking guidelines the works about Communities of Practices, even if we are not in the case of CoPs. Ce qui distingue d'une communauté de pratique c'est qu'il y a un objectif concret de réalisation d'un travail et d'un objet : la revue, ce qui est commun est la recherche d'une progression dans l'expertise individuelle par ses membres. L'apprentissage est long, il repose sur des connaissances et des savoir faire eux-mêmes très importants. It consists in « absorbing its particular mix of local rules and internalized standards » (Whittington, 2007). Rynes also underlines that this long learning consists in through various phases : at the same time for authors to learn to « read » the PRP but also in the part of reviewer and editor.

Explicit and tacit rules are used in that RIO in PRP. We shall not debate of explicit rules which are present in the routine. We shall examine their evolving process by the change of practices in the § Evolving routine Henry. The processes that we have described lye on tacit rules about what is a good paper and how to evaluate a paper. The review grids of automatic tools which are often used for conferences (and on reviews adding), shorten precisely that transformation RIO. As we know, they have a few uses in journals because the evaluating work is more complete and deep.

One part of tacit rules consists also in knowing in « being » a redactor in an editing board ; He or she has to answer for his or her evaluation in front of his peers. So, he has to use it in an active manner, as a layer or a procure ; it's a much more active posture than that one of a simple reviewer. The redactors take care of their peers opinion, they participate to the editing board, only by reputation effect and the fact of acting their part in the journal and to progress with it, is their main personal motivation.

Shorted the cycle submitted/ published : Evolving routine Henry Assignment of a reporter.

The routine assignment of a reporter, first step of the papers' selection has evolved during our observation period with the opportunity of a particular situation: several papers were received the day before the editorial board meeting. The board secretary was on vacation. To gain time, two editors in chief propose to assign a reporter without an editor reading note (represented on figure 1), that means delete the first step and the resource "paper" is directly used par the process editorial board meeting. When the new routine was used, it was applied on 2/3 papers, the others needing a detailed analysis and presentation to be allocated. The new routine was maintained and used in the same way: only for a part of the set of papers, and only when time was missing.

The competition between scientific journals, the evolving publishing medium and the financial challenge to maintained a journal lead to strategic pressure on the editorial board to shorten the cycle submission -publishing. That pressure forced itself upon the editorial board members, but they don't mentioned it. With this pressure background an opportunity came: the reception of a lot of papers during the secretary vacations.

The assimilation of the dictate « shorten the cycle» and the accommodation of the routine assign of a reporter, produce the adaptation of the editorial board and the change of the organizational routine. The external events are the pressure and the opportunity. The redactors' knowing permit the routine to evolve.

Considering the collective task, one may think that two main processes of knowing occur together: the classification among typicality of the paper, and the fit of assignment. When the collective task develops without redactor-in-chief presentation, these two knowing processes have to develop with fuzzy resources. The task is no more to validate or to modify the editor-in-chief proposal, all participants have to do their best to have a point of the view on the type of the paper, and to propose reviewers names without any helping external artefact. There is no more the medium of an individual analysis. The group, then is reduced to evaluating from elements coming from the author: title, abstract, key words and to a quick visual glance through the paper that the editor-inchief do directly during the meeting: structure of the paper, some remarks (« there are a lot of schemas or maps about ….), some bibliographical references for enlightening title and key words.

Thus, considering collective activity, we could say the structure itself of the pre-existing task is modified, not only practices evolved, but routine was changed. Practices make the routine evolve.

Considering the organizational process, without decomposing the collective task; one can consider that only a step is delete, the routine is shorter.

Knowledge engineering and practice-based

The two ways of describing knowledge, learning and organizational processes, through organization science methods and knowledge engineering methods may be complementary, there may join in the step of characterization. In organization science, a certain discourse variation is permitted in describing practices. In knowledge engineering a unique description is researched, because it is thought as avoiding misunderstandings that is necessary to develop software.

We have only used the European knowledge engineering stream that differs the US one (Menzies and Van Harmelen, 1999) and which are founded on problem solving method [START_REF] Wielinga | KADS: A Modelling Approach to Knowledge Engineering[END_REF]. Our point of view does not include all the knowledge engineering methods.

One may see a certain contradictory to use methods founded on a cognitivist perspective and others founded on a practice-based perspective. But the use of these methods are complementary, as we have shown it in this paper and as others authors like [START_REF] Carlile | A pragmatic view of knowledge and boundaries : boundary objects in new product developpement[END_REF] have already done. It can be done, being careful of not detached abstractions from a deep practices analysis: « Abstractions detached from practice distort or obscure intricacies of that practice » (Brown and Duguid, 1991) The characterisation

We propose the process of characterisation as an essential step for practices analysis and for knowledge engineering. It seems to be a junction point between the two approaches. In the process of characterisation, the analyst chooses immediately a level of analysis, without apparent difficulty, as if the granularity imposes itself, that is that [START_REF] Goffman | Les cadres de l'expérience[END_REF] calls « that occurs here and now », that may correspond to the notion of « base level » used by Rosch. Actors know to call a routine, but they do not know to characterize activity.

The characterisation of processes and of knowledge consists in describing them in generic terms, without using a formalized method. Thus, this characterisation is a synthetic formulation, in dayto-day language, describing with the professional notions, what Feldman (2003) represents with, what she called "vignettes". The characterisation is situated to the « knowledge level ». To characterize the activity that we wish to observe or to model, is to pick it up from the other activities, to distinguish it, that the actors does not need to do.

It is also a way of making categories among observed organizational processes. That allows to make the hypothesis that these processes are belonging to process types or to process type families. Following that way, we can make a second hypothesis : if these types exist, they can be found in various organisations and professional activities. We are referring, here, to the categorisation theory (Rosch, 1978) and to the notion of models library in the problem solving methods [START_REF] Wielinga | KADS: A Modelling Approach to Knowledge Engineering[END_REF].Characterisation is thus a kind of descriptive vignette attribute to the observed activity, a sort of « type » of practice.

Characterisation is a preliminary step to the steps, which are producing detailed and formalized descriptions; the matter is just to know of what we are speaking of. That do appears immediately in the dialogue with actors or through practices observations. Characterisation implies interpretation and commitment of the observer, and it represents a possible orientation for the later development of work.

The knowledge engineering methods focus on further steps, more formal and more analytic ones and which distinguish knowledge, tasks, and communications. But some authors mention that step [START_REF] Schreiber | Knowledge Engineering and Management[END_REF], [START_REF] Aussenac-Gilles | Méthodes ascendantes pour l'ingénierie des connaissances[END_REF] and use in fact this notion without giving a great attention to it.. Thus in (Schreiber et al, 2000, p. 129) the concept is used and appears as a title to speak of the action, done on a task, nevertheless, the term of characterisation does not appear in the bright index of the book.

Schreiber and al consider three steps in the knowledge modelization process (Schreiber et al, 2000, p. 169): the step of identification which consists in a familiarisation and a first task models inventory, the step of specification which complete the set of knowledge and inferences concerned by the software, the step of refinement which consists in detailing the used models for the development, and to pair them off with use scenarios and to begin simulations. For us, the step of characterisation belongs to the first step.

The characterisation of processes may be applied at different levels, first of all at the global level of organizational activity, then at different included processes, as we had done in this paper. Until the cognitive processes characterisation granularity then it joints the tasks and inferences models library of KADS.

Conclusion

Through the example of the PRP in an academic journal, we have shown that RIO recovers strongly interelated dependencies and control loops that insure the transformation of individual work in collective work. Strategizing occurs through day to day decision about core processes of the organization. We have shown that to modelize precisely these processes allows us to situate practices and their diversity. To do that sort of analysis and to describe the routine structure, knowledge engineering modelling is useful, particularly to enlighten knowledge and knowing. In this approach of representing tasks and routines, we distinguish a common step that we call characterisation.

Aknowledgements

This work was supported by the T-CAN (Knowledge treatments, learning and new technologies) program of CNRS and was a part of the project ACTMUT : Access to the Texts and Mutation of Techniques. Thanks also to the editing board, that accepted to be "observed" for a long time.

  RIO is the foundation of PRP: Focus on a detail the practice « reviewer's assignment to a paper ».

-Modelling the reviewer's assignment to a paper.

  is quoted from the generic schema proposed by Schreiber and al for the task of assignment (2000, p 158).

	proposal FIG 3. Review note and Paper Analysis Editor in chief Transforming individual work in a collective position.
	Analysis
	And reviewer proposal	Reporter	Assign
	Review note and proposal Reviewers	Selecting candidates	Candidates reviewers
			Three reviewers	Selecting a list of reviewers
	Paper		Assign	Three reviewers Linked to paper
	Income or outcome Entities, Objects,	collective process	individual process
	Actions, Decisions


Malek Kammoun

Brigitte Picard

Thierry Astruc

Mohammed Gagaoua

Denise Aubert

Muriel Bonnet

Véronique Blanquet

Isabelle Cassar-Malek
email: isabelle.cassar-malek@clermont.inra.fr

The Invalidation of HspB1 Gene in Mouse Alters the Ultrastructural Phenotype of Muscles

Even though abundance of Hsp27 is the highest in skeletal muscle, the relationships between the expression of HspB1 (encoding Hsp27) and muscle characteristics are not fully understood. In this study, we have analysed the effect of Hsp27 inactivation on mouse development and phenotype. We generated a mouse strain devoid of Hsp27 protein by homologous recombination of the HspB1 gene. The HspB1 -/-mouse was viable and fertile, showing neither apparent morphological nor anatomical alterations. We detected a gender dimorphism with marked effects in males, a lower body weight (P < 0.05) with no obvious changes in the growth rate, and a lower plasma lipids profile (cholesterol, HDL and triglycerides, 0.001 < P< 0.05). The muscle structure of the animals was examined by optical microscopy and transmission electron microscopy. Not any differences in the characteristics of muscle fibres (contractile and metabolic type, shape, perimeter, cross-sectional area) were detected except a trend for a higher proportion of small fibres. Different myosin heavy chains electrophoretic profiles were observed in the HspB1 -/-mouse especially the presence of an additional isoform. Electron microscopy revealed ultrastructural abnormalities in the myofibrillar structure of the HspB1 -/-mouse mutant mice (e.g. destructured myofibrils and higher gaps between myofibrils) especially in the m. Soleus. Combined with our previous data, these findings suggest that Hsp27 could directly impact the organization of muscle cytoskeleton at the molecular and ultrastructural levels.

Introduction

The heat shock proteins (Hsps) are among the most conserved molecules in phylogeny. They are up-regulated in response to various adverse changes of the environmental conditions inducing cellular stress (e.g. heat shock, nutrient deficiencies, viral infections, various chemicals) [START_REF] Kampinga | Guidelines for the nomenclature of the human heat shock proteins[END_REF].

Their up-regulation in stressful conditions is a universal phenomenon, occurring in plant and animal species. In mammals, Hsps are grouped into six main families according to their molecular weight-Hsp110, Hsp90, Hsp70, Hsp60, Hsp47 (Large Hsp)-and the Hsp20 family (Small Hsps, 12-43 kDa). The small Hsp family comprises eleven members (including Hsp27) with various tissue distributions that share a conserved α-crystallin domain [START_REF] Fontaine | Interactions of HSP22 (HSPB8) with HSP20, alpha Bcrystallin, and HSPB3[END_REF].

The small Heat shock protein 27 (Hsp27, also denoted Hsp25 in mouse) is constitutively present in a wide variety of tissues and cell lines. Hsp27 belongs to the Hsp chaperone network and plays a key role in the cellular response to stress including heat shock, oxidative and chemical stresses. It is constitutively expressed in small concentrations in the cytosol during unstressed conditions and translocates into or around the nucleus under stress conditions [START_REF] Arrigo | Dynamic changes in the structure and intracellular locale of the mammalian low-molecular-weight heat shock protein[END_REF]. This protein is encoded by the HspB1 gene and is transcriptionally regulated by HSF-1. Its post-translational phosphorylation allows for up to 4 isoelectric variants [START_REF] Landry | Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 kinase II[END_REF]. It is one of the most induced Hsps in response to stress, reaching 1% of the total cellular protein, but accumulates in a slower kinetics than that of Hsp70 the other highly stress responsive Hsp [START_REF] Garrido | Heat shock proteins 27 and 70[END_REF]. There is increasing evidences to substantiate Hsp27 as a molecular protector against disease [START_REF] Kargul | Small heat shock proteins: Molecular protectors against the disease[END_REF] thus used as a biomarker in several disease states [START_REF] Vidyasagar | Heat shock protein 27 (HSP27): biomarker of disease and therapeutic target[END_REF].

Muscle-specific transcriptional regulatory sequences are found in the HspB1 gene and the abundance of Hsp27 is the highest in skeletal muscle [START_REF] Neufer | Differential Expression of alpha B-Crystallin and Hsp27 in Skeletal Muscle during Continuous Contractile Activity. Relationship to myogenic regulatory factors[END_REF] indicating a crucial role for muscle physiology. HspB1 expression in muscle is induced by a variety of stimuli [START_REF] Welch | Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease[END_REF] e.g. increased physical activity [START_REF] Morton | The Exercise-Induced Stress Response of Skeletal Muscle, with Specific Emphasis on Humans[END_REF] [START_REF] Huey | Effect of HSP25 Loss on Muscle Contractile Function and Running Wheel Activity in Young and Old Mice[END_REF] including lifelong training [START_REF] Cobley | Lifelong training preserves some redox-regulated adaptive responses after an acute exercise stimulus in aged human skeletal muscle[END_REF], endurance training [START_REF] Folkesson | The expression of heat shock protein in human skeletal muscle: effects of muscle fibre phenotype and training background[END_REF], and eccentric exercise [START_REF] Paulsen | Subcellular movement and expression of HSP27, αB-crystallin, and HSP70 after two bouts of eccentric exercise in humans[END_REF]. Some protective functions of Hsp27 are suggested by its translocation from the cytosolic to myofibrillar fraction and binding to cytoskeletal structures such as Z-disk proteins [START_REF] Koh | Cytoskeletal disruption and small heat shock protein translocation immediately after lengthening contractions[END_REF] [START_REF] Paulsen | Subcellular movement and expression of HSP27, αB-crystallin, and HSP70 after two bouts of eccentric exercise in humans[END_REF]. The chaperone-like properties of Hsp27 may stabilize myofibrillar proteins during stress conditions and prevent them from loss of function. The relationships between expression of HspB1 and muscle phenotype have not yet been fully understood apart from a muscle type specific pattern of expression [START_REF] Neufer | Differential Expression of alpha B-Crystallin and Hsp27 in Skeletal Muscle during Continuous Contractile Activity. Relationship to myogenic regulatory factors[END_REF]. Hsp27 expression is further detected in zygotes and early embryos [START_REF] Liu | Expression of heat shock protein 27 in gut tissue of growing pigs fed diets without and with inclusion of chicory fiber[END_REF] and is regulated during mammalian development [START_REF] Morrow | Small heat shock protein expression and functions during development[END_REF]. To date the role of constitutively expressed Hsp27 is still questioned during development. In this study we have examined the role of constitutive Hsp27 in mouse development and physiology, especially in muscle, by engineering an HspB1-null mouse and examining the associated phenotype.

Material and Methods

Animals and samples

Generation of an HspB1-null mouse. To investigate the role of Hsp27, we generated a constitutive HspB1-null mouse using embryonic stem (ES) cells. The cells were provided by the International Knockout Mouse Consortium in which 1.381 bases of the nucleotide sequence of the HspB1 gene (http://www.velocigene.com/komp/detail/13573) were replaced by a bacterial artificial chromosome (BAC) based vector obtained from BMQ BAC library (Mouse Micer vector set 369N20) (Fig 1A). In the targeting vector, the reporter LacZ gene was fused in frame with the start codon of HspB1 to replace the HspB1 coding sequence. The mutated ES cells were derived from VGB6 ES cells (C57BL/6NTac background). Three different mutated clones were injected into Balb/c blastocysts to generate 13 chimeras at the SFR Biosciences-Plateau de Biologie Expérimentale de la Souris (PBES, Lyon, France). Male chimeras transmitting the targeted mutation were mated with females from C57BL/6J strain to generate F1 offspring at the experimental facility of nutrition and microbiology of the French National Institute for Agricultural Research (INRA-France). Heterozygous animals (HspB1 +/-) were identified by PCR analysis from tail DNA using different primers as depicted in Table 1. They were intercrossed to generate homozygous F2 mice (HspB1 -/-) (Fig 1B). The F2 offspring were mated in order to grow the heterozygous and homozygous strains. Data from heterozygous mice with intermediary phenotype between controls and mutants are not given here.

HspB1-null mice handling

Mouse strains were housed at the technical platform of animal experimentation of UMR 1019 Human Nutrition (Theix, France). They were maintained in a light/dark cycle of 12 hrs, under a controlled room at a temperature of 22°C and humidity of 45-55%. They were fed ad libitum with a conventional diet. Experimental procedures and animal handling were done according to the French animal protection legislation including licensing of experimenters. They were reviewed and approved by the French Veterinary Services and the ethics committee, named "Comité Régional d'Ethique en Matière d'Expérimentation Animale Région Auvergne" (agreement number CE 84-12). All efforts were made to minimize suffering. None of the animals utilized for this work became ill or died prior to the experimental endpoint. For plasma and tissue collection, the animals were anesthetized prior to euthanasia by cervical dislocation. Anaesthesia was induced by placing each mouse in an inhalation chamber with 4% isoflurane regulated with calibrated vaporizer. The mice were weighed once a week during the first 12 postnatal weeks (wk) in order to follow the evolution of their body weight (BW). The average daily gain (ADG) during this period was calculated. Body weight and length were recorded at 8-wk (24 HspBI -/-and 19 HspBI +/+ ), 12-wk (25 HspBI -/-and 20 HspBI +/+ ) and 52-wk (6 HspBI -/-and 6 HspBI +/+ ) of age. The body composition was also determined using the EchoMRI™ method that offers measurements of body fat mass and lean mass in live animals.

Blood sampling. Blood samples were collected from the retro orbital sinus under general anaesthesia at 4, 8 and 12 wk. Blood were sampled in tubes containing heparin to prevent the formation of clots or extension of existing clots within the blood. Then samples were centrifuged at 4°C (4000 rpm, 10 min) for plasma separation. Plasmas were kept as aliquot fractions at -80°C until analysis.

Tissue collection. Mice were sacrificed at 8-wk, 12-wk, and 52-wk of age after blood sampling. Three muscles differing in their fibre types composition were sampled immediately after death, namely the m. Soleus (Slow and Fast oxidative), m. Gastrocnemius (Fast oxido-glycolytic) and m. Tibialis anterior (Fast glycolytic). The liver and three adipose tissues (AT) namely inguinal AT, interscapular AT, and epididymal AT for males were sampled. Tissues were collected at 8-wk, 12-wk, and 52-wk of age. These ages were chosen in order to analyse the effect of Hsp27 inhibition at different stages of development. All the tissues were weighted, frozen in liquid nitrogen for molecular biology analyses or in an isopentane bath cooled by liquid nitrogen for histochemical analyses, and kept at -80°C until use. Ultrastructure analysis, was performed on 2 HspBI -/-and 2 HspBI +/+ mice of 12 weeks old. (Soleus and Tibialis anterior muscles were removed immediately after death from posterior members of mice. Muscles were pinned to each tendon on a plate cork preserving their lengths before dissection. Muscles were fixed overnight at 4°C by immersion in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2.

Measurements

Plasma analyses. The plasma concentrations of 26 parameters representative of muscular, hepatic, lipidic, and energetic metabolism (urea, glucose, amylase, uric acid, phosphorus, calcium, cholesterol, triglycerides, low-density lipoprotein (LDL), high-density lipoprotein (HDL), cholinesterase, total bilirubin, total proteins, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, iron, albumin, creatinine, creatine kinase, creatine kinase MB, lactate, lactate dehydrogenase, magnesium, transferrin and hydroxybutyrate dehydrogenase) were measured using a KONELAB 30 automat (Thermo scientific, Cergy Pontoise, France) at the Faculty of Science and Technology of Limoges (France) as described by Magnol et al. [START_REF] Magnol | A sensitised mutagenesis screen in the mouse to explore the bovine genome: study of muscle characteristics[END_REF].

Histology, immunohistochemical and β-galactosidase staining. Serial sections of 10 μm thick, were cut on a cryostat (Cryo-star HM 560 Microm International GmbH, Germany) at -26°C, perpendicular to the muscle fibres. Adjacent sections were used for immunohistochemical and histochemical analyses and for β-galactosidase staining.

A semi-automatic classification of fibres was used for classification of muscle fibre by using a combination of only two anti-Myosin heavy chain (MyHC) antibodies [START_REF] Kammoun | A reliable immunohistochemical classification of skeletal muscle fibres in mice[END_REF]. For m. Soleus BA-D5 (specific of slow MyHC I) and SC71 (specific of IIa MyHC) were used on serial sections. For m. Tibialis anterior BF-F3 specific of IIb MyHC and SC71 were used on serial sections according to Kammoun et al. [START_REF] Kammoun | A reliable immunohistochemical classification of skeletal muscle fibres in mice[END_REF]. The anti-MyHC antibodies were purchased from AGRO-BIO (La Ferté Saint-Aubin, France). After immuno-staining of MyHC isoforms, histological sections were visualized under an Olympus fluorescence microscope BX 51, using a 10X objective (NA = 0.3) and adequate band pass filter (Alexa 488: excitation filter 460-495, emission filter 510-550, dichromatic mirror 505LP; Cy3: excitation filter 530-550, emission filter 575-625, dichromatic mirror 565LP) as described by Meunier et al. [20]. This technique allowed us to determine the muscle fibre type proportions, meaning cross-sectional for an average 300 fibres per serial image. The metabolic fibre type was determined by revealing the succinate dehydrogenase (SDH, a mitochondrial enzyme representative of oxidative metabolism) activity according to Ashmore [START_REF] Ashmore | Comparative aspects of muscle fibre types in fetuses of the normal and double muscled cattle[END_REF].

For HspB1 -/-mice muscles, serial sections were also stained for β-galactosidase (β-gal) activity in order to detect reporter gene expression. Individual images were assembled into composite panoramic images of MyHC analysis, SDH activity and β-gal staining.

Electrophoretic separation of MyHC isoforms. Myofibrillar proteins were extracted using a buffer containing 0.5 M NaCl, 20 mM NaPPi, 50 mM Tris, 1 mM EDTA and 1 mM DTT according to the protocol described by Picard et al. [START_REF] Picard | Protocol for high-resolution electrophoresis separation of myosin heavy chain isoforms in bovine skeletal muscle[END_REF]. After homogenization using a Polytron (x22 000), the muscle extract was subjected to a centrifugation at 2500 x g for 10 min at 4°C. Then the supernatants were diluted 1:1 (v/w) with glycerol and stored at -20°C until use. The protein concentrations of the extracts were determined according to the Bradford method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] using the Bio-Rad Protein assay. Bovine serum albumin (BSA) at a concentration of 1 mg/mL was used as standard.

The samples were suspended in 1:1 v/v in a basic 2 x Laemmli solution containing 4% w/v SDS, 10% v/v β-mercaptoethanol, 20% v/v glycerol, 125 mM Tris (pH 6.8) and 0.01% w/v pyronin Y, incubated at room temperature for 10 min and then heated (70°C) for 10 min. The MyHC isoforms were separated with SDS glycerol gel electrophoresis according to Picard et al. [START_REF] Picard | Protocol for high-resolution electrophoresis separation of myosin heavy chain isoforms in bovine skeletal muscle[END_REF]. The stacking gel contained 4% polyacrylamide with a cross-link of 1.96%. The separating gel was an 8% polyacrylamide gradient with a cross-link of 1%. Three micrograms of protein extracts were loaded per well onto 0.75-mm-thick gels. The electrophoresis was run a Mini-PROTEAN 1 II vertical chamber (Bio-Rad, USA) at a constant voltage of 140V for 15 hours at 4°C. After staining in a Coomassie Blue dye solution, the gels were scanned using the Expression 10000XL Pro scanner (Epson) and the proportions of the different MyHCs bands were quantified by densitometry with ImageQuant Software 5500 (Amersham Biosciences/GE Healthcare).

Western-blot analyses. Invalidation of HspB1 gene was validated in the muscle of HspB1 -/-vs controls using a quantitative western-blotting technique as described by Chaze et al. [START_REF] Chaze | In vivo proteome dynamics during early bovine myogenesis[END_REF]. Fifteen or thirteen μg of total protein extracts were separated by gel electrophoresis using SDS-PAGE [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. After migration, the proteins were transferred onto PVDF membrane (Millipore, Bedford, MA01730 U S A). Membranes were blocked using a TBS 1x buffer containing 5% skimmed milk and incubated under gentle agitation overnight at room temperature in the presence of the Hsp27 primary antibody (Santa Cruz: SC13132; 1/1000). The morning, the membranes were incubated at 37°C for 30 min. with the secondary fluorochrome-conjugated LICOR-antibody. Infrared fluorescence detection was then used for protein quantification Membranes were scanned by the scanner Odyssey (LI-COR Biosciences) at 800 nm. Band volumes were quantified in the images using ImageQuant TL v 2003 software (Amersham).

Ultrastructure and image analysis. Small blocks (1 to 3 mm 3 with fibre direction identified) were prepared from the glutaraldehyde fixed muscles and were post-fixed in 1% osmium tetroxide in 0.1 M SCB with adapted pH (7.2 or 5.8) for 1 hr at room temperature. The blocks were dehydrated through a graded series of ethanol and embedded in epoxy resin (TAAB, Eurobio France) by using an automatic microwave tissue processor for electron microscopy (EM AMW, Leica company,Vienna, Austria). Ultra-thin sections (90 nm) were stained with uranyl acetate and lead citrate and observed using a transmission electron microscope (Hitachi HM 7650) at 80 kV acceleration voltage. Micrographs were obtained using a Hamamatsu AMT digital camera system coupled to the microscope. All samples were prepared and observed at the Cellular Imaging Center for Health (CICS) lab at Clermont-Ferrand University (France).

The width of myofibrils and the intermyofibrillar spaces were determined on the images of soleus and tibialis longitudinal sections of 12 weeks old mice using the open source ImageJ software (http://rsb.info.nih.gov/ij/). The grey scale ranges extend from 0 (black) to 255 (white). The image analysis included automatic thresholding, and morphological measuring. For each numerical object labelled the area estimation was based on pixels number count, the coloration intensity corresponded on an 8 bit grey level mean value. The analysis was performed on a total of 210 images distributed in 96 images for wild-type (n = 2 mice) and 114 images for HspB1 -/-mice (n = 2). Four fields were analysed per image and therefore the measurements were performed in 480 electron microscopy fields.

Statistical analysis. Statistical analyses were performed using the PROC GLM procedure (SAS Inst. Inc., Cary, NC) and mean comparisons by using post hoc procedures (Newman-Keuls) were performed to analyse differences (P < 0.05). The first model tested fixed effects of genotype, age, and gender, and all interactions for growth, body composition, tissue weights and plasma chemistry parameters. The second model tested fixed effects of genotype, muscle, and gender, and all interactions for muscle ultrastructure. Least squares means were generated for all interactions and main effects. For electronic microscopy micrographs the statistical analysis was carried out according to [START_REF] Peca | Shank3 mutant mice display autistic-like behaviours and striatal dysfunction[END_REF] using a two-way ANOVA with repeated measures followed by multiple comparisons post-hoc Tukey correction for analyzing the interactions. The analyses were performed using the repeated measure option of the PROC GLM of SAS software, and results were considered significant at p < 0.05. Values are presented as means ± sem.

Results

Broad general phenotyping of the HspB1-null mouse

The generated HspB1 -/ -mice were viable, fertile and born after 21 days of gestation at the expected Mendelian distribution. Revelation of β-gal activity in muscle confirmed that the endogenous HspB1 promoter driven construction was expressed in the HspB1 -/-mice (Fig 1C). Western-blotting analysis of muscles homogenates confirmed the absence of Hsp27 (Fig 1D).

The HspB1 -/ -mutant mice showed no apparent morphological or anatomical alterations compared to homozygous controls until up to 18 months. The weights of new born mice were lower compared to controls (1.3 g for HspB1 -/-vs 2 g for HspB1 +/+ , P < 0.001) at one day after birth. As expected, during postnatal development (S1 Dataset), there was a significant effect of age (0.001 < P < 0.01, Table 2) and of gender (0.001 < P < 0.05, Table 2) on growth and body composition parameters however a genotype effect was observed only for body weight (BW) (P < 0.05, Table 2). There was no genotype effect on tissue weights expressed as a proportion of animal BW (Table 2). Significant interactions were detected and reported in Table 2 for BW and relative weight of fat (gender × genotype, P < 0.05) liver and m. Soleus tissues (age × gender × genotype, P < 0.05).

Fig 2A illustrates BW evolution from 1 to 12 weeks of age in both genotypes. The BW of HspB1 -/ -mice were significantly lower than that of controls from week 2 to 12 of age (P < 0.01) but the average daily gain (data not shown) did not differ between the two groups (0.21 to 0.23, P > 0.05). The HspB1 -/ -males were lighter from 12-wk-old onwards (Fig 2B). Similar differences were recorded for females only at 12-wk of age. No differences were observed in length at the three ages analysed.

Of the 26 plasma parameters measured (Table 3), significant differences were detected between genotypes for the lipids profile (cholesterol, HDL, and triglycerides, 0.001 < P < 0.05), total bilirubin (P < 0.05), calcium (P < 0.01) and iron (P < 0.05). The data showed a decrease in lipidic metabolites for the HspB1-null mouse. Significant age × gender × genotype interactions (0.01 < P < 0.05) were also detected for glucose, total bilirubin and aspartate aminotransferase (Table 3) indicating that the liver functions (probably lipogenesis and detoxification) has been likely affected by HspB1 gene invalidation.

Muscle changes associated with Hsp27 deficiency

Muscle properties. As illustrated in Table 2, the skeletal muscles analysed, whether oxidative or glycolytic types, showed no differences with respect to weights between HspB1 -/-mice and wild-types. To investigate muscle phenotyping, we focused on 12-wk old adult males. Examination of fibre types (Fig 3 ) showed that the expression of MyHC isoforms, cross-sectional areas, shapes and perimeters of fibres did not differ between genotypes in the Soleus and Tibialis anterior muscles (Fig 3C -3F). However the frequency profiles of fibre sizes were different between genotypes, with a tendency to higher numbers of small fibres in the HspB1 -/- mouse than wild-type for both Soleus and Tibialis anterior muscles (Fig 3A and3B). SDH activity, an indicator of mitochondria content, revealed no obvious differences between the HspB1 -/-mouse and wild-type mouse (data not shown). In the muscles of HspB1 -/-mouse, 2 HspB1 -/-: HspB1-null mouse; HspB1 +/+ : Wild type littermates. 3 Significance: *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns: not significant. 4 LS-means with different superscripts (a, b, c) within a column for age effect are significantly different (P < 0.05). 4) and altered sarcomere (longitudinally repeating subunit of myofibrils) structure with Z lines deformation, A band and I band junctions not well defined. The sarcomere length (1.70 μm ± 0.3) was not changed. The costamers were stretched (Fig 5B vs. 5A). Some disintegration of the lateral attachments between myofibrils and the sarcolemma were also observed (Fig 5B). The morphology of nuclei appeared to be normal. They were localized at the periphery of myofibrils (data not shown). There appeared to be no differences in mitochondria numbers between HspB1 -/-mice and controls (Fig 4C and4D). However, an alteration was observed in the morphology of some mitochondria in HspB1 -/-muscles. Several fibres showed unaltered ultrastructure suggesting a high heterogeneity in the ultrastructure with altered and non-altered regions (Fig 4) ultrastructure was apparently not affected in the mutant mouse. HspB1 -/-oxidative fibres exhibited larger space between myofibrils than controls (Table 4). Lastly HspB1 -/-fibres showed more altered mitochondria.

Discussion

Small heat shock proteins (sHsps) play a fundamental role in multiple organisms through pleiotropic and more specific effects [START_REF] Bakthisaran | Small heat shock proteins: Role in cellular functions and pathology[END_REF]. They are tightly regulated during development [START_REF] Morrow | Small heat shock protein expression and functions during development[END_REF].

To investigate the role of constitutive Hsp27 in vivo, we generated a mouse strain carrying targeted invalidation of the HspB1 gene. We examined the consequences of Hsp27 deficiency on the broad phenotype of mutant mouse, and specifically on muscle development and fine characteristics. The final objective was to look for molecular mechanisms regulated by Hsp27 that could account for muscle development, structure, and physiology. The invalidation of HspB1 appeared to have only a moderate effect on the development and physiology since the mutant mice were viable, fertile and apparently normal offspring were generated. Since Hsp27 is constitutively present in a wide variety of tissues as part of the chaperone network, the occurrence of a moderate phenotype in the HspB1 -/-mouse was quite unexpected. Observation of HspB1 -/-mice over a period of 12 months did not reveal any signs of major morphological or physiological disturbance nor locomotion troubles or disease including neuropathy as predicted by Kim et al. [START_REF] Kim | Expression pattern of HSP25 in mouse preimplantation embryo: Heat shock responses during oocyte maturation[END_REF]. Thus, Hsp27 on its own did not appear to be essential for development as also reported in mouse [START_REF] Huang | Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption[END_REF][START_REF] Crowe | Heat Shock Protein B1-Deficient Mice Display Impaired Wound Healing[END_REF][32], fly [START_REF] Hao | The Hsp27 gene is not required for Drosophila development but its activity is associated with starvation resistance[END_REF], and zebrafish [START_REF] Middleton | Small heat shock protein HSPB1 regulates growth of embryonic zebrafish craniofacial muscles[END_REF] but in contrast to a study by Eroglu et al. [START_REF] Eroglu | An Essential Role for Heat Shock Transcription Factor Binding Protein 1 (HSBP1) During Early Embryonic Development[END_REF]. The moderate phenotype of the HspB1 -/-mouse indicates that the mutant has coped with the loss of Hsp27. This may be have been achieved through compensatory changes in the expression of cognate members of the sHsp family [START_REF] Fontaine | Interactions of HSP22 (HSPB8) with HSP20, alpha Bcrystallin, and HSPB3[END_REF] sharing common functions with Hsp27 and transcriptionally regulated by HSF-1. Compensation which may be elicited by other small Hsps has probably occurred during early development and postnatal life. Consistently we previously reported changes in the status of sHsps in the muscles of the HspB1 -/-mouse [START_REF] Kammoun | A network-based approach for predicting Hsp27 knock-out targets Computational and[END_REF][START_REF] Picard | Calcium Homeostasis and Muscle Energy Metabolism Are Modified in HspB1-Null Mice[END_REF] that may support functional redundancy. However we report here specific characteristics of the HspB1 -/-mouse: a gender dimorphism with marked effects in males, an effect on body weight with no obvious changes in the growth rate, a lower plasma lipids profile, and an alteration of muscle ultrastructure. These gender differences agree with previous reports that the heat shock induction of HspB1 is greater in males [START_REF] Morrow | Small heat shock proteins: big folding machines[END_REF] and that Hsp27 regulates the activity [START_REF] Zoubeidi | Cooperative interactions between androgen receptor (AR) and heat-shock protein 27 facilitate AR transcriptional activity[END_REF] and expression [START_REF] Stope | Differentially expressed proteins associated with myogenesis and adipogenesis in skeletal muscle and adipose tissue between bulls and steers[END_REF] of the androgen receptor which plays a role in testosterone-related myogenesis [41].

Although Hsp27 is expressed at the highest levels of any tissues in muscle in basal condition [START_REF] Neufer | Differential Expression of alpha B-Crystallin and Hsp27 in Skeletal Muscle during Continuous Contractile Activity. Relationship to myogenic regulatory factors[END_REF], there was no specific apparent macroscopic or microscopic muscle phenotype associated with Hsp27 loss. There was no main impact of HspB1 disruption neither on the relative weight of muscles nor on the size or types of muscle fibres. Similar observations were made in other vertebrate models, at the exception of the zebrafish for which Hsp27 was essential for optimal growth of craniofacial myofibres [START_REF] Middleton | Small heat shock protein HSPB1 regulates growth of embryonic zebrafish craniofacial muscles[END_REF]. However, as we examined animals only postnatally we cannot exclude that the muscles of the mutant mouse could have developed slower than those of wild-type during foetal development. Interestingly, very specific differences between mutants and wild-type controls were detected at the protein level [START_REF] Picard | Calcium Homeostasis and Muscle Energy Metabolism Are Modified in HspB1-Null Mice[END_REF]. The electrophoretic profiles of m. Soleus proteins showed differences in myofibrillar proteins, especially the presence of a putative developmental MyHC isoform which remains to be confirmed. These differences may be explained by differences in the kinetics of myofibrillar protein expression between the HspB1 -/-mutant and the wild-type mouse during myogenesis. Thus they probably illustrate a slower development of mutant muscles as suggested above. Accordingly, the higher number of small fibres in the HspB1 -/-mouse may result from delayed muscle growth.

Most striking is the ultrastructure alteration of the skeletal muscles in the HspB1 -/-mouse. As a result of transgenic insertion to disrupt HspB1 gene expression, introduction of LacZ gene via the targeting vector produces constitutive beta galactosidase expression. Albeit in the absence of substrate at the optimal pH in situ, this may be expected to compromise development or structure of tissues and organs. LacZ is broadly used as a reporter gene in transgenic studies with not any dedicated effect nor demonstrated cytotoxicity of beta galactosidase even after long term expression. Overexpression of this enzyme following injection or transgenic expression was demonstrated by immunocytochemistry in several studies while not any obvious change was detected in the heart and muscle cell ultrastructure in vivo [START_REF] Fuoco | In vivo generation of a mature and functional artificial skeletal muscle[END_REF][START_REF] Hoofnagle | Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes[END_REF][START_REF] Mackenzie | Transduction of satellite cells after prenatal intramuscular administration of lentiviral vectors[END_REF][START_REF] Miana-Mena | Neuronal activity-dependent membrane traffic at the neuromuscular junction[END_REF][START_REF] Pappas | Knockout of Lmod2 results in shorter thin filaments followed by dilated cardiomyopathy and juvenile lethality[END_REF]. However this does not disprove an effect of the LacZ reporter in the muscle ultrastructure in the HspB1 mouse but makes it very unlikely. We rather assume that change in muscle ultrastructure is a consequence of HspB1 invalidation. Indeed sHsps are important in maintaining cytoskeletal integrity [START_REF] Bakthisaran | Small heat shock proteins: Role in cellular functions and pathology[END_REF][START_REF] Tucker | Hsp27 associates with the titin filament system in heat-shocked zebrafish cardiomyocytes[END_REF]. Especially during intense exercise and associated hyperthermia, high levels of sHsps are needed to prevent cellular damage in skeletal muscles [START_REF] Huang | Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption[END_REF]. Hsp27 interacts with key cytoskeletal protein elements and is associated with Z-disk maintenance [START_REF] Dubińska-Magiera | Contribution of small heat shock proteins to muscle development and function[END_REF]. This has been described at the level of microfilaments and intermediate filaments [START_REF] Perng | Intermediate filament interactions can be altered by HSP27 and alpha B-crystallin[END_REF]. Under excise damage Hsp27 and αB-crystallin translocate from the intermyofibrillar space to the cytoskeletal/myofibrillar structures after a repeated bout of exercise [START_REF] Paulsen | Subcellular movement and expression of HSP27, alpha beta-crystallin, and HSP70 after two bouts of eccentric exercise in humans[END_REF]. They accumulate in myofibrillar structures, especially in the Z-line and intermediate filament structures, and in areas of disrupted sarcomeres. Cytoplasmic Hsp27 is thought to interact primarily with actin microfilaments [START_REF] Mounier | Actin cytoskeleton and small heat shock proteins: how do they interact?[END_REF], while cytoplasmic αB-crystallin appears most frequently to interact with intermediate filament proteins e.g. desmin [START_REF] Perng | Intermediate filament interactions can be altered by HSP27 and alpha B-crystallin[END_REF]. Thus Hsp27 likely modulates stability, structure and dynamics of actin filaments [START_REF] Guay | Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27[END_REF]. In Zebrafish, localization of Hsp27 in resting length myofibrils correlates with regions adjacent to the Z-line. However in stretched myofibrils Hsp27 localization does not depend on desmin, α-actinin, myosin, or filamentous actin, but is connected with titin filaments [START_REF] Tucker | Hsp27 associates with the titin filament system in heat-shocked zebrafish cardiomyocytes[END_REF]. A fine control of muscle architecture is of paramount importance during muscle development and myoblast differentiation, or when facing excessive damage. In Xenopus embryos lacking Hsp27 after morpholino injection, defects in myofibril architecture were observed in heart and skeletal muscle [START_REF] Brown | Small heat shock protein hsp27 is required for proper heart tube formation[END_REF]. There were evidences of failure of myofibril assembly and sarcomere formation. Thus, the primary function of Hsp27 may be the regulation of myofibril structure. In our gene knock-out study, constitutive loss of Hsp27 also resulted in myofibril ultrastructural abnormalities in basal conditions as shown by disturbances in the cross-striation band pattern, deformation of myofibrillar structures, enlarged intermyofibrillar space, presence of some distorted mitochondria, and grouping of subsarcolemal mitochondria. Several hypotheses can be formulated to account for the muscle ultrastructural phenotype of The measurements were performed in 480 electron microscopy fields.

1 HspB1 -/-: HspB1-null mouse; HspB1 +/+ : Wild type littermates.

the HspB1 mutant mouse including misfolded proteins (loss of protective function through protein triage [START_REF] Garrido | The small heat shock proteins family: The long forgotten chaperones[END_REF]), abnormal myofibrillogenesis and disorganization / loss of costameric proteins (e.g desmin) (loss of protective function from fragmentation) in the absence of Hsp27. Moreover, the alterations observed may be linked to altered maintenance of muscle homeostasis and activation of proteolysis through different mechanisms including proteolytic systems, apoptosis, proteasomal or autophagosomal protein degradation [START_REF] Acunzo | Small heat shock proteins HSP27 (HspB1), alpha B-crystallin (HspB5) and HSP22 (HspB8) as regulators of cell death[END_REF]. Interestingly, the specific phenotype of mutant muscle was associated with a lower abundance of desmin (a potential substrate of the calpain system and of caspase 3) and changes in the abundance of others proteins involved in muscle structure [START_REF] Kammoun | A network-based approach for predicting Hsp27 knock-out targets Computational and[END_REF]. Since (i) under physiological conditions Hsp27 loss does not impair mouse development and physiology and (ii) the protein is stress-inducible, the phenotype of mouse is questioned under stress conditions. Indeed, the analysis of Hsp27 deficient muscle in stress conditions (exercise or hyperthermic challenge) will provide key elements on the function of Hsp27 and its interactors during pre-and post-natal development. This has been partly examined by Huang et al [START_REF] Huang | Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption[END_REF] who did not detect any change in the levels of several Hsps following hyperthermic challenge. However, as stated by these authors multiple sHsp targeted disruption might be helpful for further understanding of Hsps functional redundancy in mouse development.

Conclusions

Overall, this study showed that mouse development and morphometric characteristics of their skeletal muscles were not impaired in the absence of the HspB1 gene. These data suggest a functional redundancy between Hsps during development with subsequent apparently normal phenotype. However, the invalidation of the HspB1 gene resulted in alteration of muscle ultrastructure. Therefore redundancy was not detected at ultrastructural levels suggesting a crucial role for Hsp27 in the regulation of protein make-up for muscle ultrastructure.

Fig 1 .

 1 Fig 1. Generation of the HspB1 -/--null mouse. (A) The targeting vector was constructed by replacing 1.381 bases of the genomicDNA A, including the three exons of the HspB1 gene, with a neomycin resistance (Neo) gene and LacZ reporter gene. The sequences of the primers used are given in Table 1. (B) PCR-based genotyping amplifies targeted construction of LacZ reporter gene and Neo gene by using primers P5 and P6 respectively. (C) β-gal activity encoded by the LacZ reporter gene was used to monitor gene expression in the m. Tibialis anterior of the HspB1 -/-mouse. (D) Absence of Hsp27 protein as confirmed by western-blotting on muscle homogenates prepared from HspB1 -/-mouse and the wild-type (HspB1 +/+ ) mouse. doi:10.1371/journal.pone.0158644.g001

doi: 10 .

 10 1371/journal.pone.0158644.t002 expression of the HspB1 promoter driven construction was found to be fibre-type specific (type IIB < type IIA < type I and IIX) (Fig 1 and data not shown). An adequate electrophoresis technique to quantify proportions of MyHC revealed distinct profiles between the HspB1 -/-mouse and the wild-type mouse in the m. Soleus (Fig 3G right panel). A third MyHC was detected in HspB1 -/-mice comparatively to their controls. As illustrated in Fig 3G (left panel) this MyHC could be similar to the developmental isoform detected at 4 weeks of age in the wild-type mouse. However, this could not be validated by immunohistochemistry likely because the fast antibody cross-reacted with developmental MyHC [27]. Muscle ultrastructure. To examine the in-depth phenotype of muscle, we performed electron microscopy of longitudinal muscle sections and measurements. Figs 4-6 illustrate changes in muscle ultrastructure following Hsp27 invalidation. Compared to the wild-type muscle (Fig 4A, 4C and 4E), the soleus muscle of the HspB1 -/-mouse (Fig 4B, 4D and 4F) showed increased intermyofibrillar spaces (Table

  . The ultrastructure of the m. Tibialis anterior in the mutant mouse is presented in Fig 6. Four fibre types (I, IIA, IIB and IIX) are observed. The oxidative fibres (Fig 6A, 6B, 6C and 6D) are characterized by a high content of mitochondria while glycolytic fibres (Fig 6E and 6F) are poor in mitochondria. Changes in m. Tibialis anterior ultrastructure were less marked than in the Soleus muscle. Glycolytic fibres showed very small intermyofibrillar spaces and their

Fig 2 .

 2 Fig 2. Influence of HspB1 gene disruption on the growth and size of animals. (A) Evolution of body weight from 1 to 12 weeks of age. (B) Body weight at 8-wk, 12-wk, and 52-wk of age; HspB1 -/-: HspB1-null mice; HspB1 +/+ : Wild type littermates; F: Females; M: Males. LS-means (± SD) with different superscripts ( a, b, c, d, e ) are significantly different (P < 0.05). doi:10.1371/journal.pone.0158644.g002

  Abbreviation: aminotransf: aminotransferase; dehyd: dehydrogenase 1 HspB1 -/- : HspB1-null mouse;HspB1 +/+ : Wild type littermates.

Fig 3 .

 3 Fig 3. Influence of HspB1 gene invalidation on muscle fibre characteristics. (A, B) Frequency profiles of muscle fibre cross sectional area at 12 weeks of age in the m. Soleus (A) and the m. Tibialis anterior (B). (C-F) Myosin heavy chain (MyHC) immunostaining in the m. Soleus (C, D, BAD5 staining) and m. Tibialis anterior (E, F, BF-F3 immuno-staining) at 12 weeks of age. (C, E) wild type controls; (D, F) HspB1 -/-mice. Fibre types: I, slow oxidative fibre; IIA and IIX, fast oxido-glycolytic fibre; IIB, fast glycolytic fibre. Scale bar: 50 μm; (G) electrophoretic separation of MyHC at 3 ages in wild type controls (HspB1 +/+ ) and HspB1 -/-mutant mice. doi:10.1371/journal.pone.0158644.g003

Fig 4 .

 4 Fig 4. The loosely ultrastructure of the Soleus muscle associated with invalidation of Hsp27. (A, C, and E): HspB1 +/+ (Wild type control); (B, D, and F): HspB1 -/-(HspB1-null mouse). Fi: muscle fibre, ECS: extra cellular space, My: Myofibril, EMyS: Extramyofibrillar space, Mi: Mitochondria, Sr: Sarcomere, A: A band, I: I band. doi:10.1371/journal.pone.0158644.g004

Fig 5 .

 5 Fig 5. Details of the ultrastructure of Soleus muscle of the HspB1 -/-mouse. Desmin lateral attachments between myofibrils to the sarcolemma are shown in the HspB1-null mouse. (A) Well defined sarcolemma in the wild type control; (B) disintegration of the lateral attachments between myofibrils to the sarcolemma. Fi: muscle fibre, My: Myofibril, Mi: Mitochondria, C: Costamers, D: Desmin Filaments, S: Sarcolemma, EMyS: Extramyofibrillar space. doi:10.1371/journal.pone.0158644.g005

Fig 6 .

 6 Fig 6. Ultrastructure of the Tibialis anterior muscle of the HspB1 -/-mouse. (A, C, and E): HspB1 +/+ (Wild type mouse); (B, D, and F): HspB1 -/-(HspB1-null mouse). My: Myofibril. Arrows: Altered mitochondria. (A, B, C, D): oxidative fibres characterized by high mitochondria content, (E, F) glycolytic fibres with small amount of mitochondria. doi:10.1371/journal.pone.0158644.g006

Table 4 .

 4 Influence of HspB1 gene disruption on muscle ultrastructure.

Table 1 .

 1 Primer sequences used for PCR-based genotyping.

	Primer pair	Forward sequence	Reverse sequence
	P1	CTCGGAGATCCGACAGACG	TCTGGGCAC AAGGGAAACTC
	P2	GGGCTGCTTCTGACCTTCTG	CCATGTTCGTCCTGCCTTTC
	P3	TCCAGCTACCGGTATTACGC	CTCTGCATT CCTCCCCCTAA
	P4	GGTTATCTCTTTTCCCACAG	ATTATGGTGGGCTAGGGATG
	P5	TTGACTGTAGCGGGCTGATGTTG	GGTAAACTGGCTCGGATTAGGG
	P6	TACTTTCTCGGCAGGAGCAAGGTG	TGGGATCGGCCATTGAACAAGATG

The location of the different primers are presented in Fig 1A doi:10.1371/journal.pone.0158644.t001

Table 2 .

 2 Influence of HspB1 gene disruption on growth parameters, body composition and relative tissue weights 1 .

	Main effect	BW, g Length,	Fat,	Lean	Liver	m.	m.	m. Tibialis	AT.	AT.	AT.
			cm	mg/g	mg/g	mg/g	Soleus	Gastrocnemius	anterior	Epididymal	Inguinal	Interscapular
							mg/g	mg/g	mg/g	mg/g	mg/g	mg/g
	Genotype (Ge) 2
	HspB1 +/+ (n = 45)	23.20	6.71	9.74	85.93	5.64	0.07	0.87	0.31	1.29	0.79	0.65
	Male (n = 25)	25.24	6.85	9.14	86.42	5.83	0.07	0.89	0.31	1.29	0.74	0.62
	Female (n = 20)	20.64	6.53	10.50 85.32	5.39	0.07	0.86	0.32	/	0.84	0.69
	HspB1 -/-(n = 55)	22.0	6.59	9.22	86.66	5.40	0.07	0.89	0.35	1.21	0.75	0.60
	Male (n = 33)	23.43	6.71	8.15	87.71	5.40	0.07	0.92	0.37	1.21	0.68	0.55
	Female (n = 22)	19.84	6.41	10.84 85.08	5.39	0.07	0.84	0.32	/	0.86	0.67
	P-value 3	*	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
	Gender (G)
	Male (n = 58)	24.21	6.77	8.57	87.15	5.59	0.07	0.90	0.35	1.24	0.71	0.58
	Female (n = 42)	20.22	6.46	10.68 85.20	5.39	0.07	0.85	0.32	/	0.85	0.68
	P-value	***	**	**	*	ns	ns	ns	ns	/	**	*
	Age (A) 4
	8 wk (n = 43)	19.59 c	6.36 c	7.75 b 88.06 a 5.80 a 0.07 a,b	0.88	0.37	0.87 c	0.67 b	0.58 b
	12 wk (n = 45)	23.03 b	6.61 b	7.95 b 87.65 a 5.44 a 0.074 a	0.90	0.32	1.18 b	0.67 b	0.53 b
	52 wk (n = 12)	31.25 a	7.71 a	21.23 a 75.17 b 4.80 b 0.065 b	0.83	0.29	3.13 a	1.46 a	1.11 a
	P-value	***	***	***	***	**	**	ns	ns	***	***	***
	Interactions
	Ge x G	*	ns	*	ns	ns	ns	ns	ns	/	ns	ns
	Ge x A	**	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
	G x A	***	ns	ns	ns	ns	ns	ns	ns	/	**	ns
	Ge x G x A	*	ns	ns	ns	*	*	ns	ns	/	ns	ns

1

Muscle weight, adipose tissues (AT) weight, Liver weight, Fat and Lean are relative to animal body weight.

Table 3 .

 3 Influence of HspB1 gene disruption on the plasma chemistry parameters during postnatal growth.

		Urea	mmol/	L	9.84	9.76	9.97	9.40	8.80	10.14	ns		9.33	10.06
		Uric	acid μmol/L		78.86	79.14	78.45	77.25	71.69	84.19	ns		75.75	81.32
		Creatinine	UI/L		21.86	2 0 .97	2 3 .20	21.52	2 1 .35	2 1 .74	ns		21.14	22.50
		Creatine	Kinase	MB, UI/L	396.6	387.9	409.6	348.1	351.9	343.5	ns		371.5	376.5
		Creatine	Kinase C UI/	L	439.5	439.4	439.7	460.9	368.9	575.8	ns		507.8	407.4
		Cholinesterase	UI/L		1452.8	1274.1	1721.0	15.28.2	1320.5	1787.7	ns		1295.2	17.54.3
		Amylase	UI/L		2742.6	2914.14	2485.30	2812.2	2986.60	2594.33	ns		2947.1	2539.8
		Total	bilirubin μmol/	L	1.24	1.11	1.42	1.48	1.63B	1.29	*		1.35	1.35
		Aspartate	aminotransf.	UI/L	105.7	101.73	111.62	107.4	101.53	114.68	ns		101.64	113.15
		Alanine	aminotransf.	UI/L	41.51	48.96	30.33	43.10	55.70	27.35	ns		52.02	28.84
		Alkaline	phosphatase	UI/L	124.52	121.14	153.29	119.25	116.49	134.77	ns		106.63	137.15
		Transferrin	g/L		1.01	1.00	1.02	1.00	1.01	0.99	ns		1.01	1.00
		Albumin	g/L		27.0	26.64	27.54	26.63	26.29	27.05	ns		26.5	27.3
	A	Main effect		Genotype (Ge) 1	HspB1 +/+ (n = 30)	Male (n = 18)	Female (n = 12)	HspB1 -/-(n = 27)	Male (n = 15)	Female (n = 12)	P-value 2	Gender (G)	Male (n = 33)	Female (n = 24)	P-value

Table 3 .

 3

	Triglyceride.	mmol/L
	LDL mmol/	L
	HDL	mmol/L
	Cholesterol	mmol/L
	hydroxybutyrate	dehyd. UI/L
	Lactate	dehyd.	UI/L
	μmol/
	Lactate	L
	Glucose	mmol/L

PLOS ONE | DOI:10.1371/journal.pone.0158644August 11, 2016

Significance: *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns: not significant.

SEM: standard error of the mean.

LS-means (± SD).doi:10.1371/journal.pone.0158644.t004

Acknowledgments

The authors thank F. Guillou for helpful scientific discussions. We thank the Plateau de Biologie Expérimentale de la Souris (AniRA-PBES) of the UMS344/US8 SFR Biosciences for transgenic mouse production., G. Gentes, C. Barboiron, V. Largeau, N. Allegre, and J. Hermet for technical support for animal handling, sample collection and analysis, C. Szepaniack for electronic microscopy samples preparation, and A. Delavaud, for measurement of ultrastructure parameters.

INRA Phase Division funded the study (http://www.phase.inra.fr/en/).

All relevant data are within the paper and its Supporting Information files.

Supporting Information

Coordinated the redaction of the manuscript: ICM BP.