Carine Edith Toure
email: carine-edith.toure@liris.cnrs.fr

Christine Michel
email: christine.michel@liris.cnrs.fr

Jean-Charles Marty
email: jean-charles.marty@liris.cnrs.fr

Refinement of Knowledge Sharing Platforms to promote effective use: A use case

Keywords: knowledge management methods, knowledge repositories, computer supported collaborative work, humancomputer interactions, user-centered design, information architecture, enterprise social networks I

This work focuses on the improvement of a line of corporate knowledge management systems, knowledge repositories. For the implementation of such systems, companies can deploy important means for small gains. Indeed, management services often notice very limited use compared to what they actually expect. We present a use case of a five-step re-designing user-centered approach that combines a top-down content architecture, integration with other corporate information systems and adapted interfaces and interactions design to increase the use of these artifacts. Our implementation is done on the knowledge repository used by operators from Société du Canal de Provence (SCP), a French water management company. Their system was taken into production but very occasionally used. We describe the reasons for this limited use and we propose a design methodology adapted to the context. Promoting the effective use of the system, our approach has been experimented and evaluated with a panel of users working at SCP. Results of our semi-structured questionnaire reveal that more than half of the participant are satisfied and present a positive attitude towards the re-designed system.

INTRODUCTION

Knowledge management systems (KMS) are a line of information technologies that focus on creating, gathering, organizing, and disseminating an organization's "knowledge" for managerial and professional workers [START_REF] Alavi | Knowledge management systems: Issues, Challenges, and benefits[END_REF]. Their success depends on their use by knowledge workers. As a matter of fact, it is only by an actual and continuous use that we can observe artifact and information appropriation, technical support, exchanges among participants and change of practices. For various reasons [START_REF] Prax | Le Manuel du Knowledge Management -Une approche de 2ème génération[END_REF], it is common to find that KMS are slightly used and fail within organizations. The causes are of functional order (too complex technology, not meeting the workers' needs, inappropriate formalization of knowledge, etc.), of managerial order (inadequate or non-existent management support), or of social order (inappropriate choice of technology according to the cultural context or work practices). Several major research streams strive to propose solutions for successful knowledge management initiatives within companies. For example, the research area of Management and Information Sciences propose a large panel of models for knowledge management (KM) success. These models are largely normatively and prescriptively oriented but with a lack of technologydesign orientation as well as a micro-scale perspective of knowledge sharing practices [START_REF] Ackerman | Sharing Knowledge and Expertise: The CSCW View of Knowledge Management[END_REF]. In Computer Science, specifically in the stream of artificial intelligence and knowledge engineering, tools and methods have been proposed to formalize workers' know-how, complex sets of data and procedures, and make them reusable. However, this is done most of a time in a strictly technical manner that lead to very complex systems, far from users' expectations and practices. In [START_REF] Folcher | Appropriating artifacts as instruments: when design-for-use meets design-in-use[END_REF], the author shows that appropriating knowledge sharing artifacts must be supported by a design and a structuring of contents manipulated but we can observe that very little is done in terms of content organization, integration with other organizational information systems, integration of professional/social practices and interfaces design and interactions.

The objective of our work is, ultimately, to propose a design methodology that produces more usable and adapted systems; that is artifacts with content organization and interfaces matching knowledge workers' needs and professional practices and integrated with other organizational information systems what we believe will minimize the risks of knowledge sharing systems failure. This paper will present a use case in a French hydraulics company Société du Canal de Provence (SCP) where the knowledge has been formalized into a knowledge repository (KR). We focus our proposal on a method for re-designing an existing KR. This approach has been implemented in real context and pre-tested in order to provide elements of evaluation and feedback for further works.

The general research question in this article is how to re-design a KR to make it more usable? More specifically how designing for a better context adapted content organization and artifact integration into professional practices can promote a better acceptation by corporate users? To do this, we propose in this work to use a usercentered design method to understand the corporate organization and find the most appropriated way to combine conventional knowledge design engineering methods with Architecture Information, a method proposed in Human-Computer Interaction (HCI).

The organization of this paper is as follows. In paragraph 2 we present a review of literature. Paragraph 3 describes our re-designing approach. In paragraph 4, we show how it has been implemented within SCP. We then conduct an a priori assessment of our approach through a semi-structured questionnaire to users. Finally, we conclude and present our further research works.

II. REVIEW OF LITERATURE

A. Overview of Knowledge Engineering methods

KMS design in Knowledge engineering consists most of the time in formalizing of all procedures and know-how that structure the worker activity. In [START_REF] Ermine | Management et ingénierie des connaissances -Modèles et méthodes[END_REF] for example, the KOD method presented attempts to codify the knowledge of an expert from his free speech. The MEREX method [START_REF] Prax | Le Manuel du Knowledge Management -Une approche de 2ème génération[END_REF] developed at Renault, proposes to make returns on operating experience in the form of sheets. The MASK method [START_REF] Ermine | Management et ingénierie des connaissances -Modèles et méthodes[END_REF] consists in structuring the knowledge of experts into corpus of documents and information, the dissemination of this knowledge in the company and their integration into company processes is based on a set of models to address and describe the activity knowledge. The process leads to the publication of a structured synthesis of knowledge into a Knowledge Repository (KR). In their final state, these KR are multimedia centers of documentation on knowledge. They allow finding, understanding and using information easily.

Nevertheless, there are in these different methods, low consideration of the end user in the design process, and in the design of access modes and organization of information that may impede the actual exploitation of knowledge.

B. Design methods from Human-Computer Interactions 1) User-centered design methods

The HCI vast research area is dedicated to study, plan and design modalities of interaction between the user and the computer. The challenge is not only to produce useful, usable and acceptable interactions but also to improve user experience according to its value system, its context of activity and its objectives. User experience is defined as the set of perceptions of a user when interacting with a product [START_REF] Garrett | The elements of user experience -Centered Design for the Web and Beyond, 2nd Edition[END_REF]. User-centered design is a philosophy and an approach that takes into account needs, expectations and characteristics of end users at every stage of the design process [START_REF] Nielsen | Usability Engineering[END_REF]. Reference [START_REF] Mathis | Designed for Use -Create Usable Interfaces forApplications and the Web, Pragmatic[END_REF] proposes job shadowing and contextual interviews as approaches to better understand users' expectations. They consist for the designer to accompany and observe the worker during his activity for a certain period of time (job shadowing) or interview the worker about his core business activities. Focus groups are another mean to understand users' professional context and collect their needs. Reference [START_REF] Tremblay | Focus Groups for Artifact Refinement and Evaluation in Design Research[END_REF] shows that focus group can be a powerful tool for artifact refinement and proposes a detailed description of how to conduct them.

2) User-centered design methods in Knowledge Management

A couple of researchers applied other user-centered design tool for designing KMS [START_REF] Karner | Usage-Centered Interface Design for Knowledge Management Software[END_REF] [START_REF] Rinkus | Human-centered design of a distributed knowledge management system[END_REF]. They generally use a 3 steps methodology based on models: user model (identification of end-users and their relationships), tasks model (identification of general features of the system) and content model (content and organization of the user interface). These works have been successful in developing usable knowledge sharing artifacts, but have not yet been proven in the field of collaborative sharing and seeking of knowledge. Paper [START_REF] Cabitza | On a QUESt for a web-based tool promoting knowledge-sharing in medical communities[END_REF] presents the designing process of a web-tool for collaborative knowledge sharing within medical communities using focus groups. His paper focuses on the description of user needs and their transcription but give less information about the other stages of the design process.

Because our paper aims to describe a complete step-bystep approach integrating, among other features, content organization, we need, on the sidelines of the user needs gatherings, to define information structure, interactions and access modes to the artifact.

3) Information Architecture The stream of Information Architecture's aim is the presentation of information in the most appropriate way, according to end-users and their use context. A good information architecture has to be browsable (the user is not confused), coherent (semiotic adapted to the context of use), adaptable, simple (sufficient amount of presented information), able to make recommendations for information consultation [START_REF] Resmini | Pervasive Information Architecture -Designing Cross-Channel User Experiences[END_REF]. Paper [START_REF] Garrett | The elements of user experience -Centered Design for the Web and Beyond, 2nd Edition[END_REF] proposes a general conceptual framework to structure the design, particularly suited for Web applications. This concrete and detailed approach is based on five stages: strategy, scope, structure, skeleton and surface. These steps describe how to pass abstract elements of the product design to the most concrete elements. To structure the information, the author suggests using either a top-down approach (from the needs and objectives of the system to organize information) or a bottom-up approach (use categories and sub categories that spontaneously emerge from designer's analyses of information). In our case, the top-down approach seems to be the most adapted because the artifact organization and contents manipulated must match the organizational structure of the enterprise and the user's schemes of professional activity.

C. Summary

Conventional knowledge management methods are incomplete because they often lead to KMS that are far from user expectations. Participatory design techniques and structuring of information offer ways for designers to better understand expectations of end users. In the next paragraph, we describe the methodology used.

III. A FIVE STEPS APPROACH

Companies are already sensitized to the interest of formalizing business knowledge and build KMS. Most of them have already invested in these strategies but could not get a really usable system or accepted by those in the field. That is why we propose a re-design approach based on an existing knowledge capitalization. We use as a basis for our approach the 5 elements of the conceptual framework of [START_REF] Garrett | The elements of user experience -Centered Design for the Web and Beyond, 2nd Edition[END_REF] which is adapted to web platforms and pretty much easy to apply in real context. The phases are composed of different methods from the literature that was adapted to and implemented in our context. The steps may overlap if necessary. The process is iterative. An evaluation of the resulting prototype is done and may lead to a redefinition of previous choices.

A. Need analysis

In this step, we propose to analyze the initial situation of the KMS, the users' needs, his professional practices and the system objectives. This information gathering allows us to work on capitalized knowledge, the business context and the expectations of the users and of the company. The objective is to observe difficulties the users may encounter in front of the existing KMS and find appropriate solutions. For a knowledge book, it is interesting to observe users' reaction in front models of organization, media or forms of navigation. Concerning the other aspects, an immersion in the industrial environment is required; matching methods and contextual interviews are appropriate means to understand the environment of use and the needs [START_REF] Mathis | Designed for Use -Create Usable Interfaces forApplications and the Web, Pragmatic[END_REF]. In addition, mindmapping exercises [START_REF] Prax | Le Manuel du Knowledge Management -Une approche de 2ème génération[END_REF] used during focus groups can help to better understand the business vocabulary through games characterization. Mind mapping methods are ludic ways to classify the important business concepts into categories and super categories that will help us to understand workers' activity and the information structuring.

B. Definition of the new KMS

This step is used to define the features of the artifact that meet the needs. Nowadays, new technologies of information and knowledge (Social Networks, Blogs, Wikis, etc.) are becoming increasingly present in daily people habits. Hence, companies and workers are increasingly interested in easy sharing and exchanging of information via Enterprise Social Networks (ESN) [START_REF] Zammit | Social Networks for Knowledge Management[END_REF] and are turning out to these new forms of exchange and communication [START_REF] Dudezert | Vers le KM 2.0: Quel management des connaissances imaginer pour faire face aux défis futurs?[END_REF] . They look for ways to exploit them. Paper [START_REF] Zammit | Social Networks for Knowledge Management[END_REF] justifies the effectiveness of ESN in knowledge management by carrying out an analysis based on the knowledge creation model of Nonaka [START_REF] Nonaka | A dynamic Theory of Organizational Knowledge Creation[END_REF]. This is why we assume that discussing with users about refining the KR into an ESN may be a valuable choice. This assumption will be evaluated in future works to see if such a solution could be better than another collaborative way of sharing knowledge.

Hence, designers will discuss during focus group the usefulness and the format of common features within ESN which are members' directory, blogs, news, statistics, newsletter, photo album, dialogue groups, events, polls, discussion forums. User roles and social features are essential in ESN. The definition of roles will also be discussed with users. Most of the time, collaborative blogs within ESN propose a repartition of roles as follows: a redactor, a commentator, a validator and a general manager.

C. Design of the information structure

This step addresses the specification of interaction formats adapted to the features and information architecture of the system. This phase is closely related to the results of the analysis in the first step. It consists in designing patterns of interaction and structuring information models that are familiar to users. If the forms of interaction in the ESN are already pervasive in users' habits, we recommend keeping them. Structuring the content on the other hand, should be investigated. Among the two approaches proposed by [START_REF] Garrett | The elements of user experience -Centered Design for the Web and Beyond, 2nd Edition[END_REF] and presented above, we propose to implement the top-down approach. Content will be structured according to the corporate organization (departments, services, teams, projects, etc.) workers are familiar with. Mind mapping sessions conducted during focus groups will help identify key concepts as well as their different facet and forms of information; they will then be classified within the defined organization. Access modes to and from other information systems will also have to be studied and integrated into the knowledge sharing artifact so that users can keep a link with of other corporate artifacts they currently use.

D. Design of the skeleton

This step is used to design the main functional areas and how they are interconnected. The use of the personas methodology [START_REF] Boucher | Ergonomie Web -Pour des sites web efficaces[END_REF] may be helpful at this stage. Personas are virtual characters that correspond to the end users, they allow designers to really get into the skin of end users and make proposals that best fit them. They are important to specify the navigation schema connecting the different functionalities.

E. Visual Design

The general graphical appearance and textual fonts will be determined in this phase. We recommend setting the visual design in accordance with the Charter of communication and graphical design of the company to maintain familiarities with interfaces. Moreover, improvements in usability can be made if necessary. One possibility is to stage the main use cases, and discuss with the users on proposed models. We therefore recommend a prototyping approach which allows the improvement of the visual design.

The last two steps of our methodology, Design of the Skeleton and Visual Design, are highly sensory and will impact the user experience. The designer will have to be particularly careful when developing the platform in terms of ergonomics; He will also be able to develop motivators, all in accordance with user's expectations. This may require browse regularly through these steps.

IV. IMPLEMENTATION

Société du Canal de Provence is a company in the south of France, specialized in services related to the treatment and distribution of water for companies, farmers and communities. It employs a significant number of collaborators called Operators. They are responsible for maintenance of hydraulic infrastructures (canals, pumping stations, water purification stations, etc.). Their intervention territory is divided into ten geographic areas called Operating Centers (OC). The water infrastructures that composed the different OC are complex, various and require to be maintained in ways corresponding to the geographical area. The administrative contact and maintenance procedures are also specific to each infrastructure. Any error on one of them infers consequences for the entire downstream hydraulic network. To help them in their daily work, they often use a geographic information system (NetView) and an event management system (Supervision) for distant monitoring of incidents that may occur on hydraulic infrastructure. The operators have a knowledge intense work that is particularly difficult. SCP in 1996, invested in a KR named ALEX (Aide à L'EXploitation). ALEX makes its information accessible through return on experience sheets in HTML format located within a directory on a dedicated server in each OC. SCP however found that it was little used despite the fact that there is, among collaborators, a real awareness of the usefulness of such a system. A preliminary study allowed us to identify problems at the level of ergonomics and at the level of access processes and information update (experience sheets was very long to create since they have to write them first in a .doc format and then turn it into HTML format, only a few workers had the time and the technical capabilities to execute the process). This context is particularly representative of design issues that can reveal KMS. We applied our methodology to redesign their system. The redesign was carried out with a working group of twelve employees, representing in terms of position and skills of all the users. It took six months and six working meetings to deliver a first prototype to users.

A. Deployment of the methodology 1) Need analysis:

The study of the initial system showed that the information filled in on business procedures was generally of good quality, which is fine since we haven't had to rework on the formalization of the knowledge itself. Users' needs collected during focus group can be summarize as follows: workers wish to have an accessible platform from both the office and the outside (web platfom), that is easy to learn but effective (casual and responsive functionnalities), which reduces the time for entering information (content easily updatable), facilitates data search (a good search engine) and allows exchanges between employees (with a social area). Our proposition of implement an ESN therefore was well accepted since it met the needs.

2) Specification of the ESN: During focus groups, the following roles have been defined: the administrator, the validator, the contributor and the commentator. Employees, depending on the role they have in the system, are more or less involved in the animation of the platform and content validation. These roles helped develop access features for system security levels and promote empowerment of actors. The features discussed and chosen to be integrated in the ESN are the submission, moderation and publication of pre-formatted forms based on roles, possibility for users to submit comments on the experience sheets, pictures of new activity performed in each operating center, photo galleries, links from and to other corporate information systems (NetView, Supervision). The architecture of the new system is proposed on Fig. 1. The submission of experience sheet is made by the contributor under moderation of the validator or the administrator. It is done using forms with large areas of open writing because the activity is too complex to be defined by a structure; employees without any role distinction communicate by commentaries left on the sheets. These comments serve to convey the appreciation of the reader regarding the record, as its content is a good idea to generalize or if there is a need for additional information; research content is natural language or keywords; photos of the galleries will be indexed according to the operating structures and equipment in order to facilitate research.

3) Design of the information structure: We organized the ESN according to the organization of OC in SCP. Each OC is represented as 'Collection' containing several hydraulic infrastructures. Each infractructure contains one or several equipments on which several operations can be executed. These operations done by the collaborators on infrastructures are described in experience sheets, with descriptions of equipment, operating instructions, processes and alarms. This resulted in the definition of eleven types of sheets: presentation, equipment, set operating instructions, hydraulic diagram, spreadsheet, process, operation, alarm, contract / contact and detail. Each item is described by a presentation sheet that can be associated with one or more sheets of the other types. Each sheet is presented as a form formatted according to its type-specific metadata (type of equipment for equipment sheets or instruction name for operating instruction sheets), by cons, the content of fields is open written as shown on Fig. 2.

4) Skeleton Design: This phase was animated by discussions on the basis of a proposal of a skeleton made with a content management system (CMS) named Drupal. The use of a CMS allowed us to accelerate the development and modifications of the prototype according to the users' feedback. In the light of the opinions that have been collected, we made the proposal to reorganize the new system in different functional areas gathering the main features of ALEX that are information, submission, navigation and communication. The content is classified in accordance with the operating centers. Each OC has a dedicated subsite in which each page contains the 5 functional areas described previously. Experience sheets are classified and indexed in accordance with their type.

5) Visual Design: On the visual aspects of the site, we started with a proposal of "theming" and, according to different users' feedback, we adapted the different choices. In this phase, we had to get into the skin of each user type and depending on the use case, to make choices that facilitate the use of the tool. As examples, we can cite the search area that has been enlarged and positioned prominently at the top right corner, the default font for the fill of sheets that has been standardized to Arial, size 12, or the area for data entry that has been enlarged.

6) ALEX+: At the end of the previous works, we delivered a new version of ALEX named ALEX+. We present you the front page of an OC on Fig. 2. Features have been proposed in order to improve the user experience in terms of complexity and duration, during the activity of corporate knowledge. An assessment has been made and is detailed in the next section.

B. Evaluation

The objective of this assessment was to see if we had managed to transcribe the latent needs of SCP operators and developed an acceptable system. Indeed, quality of a system can be measured by its usefulness (does the system meet the user's needs?) and its usability (is the system functional according to the use context?) [START_REF] Février | Vers un modèle intégrateur 'Expérience-Acceptation' : Rôle des affects et de caractéristiques personnelles et contextuelles dans la détermination des intentions d'usage d'un environnement numérique de travail[END_REF].

1) Evaluation criteria: Models of technology acceptance divide the acceptance process into two stages. Before the first use, the user built beliefs based on his perceptions of the system. These beliefs leverage his attitude and use intention. The actual acceptance occurs while using the system and infers an appropriation or a rejection. Initial acceptance, first condition for technology success was widely studied in literature. As major models, we can cite TAM [START_REF] Davis | User acceptance of information technology : system characteristics, user perceptions and behavioral impacts[END_REF] or UTAUT [START_REF] Venkatesh | User acceptance of information technology : Toward a unified view[END_REF]. The first one considers perceived usefulness and perceived ease of use avec basic elements for attitudes and users' behaviors. The second one refines the criteria by considering users social characteristics. The ISSM [START_REF] Delone | The DeLone and McLean model of information systems success: a ten-year update[END_REF] opens up the criteria and considers quality of the system, of the information and of the service as determinants of positive intention and use. The ISO 9241-11 model considers usability (efficacy, efficiency and satisfaction) as a required condition of use [START_REF] Février | Vers un modèle intégrateur 'Expérience-Acceptation' : Rôle des affects et de caractéristiques personnelles et contextuelles dans la détermination des intentions d'usage d'un environnement numérique de travail[END_REF]. Efficacy and efficiency are first perceived and infer in the construction of satisfaction and positive attitude towards the technology. During the use of the technology, these opinions consolidate or evolve according to the perceived benefits; they condition long-term use with other determinants like Use Context. The whole acceptance process is represented on Fig. 4.

A successful deployment of ALEX+ in real context is related to a positive first acceptance that is why we focused on measuring these criteria first. Measurement of actual benefits and satisfaction being long to construct, we were not able to assess them in this study. Usage analysis depends in addition on many other organizational factors. The quality of our methodology was therefore assessed on the basis of an a-priori acceptance.

2) Data gathering: The evaluation was conducted with the members of the working group. This was about completing a semi-structured questionnaire organized into nine sections describing the nine factors listed in the previous model: System Quality (QS), Quality of information (QI), perceived Ease of use (FUP), Attitude toward the use of the application (ATT), Intention to use (IC), Service Quality (How is the system proposed by the SCP?), professional perceived Usefulness related to the use of the application (UPR), Personal perceived Usefulness related to the use of the application (UPE) and Satisfaction (SA). Each section contains a series of 4 to 6 questions [START_REF] Touré | Mise en place d'un dispositif de gestion des connaissances pour le soutien à l'activité industrielle[END_REF]. For each section, we left blank textboxes so that participants can express themselves on open questions. 3) Results: The analysis of the results reveal a majority of positive answers to questions about Quality of System, of Information and Satisfaction. About Perceived Ease of Use, more than half of the participants wrote in substance that "Alex+ should be simpler to use than its previous version but we could judge it actually with further use". Concerning the attitude towards system criteria, one of the participants pointed out that "he thinks he could use the system by now without major problems but this feeling could change with time". Generally, almost all the feedbacks are positive regarding user satisfaction. However, the comments left by the users about long-term use point out the limits of this assessment that just consider the a-priori acceptance. The risk that users may lose motivation to use the system on a long term usage is present. First acceptance does not guaranty continuous and sustainable use [START_REF] Touré | Mise en place d'un dispositif de gestion des connaissances pour le soutien à l'activité industrielle[END_REF].

V. CONCLUSION AND DISCUSSIONS

Given the limits of conventional knowledge management methods that focus specifically on knowledge formalization at the expense of the final interface, we proposed a concrete user-centered redesigning approach that turns knowledge repositories into enterprise social networks. Our approach combines methods from knowledge engineering and information architecture. We implemented our approach in SCP and as a result we obtained a prototype, ALEX +. ALEX + was evaluated by a panel of users. It shows that collaborators are generally satisfied with the proposals that were made in the final system and will tend more to use it. We can however identify a couple of limitations in our approach. Firstly, the limited number of participants in the workgroup allows us to only have the viewpoints of a small part of the actual user population; an assessment of a larger amount of people in SCP and also in other companies would help us have a better insight of the impact of our methodology on the KMS use in the company. Secondly, an ideal experimental approach would be to do a comparative evaluation of our methodology with others proposed by literature in the domain of design of corporate KMS. More generally, with our approach, we can just have an overview of the users' intentions but not of the effective use. The choice of ESN as a solution for more usable knowledge management artifacts also need to be investigated to evaluate if such a solution is generally better than another one. These points are planned for future work. Indeed, KMS are really effective when users appropriate it, consult the available information, discuss, comment and update contents. This is what we call sustainable knowledge management. To achieve this, we need to add other features in the system. We believe that metacognitive assistance features like indicators of awareness may be useful [START_REF] Marty | Observation of Collaborative Activities in a Game-Based Learning Platform[END_REF]. Indeed, by proposing activity indicators, we can promote a reflexive dynamic of learning by user selfregulation processes [START_REF] George | Usages réflexifs des traces dans les environnements informatiques pour l'apprentissage humain[END_REF]. For example, users by visualizing the impact of their contribution on other actors in the company may be more motivated to use the system. Conversely, by identifying the comments that were made on experience sheets related to their professional field, they may become aware of new procedures or changes in business practices and thus increase the credit given to the developed tool. As such, comments could be seen as a recommendation to consult. On the sidelines of this work, we plan to monitor the activity and assess these new features by analyzing traces of activity [START_REF] Karray | PETRA: Process Evolution using a TRAce-based system on a maintenance platform[END_REF]. These traces provide much more diagnostic of use by sector and functionality. What we are doing now is therefore to identify, still with an incremental approach, which indicators and interaction modalities may be most suitable. Phases 4 and 5 of our method are mainly concerned; the design that affects the sensory and user experiences.

Figure 1 .

 1 Figure 1. View of ALEX+ architecture. The plaform is organized according to the different operating centers. Integration with other information systems NetView and Supervision is done by the mean of links.

Figure 2 .

 2 Figure 2. View of two types of sheets. Form 1 is an equipment sheet and form 2 a worksheet. Organization of the content depends on the type: equipement sheets are arranged in predefined paragraphs while a worksheet contains an attached file with a free content textbox.

Figure 4 .

 4 Figure 4. Process for acceptance of technologies

Figure 3 .

 3 Figure 3. View of ALEX+ Trevaresse OC front page. Boxes 1 et 2 show different type of browsing: with tabs on the top or with a menu on the side. Box 3 present a slideshow of pictures posted in the OC library. In box 4, we placed the submission options. Box 5 shows the research form

TABLE I

 I

			.	RESULTS SUMMARY
	QS and QI	UP		ATT and IC
		QS QI UPR UPE	FUP	ATT	IC	SA
	PA(%) 96,3 83,3 64,4 66,7	88,9	85,2	71,1	86,1
	NA(%) 0	2,8 26,7 22,2	0	0	17,8	0
	A(%) 3,7 13,9 8,9	11,1	11,1	14,8	11,1	13,9

ACKNOWLEDGMENT

This research work is initiated by the Operation Service within the Technique and Water Direction of SCP. We would like to thank, Jean-Luc DELTOUR and Frederic ROMITTI, who are responsible for the ALEX project at SCP, and all the collaborators who participate to the project.


Bristeau Mauffretaourell

Mouvet Christophe Sebastien

Biodegradation of both chlordecone and degradation productsresulting from chemical treatment

Keywords: Chlordecone, biodegradation, highly hydrophobic compound

come

Biodegradation of both chlordecone and degradation products resulting from chemical treatment

Aourell Mauffret, Sébastien Bristeau, Christophe Mouvet

Chlordecone (CLD: C 10 Cl 10 O) is an organochloride insecticide used to control the banana weevil from 1972. Even its withdrawal in 1993, CLD is still present in drinking water and food crop products and impacts on human health have been documented since 2001. Insoils, concentrations up to 15 mg/kg have been reported which also stress its high persistency.The depollution process of In Situ Chemical Reduction (ISCR) tested in laboratory conditions by BRGM enabled to reduce by 60-70% the CLD concentration in soil. Ithas successfully gone through a pilot test in a banana field in martinqiue (French west Indies). This process breaks down the parent molecule in dechlorinated molecules (mainly mono, di and tri-hydro CLD), for which ones it could be hypothesized that bioaccessibility via the dechlorinated region of the molecule may be facilitatedas compared to the parent molecule. We have selected various inocula, based on their historical exposure to CLD, ability to degrade other highly chlorinated compounds (PCB…), or high bacterial activity. Their ability to degrade CLD and dechlorinated CLDwas tested in feed-batch systems spiked with CLD and tri-hydro-CLD, for which ones we have standards in sufficient quantity to run such tests; synthetises of purified dechlorinated CLD still being a challenge. We have a methodology based on liquid extraction and GCMS to quantify simultaneously the parent compound and several of its metabolites to ensure distinguishing loss by sorption from degradation in the feed batches with solid support on which one the highly hydrophobic compounds can sorb on. Very preliminary results after 7-month incubation did not point out a clear distinction between biodegradation kinetics for CLD and the tri-hydro CLD. We also observe a biodegradation product for CLD resulting from the opening of carbonated cageof the molecule (C 9 Cl 5 H 3 ), which ones biodegradation steps are still unclear. Biodegradation tests are undergoing.


Nadine Bouby
email: nadine.bouby@crc.jussieu.fr

Keywords: Bradykinin receptors, Type 1 diabetes, Type 2 diabetes, Skin wound healing, Kallikrein-kinin system

Impaired skin wound healing is a major medical problem in diabetic subjects. Kinins exert a number of vascular and other actions limiting organ damage in ischemia or diabetes but their role in skin injury is unknown. We investigated, through pharmacological manipulation of kinin B1 and B2 receptors (B1R/B2R), the role of kinins in wound healing in non-diabetic and diabetic mice.

Using two mouse models of diabetes (streptozotocin-induced and db/db mice) and nondiabetic mice, we assessed effect of kinin receptor activation or inhibition by subtype selective pharmacological agonists (B1R and B2R) and antagonist (B2R) on healing of experimental skin wound. We also studied effects of agonists and antagonist on keratinocytes and fibroblasts in vitro.

B1 and B2 receptor mRNAs level increased one to two fold in healthy and wounded diabetic skin compared to non-diabetic skin. Diabetes delayed wound healing. B1R agonist had no effect on wound healing. In contrast, B2R agonist impaired wound repair in both nondiabetic and diabetic mice, inducing skin disorganization and epidermis thickening. In vitro, B2R activation unbalanced fibroblast/keratinocyte proliferation and increased keratinocyte migration. These effects were abolished by co-administration of B2R antagonist. Interestingly, in the two mouse models of diabetes B2R antagonist administered alone normalized wound healing. This effect was associated with induction of MCP1/TNFα mRNAs.

Thus, stimulation of kinin B2 receptor impairs skin wound healing in mice. B2R activation occurs in the diabetic skin delays wound healing. B2R blockade improves skin wound healing in diabetic mice and is a potential therapeutic approach to diabetic ulcers.

Improvement of skin wound healing in diabetic mice by kinin B2 receptor blockade

Dorinne Desposito 1,2,3 , Catherine Chollet 1,2,3 , Christopher Taveau 1,2,3 , Vincent Descamps 4,5 , François Alhenc-Gelas 1,2,3 , Ronan Roussel 1,6,7 , Nadine Bouby 1,2,3 , Ludovic Waeckel 1,2,3

Introduction

Impaired skin wound healing is a source of morbidity, disability, loss of quality of life and mortality in subjects affected by diabetes. Foot ulcers affect at least once in their life about 15% of patients with diabetes. Amputation rate is high in affected patients [START_REF] Ramsey | Incidence, outcomes, and cost of foot ulcers in patients with diabetes[END_REF][START_REF] Bartus | Reducing the incidence of foot ulceration and amputation in diabetes[END_REF][START_REF] Boulton | The global burden of diabetic foot disease[END_REF]. The pathogenesis of chronic foot ulcers is multifactorial and still incompletely understood. Loss of insulin action, hyperglycaemia, ischemia secondary to micro-and macro-angiopathy, neuropathy and infection all contribute to delayed skin repair and favour tissue necrosis, increasing amputation risk [START_REF] Brem | Cellular and molecular basis of wound healing in diabetes[END_REF]. Improving skin wound healing in patients with diabetes is an important therapeutic issue.

Skin healing after wounding involves several different biological processes and comprises an acute response phase with haemostasis and inflammation, a granulation phase with cell migration, angiogenesis, production of extracellular matrix, wound contraction and re-epithelisation and a late scar maturation phase. Various cell types are involved in these processes: platelets, inflammatory cells, endothelial cells, fibroblasts and keratinocytes [START_REF] Falanga | Wound healing and its impairment in the diabetic foot[END_REF]. In diabetes, all phases of wound healing are altered. Cellular infiltration, migration and proliferation, and granulation tissue formation are delayed, together with impaired collagen organization and reduced angiogenesis [START_REF] Falanga | Wound healing and its impairment in the diabetic foot[END_REF]. Moreover, chronic hyperglycaemia alters fibroblasts and keratinocytes phenotypes. While impairment of the different components of the healing process has been documented in diabetes, therapeutic options for improving wound healing in diabetic subjects remain currently limited.

The kallikrein-kinin system (KKS) is implicated in physiological vasodilatation [START_REF] Couture | Kinin receptors in vascular biology and pathology[END_REF][START_REF] Waeckel | Pathophysiology of genetic deficiency in tissue kallikrein activity in mouse and man[END_REF], anticoagulation [START_REF] Furchgott | Endothelium-derived relaxing and contracting factors[END_REF], inflammation [START_REF] Couture | Kinin receptors in pain and inflammation[END_REF], and angiogenesis [START_REF] Desposito | Kinin receptor agonism restores hindlimb postischemic neovascularization capacity in diabetic mice[END_REF]. Previous studies have documented the role of this system in limiting organ damage in the setting of hemodynamic, hyperglycaemic or ischemic aggression [START_REF] Waeckel | Pathophysiology of genetic deficiency in tissue kallikrein activity in mouse and man[END_REF][START_REF] Kayashima | The kallikrein-kinin system and oxidative stress[END_REF]. We hypothesized that KKS could play a role in cutaneous responses to injury, especially in diabetes. Indeed, kinins have several cellular targets involved in skin repair including platelets, endothelial cells, keratinocytes and fibroblasts. Studies have documented the presence of the different components of the KKS in healthy, traumatized or diseased human skin [START_REF] Komatsu | Expression and localization of tissue kallikrein mRNAs in human epidermis and appendages[END_REF][START_REF] Vidal | Kinin B2 receptorcoupled signal transduction in human cultured keratinocytes[END_REF][START_REF] Schremmer-Danninger | Visualisation of tissue kallikrein, kininogen and kinin receptors in human skin following trauma and in dermal diseases[END_REF][START_REF] Poblete | Tissue kallikrein and kininogen in human sweat glands and psoriatic skin[END_REF][START_REF] Milia | Differential expression of tissue kallikrein in the skin of systemic sclerosis[END_REF]. Kinin receptors, bradykinin type-1 receptor (B1R) and bradykinin type-2 receptor (B2R), have been shown to participate in hyperproliferative process in chronic skin inflammation model [START_REF] Pietrovski | B1 and B2 kinin receptor participation in hyperproliferative and inflammatory skin processes in mice[END_REF]. Kinins increase skin microvascular blood flow in rabbits and humans [START_REF] Warren | Captopril increases skin microvascular blood flow secondary to bradykinin, nitric oxide, and prostaglandins[END_REF]. These studies suggest a role for KKS in skin physiology. However, involvement of KKS in skin repair in the setting of diabetes remains undocumented.

The aim of the present study was to investigate the role of kinins and their receptors in wound healing in diabetic mice, and to compare with non-diabetic animals. Effect of potent selective pharmacological B1R or B2R agonists and/or of B2R antagonist was studied in vivo in mice and in vitro in cultured fibroblasts and keratinocytes. The study shows that a) B2R stimulation has deleterious effects in the wounded skin, unbalancing fibroblast/keratinocyte proliferation and migration and delaying cicatrisation; b) B2R activation occurs in the diabetic skin and impairs wound healing; c) B2R blockade improves wound healing in diabetic mice.

Materials and methods

Animals, experimental protocols and treatments

Effects of B1R or B2R agonists or B2R antagonist on wound healing were studied, in both non-diabetic (NonDiab) and diabetic (Diab) mice. All mice were housed with a 12 h light/dark cycle and had free access to standard mice chow and water. All experimental procedures were approved by the local Ethics Committee for Animal Experiment Charles Darwin and performed in accordance with European legislation for the care and use of laboratory animals (L 358-86/609/EEC).

Murine model of type 1 diabetes

Diabetes was induced in 10 weeks-old mice (C57BL/6J strain, male, Charles River Laboratories, France) by 5 daily i.p. injections of streptozotocin (STZ) (Sigma-Aldrich, France) (50 mg/kg body weight in 0.05 mol/L -1 sodium citrate, pH 4.5) [START_REF] Desposito | Kinin receptor agonism restores hindlimb postischemic neovascularization capacity in diabetic mice[END_REF][START_REF] Johnson | Early loss of peptidergic intraepidermal nerve fibers in an STZ-induced mouse model of insensate diabetic neuropathy[END_REF]. Diabetes was confirmed by assessing fasting blood glucose level at 0, 7 and 30 days post STZ injection. After 5 weeks of confirmed diabetes (fasting blood glucose>300 mg/dl), excisional wounds were created on the back of the mice as described below. Control NonDiab animals were treated with vehicle only.

Murine model of type 2 diabetes

Diabetic db/db mice were obtained from Janvier Labs (Saint-Berthevin, France). Experiments were performed on male mice, 12 week-old. Only mice with 4 weeks of fasting blood glucose>300 mg/dl were used.

Murine model of skin wound healing Animals were anesthetized by isoflurane inhalation (1.5% in O 2 ). A dorsal 8 mm diameter full-thickness wound was made on dorsal depilated and cleaned (povidone-iodine solution) skin using a sterile biopsy punch (Kai medical, Japan) [START_REF] Ebrahimian | Plasminogen activator inhibitor-1 controls bone marrow-derived cells therapeutic effect through MMP9 signaling: role in physiological and pathological wound healing[END_REF][START_REF] Balaji | Adenoviralmediated gene transfer of insulin-like growth factor 1 enhances wound healing and induces angiogenesis[END_REF]. Buprenorphine (0.05 mg/kg; Buprécare, Axience, France) was administered as analgesic agent ten minutes prior and 24 hours after biopsy. Wounds were harvested by taking high-resolution photos every 2-3 days and the area was quantified relative to a millimetre reference using Image Analyzer Software (ImageJ, NIH) and expressed as percentage of wound area measured at day 0. At day 11 after injury, corresponding to wound closure of NonDiab mice, animals were sacrificed by pentobarbital overdose (120 mg/kg) and a skin sample including wound and surrounding tissues was collected. One half of the sample was immediately fixed in 10% formalin for histological analysis, the other part being kept at -80°C for molecular analyses.

Treatments administration Immediately after wounding, mice were implanted i.p. with osmotic minipumps (Alzet, model 1002, Charles River, France) delivering the selective B1R agonist (SarLys[Hyp3,Igl5,DPhe8]desArg9-bradykinin, B1R-ag) or the selective B2R agonist ([Hyp(3),Thi(5),(N)Chg [START_REF] Waeckel | Pathophysiology of genetic deficiency in tissue kallikrein activity in mouse and man[END_REF],Thi [START_REF] Furchgott | Endothelium-derived relaxing and contracting factors[END_REF]]-bradykinin, B2R-ag) [START_REF] Cote | Novel kinin B1 receptor agonists with improved pharmacological profiles[END_REF][START_REF] Belanger | Structureactivity relationships of novel peptide agonists of the human bradykinin B2 receptor[END_REF], both at non-hypotensive dose of 720 nmol/kg.day -1 (B1R-ag: 796 µg/kg.day -1 ; B2R-ag: 813 µg/kg.day -1 ) [START_REF] Desposito | Kinin receptor agonism restores hindlimb postischemic neovascularization capacity in diabetic mice[END_REF], associated or not with the specific B2R antagonist (HOE140, Icatibant, B2R-ant) [START_REF] Wirth | Hoe 140 a new potent and long acting bradykinin-antagonist: in vivo studies[END_REF] at a dose of 380 nmol/kg.day -1 (500 µg/kg.day -1 ) [START_REF] Tschope | Kinins are involved in the antiproteinuric effect of angiotensin-converting enzyme inhibition in experimental diabetic nephropathy[END_REF]. B2R-ant was also administered alone at the same dosage. Control mice received only vehicle (Phosphate Buffered Saline, PBS). Treatments were pursued until sacrifice (n=7-30/group).

Histological analysis Skin biopsies fixed in 10% formalin and embedded in paraffin were cut into 7-µm section and stained with Masson's trichrome according to the manufacturer's instructions (Sigma-Aldrich, France). Photomicrographs were obtained using digital camera attached to light microscope (Leica DM 4000B and LAS v3.8 software). Epidermal thickness was measured in wounded skin and healthy surroundings area by using Image Analyzer Software (ImageJ, NIH) (3 measurements for each compartment/section, 4 sections/animal, n=7-13/group). Structure and organisation of the granulation tissue and/or the scar were observed.

Wound closure and histological studies were performed blindly with regard to treatment.

Measurement of kinin receptors and cytokines mRNA level and circulating MCP-1 and SAP Real time PCR was performed to assess B1R and B2R mRNA levels. Total RNA was isolated from the skin, collected at day 11, using TRIzol (Invitrogen, France) and reverse transcribed with superscript II reverse transcriptase as previously described [START_REF] Bodin | Kallikrein protects against microalbuminuria in experimental type I diabetes[END_REF]. The cDNAs were amplified and quantified using TaqMan Universal Master Mix and Assays-on-Demand Gene Expression Probes for gene of B1R and B2R (Applied Biosystems, France) in an ABI PRISM-7000 Sequence Detection System (Applied Biosystems, France). Each sample was tested in triplicate. Data were normalized to 18S rRNA. Changes in the target gene were calculated by the 2 -∆∆CT comparative method for each sample [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF]. Same protocol was used to determine levels of MCP-1, TNF-alpha, TGF-beta and IL-6, four major pro-inflammatory cytokines, in wounded skin, using probes from the same supplier. Plasma MCP-1 concentration was also determined in blood samples at 11 days using a mouse MCP-1 enzyme-linked immunosorbent assay kit (Invitrogen, France) according to the manufacturer's instructions. Serum Amyloid P (SAP), an inflammatory marker equivalent to C-reactive protein in man, was quantified at 11 days using a mouse Elisa test kit (Life Diagnosctic, UK).

Keratinocytes and fibroblasts cell culture

Keratinocytes (human keratinocyte cell line HaCaT; ATCC, Virginia) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Glucose concentration 4500 mg/l, Gibco, LifeTechnologies, UK) supplemented with 10% Foetal Bovine Serum (FBS, Gibco, LifeTechnologies, UK) and antibiotics (50 U/mL Penicillin, 50 U/mL Streptomycin, Gibco, LifeTechnologies, UK). Keratinocytes were used between passages 68 and 75.

Fibroblasts (NIH-3T3 mouse fibroblasts; Sigma-Aldrich, Missouri) were cultured in DMEM supplemented with 5% New Born Calf Serum (NBCS, Gibco, LifeTechnologies, UK), 5% FBS and antibiotics according to manufacturer's guidelines. Fibroblasts were used between passages 162 and 171. Keratinocytes and fibroblasts were kept at 37°C in a 5% CO 2 environment. Culture media were changed every two-day. When the cells reached subconfluence, they were harvested using 0.05% trypsin-EDTA (Gibco, LifeTechnologies, UK), and fresh culture medium was added for obtaining single cell suspensions used for further study.

Cell proliferation assay Cell proliferation was measured using Quick Cell Proliferation Assay kit (Abcam, Cambridge, MA) according to the manufacturer's instructions [START_REF] Guo | Lipopolysaccharide causes an increase in intestinal tight junction permeability in vitro and in vivo by inducing enterocyte membrane expression and localization of TLR-4 and CD14[END_REF]. This assay is based on cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. Briefly, keratinocytes or fibroblasts were seeded on 96-well plates (1x10 4 cells/well) and incubated with two different concentrations of B1R-ag or B2Rag (1x10 -6 mol.L -1 and 1x10 -7 mol.L -1 ) and/or with B2R-ant (1x10 -5 mol.L -1 ) in DMEM supplemented with 2% FBS. Cell proliferation was measured after 24, 48, 72 and 96 hours of incubation. Optical density was measured at 440 nm with a reference wavelength of 650 nm using a precision microplate reader (iMark Microplate Absorbance Reader, BIORAD).

Results were expressed as percentage of control (DMEM supplemented with 2% FBS). Each experiment was performed in duplicate per condition and repeated three times.

Wound closure assay Spreading and migration capabilities of cells were assessed using a scratch assay [START_REF] Fronza | Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts[END_REF]. Keratinocytes or fibroblasts were seeded on 6-well plates (3x10 5 cells/well). After reaching confluence, cultures were scratch-wounded with a 0.2 ml sterile pipette tip at the centre of each well and cell debris were removed by PBS washing. For keratinocytes, plates were pre-coated with collagen I (BD Biosciences, California). B1R-ag or B2R-ag (1x10 -6 mol.L -1 and 1x10 -7 mol.L -1 ) and/or B2R-ant (1x10 -5 mol.L -1 ) were added in DMEM supplemented with 2% FBS. Mitomycin C (Sigma-Aldrich, Missouri) (10µg/mL) was also added in medium to inhibit cellular proliferation. After 15 hours, photographs of the scratch width were taken every 3 hours until closure and scratch closure was quantified by monitoring the front of migration. Results were expressed in percentage of scratch width measured at hour 0. Each experiment was realized in duplicate per condition and repeated four times.

Western blotting Cells were deprived in FBS during 24 hours and then treated 0, 2, 5, 15 and 40 minutes with B2R-ag (1x10 -6 mol.L -1 ) and/or B2R-ant (1x10 -5 mol.L -1 ). Proteins were extracted from treated-cells using standard protein extraction protocols and were dosed using BCA protein assay (Pierce Protein Biology Products, USA). Fifteen-µg proteins were separated on a Mini-PROTEAN TGX gel (Biorad, USA) and membranes were blotted with phospho-and total p44/42 MAPK (Erk-1/2) (Thr202/Tyr204) rabbit antibody (dilution 1/1000; Cell signalling technologies, Danvers, USA). They were exposed to ECL plus western blotting reagents (Biorad, USA) and reactive bands were detected in ImageQuant LAS 4000 (GE Healthcare, France) and quantified using Multi Gauge software 2.0 (FujiFilm, Japan). Each blot was then stripped and re-probed with anti-β-actin antibodies for data normalization. Results were confirmed by repeating the experiment 3-5 times.

Statistical analysis

Data are presented as mean±SEM. Statistical analysis was performed using one-way or two-way ANOVA for comparing effect of diabetes and/or treatments in mice. One-way ANOVA was used for comparing effect of treatment on cell migration or proliferation and effect of diabetes on receptor mRNA level. ANOVA was followed by ad-hoc multiple comparison tests. Statistical significance was accepted at p<0.05.

Results

Treatments did not affect metabolic parameters. Body weight and plasma glucose level of the different groups of mice at the beginning and end of the experiment are presented in Table1. Treatment with B1R-ag, B2R-ag and/or B2R-ant did not affect these parameters (Table 1).

B1R and B2R mRNA levels are increased in diabetic skin. Analysis of bradykinin receptor levels in healthy skin and wounded area at day 11 demonstrated that B1R mRNA level were increased roughly 3 times in STZ-Diab mice compared to NonDiab mice (p<0.01). Similarly, B2R mRNA level were also increased 2 times compared to Non-Diab mice (p<0.01) (Fig. 1).

B1R agonist treatment had no effect on wound closure. B1R-ag treatment had no effect on wound closure in NonDiab mice (Fig. 2A) and in STZ-Diab mice (Fig. 2B). Moreover, histological analysis showed that B1R activation did not alter skin organisation and had no effect on epidermal thickness, neither in intact surrounding skin nor in wounded skin (data not shown).

B2R activation delays wound closure in mice. In NonDiab mice, B2R-ag treatment significantly delayed wound healing from the 1 st day of treatment onward (Fig. 2A). Cotreatment with the B2R-ant totally abrogated the wound healing delaying effect of B2R-ag. Kinetic profile of wound closure in mice co-treated with B2R-ag and B2R-ant was indistinguishable from PBS-treated mice (Fig. 2A). In STZ-Diab mice, wound closure was significantly delayed when compared with NonDiab mice (Fig. 2B). On day 11, wounds were healed in NonDiab mice whereas they remained open in STZ-Diab mice (12.7 ± 1.2 %, p<0.05). In STZ-Diab mice, B2R-ag treatment resulted in a further significant delay in wound healing from the 1 st day of treatment onward (Fig. 2B). On day 11, wound area was 2.3 times larger in B2R-ag-treated STZ-Diab mice compared to PBS-treated STZ-Diab mice (p<0.05).

B2R blockade improves skin wound healing in two models of diabetic mice. B2R-ant alone had no significant effect on wound healing in NonDiab mice (Fig. 2A) but significantly improved skin wound healing in diabetic mice. In STZ-Diab mice, wound area was reduced on the 1 st day of B2R-ant treatment and was indistinguishable from NonDiab mice during the 11 days follow-up period (Fig. 2B). The beneficial effect of B2R-ant on wound healing was also observed in another diabetic model, db/db-Diab mice. In these mice, B2R-ant significantly reduced wound area during the 11 th days of treatment (Fig. 2C). On day 11, wound area was 3.2 times smaller in B2R-ant-treated db/db-Diab mice compared to PBStreated db/db-Diab mice (p<0.05).

B2R activation induces epidermal thickening and skin layer disorganization in wounded skin. B2R activation induced no alteration in intact part of skin in NonDiab and STZ-Diab mice (data not shown). But, in the wound of NonDiab and Diab mice, B2R-ag treatment induced skin layer disorganisation, with a defect in skin and epidermal maturation, hypervascularisation and hypercellularisation of granulation tissue (Fig. 3A). In B2R-agtreated NonDiab mice, epidermal thickness of wound area was increased compared to PBStreated mice (111.1±8.7 µm vs 69.9±5.7 µm; p<0.05). This effect was abrogated by cotreatment with B2R-ant (56.3±8.2 µm; p<0.05) (Fig. 3B). In Diab mice, treatment with B2Rag had no further effect on epidermal thickness (Fig. 3C).

B2R antagonist treatment restores wounded skin histology in diabetic mice. After B2R-ant treatment alone, the epidermis of Diab-mice was histologically indistinguishable from NonDiab mice (Fig. 3A). B2R-ant decreased by 45% the epidermal thickness of diabetic wounds (112.4±21.9 µm vs 206.3±21.6 µm; p<0.05) (Fig. 3C).

B2R-ant treatment increases mRNA level of MCP-1 and TNF-alpha. IL-6 and TGF-beta mRNA levels were not influenced by B2R-ag or B2R-ant treatment (data not shown). MCP-1 and TNF-alpha mRNA levels were not influenced by B2R-ag treatment but B2R-ant treatment significantly increased both mRNAs in the wounded skin compared to PBS-treated Diab-mice (Fig. 4). However, treatment had no effect on plasma concentration of MCP-1 (Fig. 4). Concentration of SAP was below detection level in all groups (data not shown).

B2R activation unbalances keratinocyte and fibroblast proliferation in vitro.

Treatment with B1R-ag or B2R-ag significantly increased keratinocyte proliferation, assayed using Quick Cell Proliferation Assay kit (+36% to 46% at 48h, p<0.05) (Fig. 5A). Moreover, fibroblast proliferation was significantly decreased by the B2R-ag (-33% at 48h, p<0.05) but was not altered by the B1R-ag (Fig. 5B). These B2R-ag effects on cell proliferation were abolished by co-treatment with B2R-ant (Fig. 5). B2R-ant alone had no effect on cell proliferation.

B2R activation stimulates keratinocyte but not fibroblast migration. Figure 6 shows that keratinocyte migration, assessed using scratch assay, was significantly stimulated with B2Rag during the 30 hours scratch closure process and independently of the dose used (wound closure +32% compared to medium alone after 24h, p<0.05). This effect was abolished in presence of the B2R-ant (Fig. 6B). B2R-ant alone or B1R-ag had no effect on cell migration (Fig. 6B). B2R-ag treatment had no effect on fibroblast migration (data not shown).

B2R activation induces ERK phosphorylation in keratinocytes. B2R-ag induced phosphorylation of ERK-1/2 in keratinocytes. Peak phosphorylation occurred 5 to 40 min after stimulation (p<0.05) and thereafter, the phosphorylation level gradually decreased (60-120 min). This effect was blocked by B2R-ant co-treatment (Fig. 7).

Discussion

The main findings of the present study are that kinin B2R activation delays healing in nondiabetic and diabetic mouse skin wound models. Treatment with a selective B2R antagonist accelerates skin wound repair in diabetic mice. The healing effect of B2R antagonist was consistent in two different murine models of diabetes, close to either type 1 or type 2 human diabetes. The deleterious effects of B2R activity in the wounded skin may involve unbalancing of keratinocyte and fibroblast proliferation during the re-epithelialisation and scar remodelling phases after injury.

Previous reports indicated that B1R and B2R are present in healthy human skin and in skin of patients with cutaneous wound or proliferative skin diseases [START_REF] Komatsu | Expression and localization of tissue kallikrein mRNAs in human epidermis and appendages[END_REF][START_REF] Vidal | Kinin B2 receptorcoupled signal transduction in human cultured keratinocytes[END_REF][START_REF] Schremmer-Danninger | Visualisation of tissue kallikrein, kininogen and kinin receptors in human skin following trauma and in dermal diseases[END_REF]. We show that both receptors are present in murine skin and are upregulated by diabetes mellitus.

Kinins can exert their biological actions through these two types of G protein-coupled membrane receptors. While the B2R is constitutively present in most tissues and mediates the main effect of kinins, the B1R is considered as being mainly inducible and its functions are less well understood. Interestingly, we recently showed that B1R activation protects the heart against ischemia damage [START_REF] Potier | Selective kinin receptor agonists as cardioprotective agents in myocardial ischemia and diabetes[END_REF] and has pro-angiogenic effects in a diabetic mouse model of hindlimb ischemia [START_REF] Desposito | Kinin receptor agonism restores hindlimb postischemic neovascularization capacity in diabetic mice[END_REF]. However in the skin, B1R activation had no effect on wound healing in mice, diabetic or non-diabetic. This receptor does not seem to be importantly involved in control of skin trophicity in diabetes, despite its strong induction by chronic hyperglycaemia.

By contrast, B2R activation disorganized the architecture of the wound, increased epidermis thickness and significantly delayed wound healing in mice. The deleterious effect of B2R activation on wound repair was observed in both non-diabetic and diabetic mice and was additive to the effect of diabetes. Co-treatment with a specific B2R antagonist abrogated the effects of B2R agonist administration indicating that these effects were truly due to B2R activation.

Wound healing involves tightly regulated humoral and cellular processes. Alteration of these timely-controlled processes by chronic hyperglycaemia expands tissue damage and delays tissue repair [START_REF] Li | Pathophysiology of acute wound healing[END_REF]. After a cutaneous injury, the healing process can be divided into four successive, overlapping phases: coagulation, inflammation, cell migration and proliferation, and remodelling. Kinins, through B2R activation, could likely influence several of these phases.

The first steps of wound healing are haemostasis and inflammation [START_REF] Li | Pathophysiology of acute wound healing[END_REF]. Kinins, through vascular endothelium activation, have anti-clotting and profibrinolytic effects [START_REF] Brown | Bradykinin stimulates tissue plasminogen activator release from human forearm vasculature through B(2) receptordependent, NO synthase-independent, and cyclooxygenase-independent pathway[END_REF][START_REF] Murphey | Bradykinin and its metabolite bradykinin 1-5 inhibit thrombin-induced platelet aggregation in humans[END_REF], increase blood flow and trigger leucocyte migration [START_REF] Blaes | Targeting the 'Janus face' of the B2-bradykinin receptor[END_REF]. In our study, pharmacological manipulation of B2R activity influenced wound repair already on the first day of follow-up after surgery. This observation suggests that kinins influence the initial phase of wound repair. The absence of effect of B2R agonist treatment on inflammatory markers in plasma or on cytokine gene expression in scar suggests that inflammation does not play an important role in B2R effect in wounded skin. On the other hand, B2R antagonist induced gene expression of MCP-1 and TNF-alpha, two cytokines previously shown to be involved in wound repair that may thus mediate, at least in part, the beneficial effect of B2R inhibition [START_REF] Wood | Proinflammatory chemokine CCL2 (MCP-1) promotes healing in diabetic wounds by restoring the macrophage response[END_REF][START_REF] Rapala | The effect of tumor necrosis factor-alpha on wound healing. An experimental study[END_REF]. The mechanism of MCP-1 and TNF-alpha induction during B2R inhibition remains however undocumented.

Kinins can also influence late phases of healing. Our histological results show that B2R agonist induced skin disorganisation and increased epidermal thickness and hypercellular granulation tissue. Moreover, previous studies suggested that bradykinin enhances keratinocyte motility [START_REF] Coutant | Bradykinin induces actin reorganization and enhances cell motility in HaCaT keratinocytes[END_REF] and differentiation even if effect on cell proliferation is unclear [START_REF] Vidal | Kinin B2 receptorcoupled signal transduction in human cultured keratinocytes[END_REF][START_REF] Poblete | Tissue kallikrein and kininogen in human sweat glands and psoriatic skin[END_REF]. All these findings suggest that kinins influence proliferative and remodelling phases of healing. In this context, we studied in vitro effect of B2R activation on proliferation and migration of the two major cell types involved in scar formation: keratinocytes and fibroblasts. Our results show that B2R activation inhibited fibroblast proliferation but increased keratinocyte proliferation and migration in a cell layer injury model. Despite limitation in extrapolation of data from in vitro to in vivo settings due to use of established cell lines and different species, these effects of B2R agonist are consistent with the wound repair delaying effect observed in mice. Indeed, accumulation of fibroblasts, formation of granulation tissue, reconstitution of the dermis and reformation of an intact epidermis via keratinocyte migration and proliferation are important features of the wound healing process [START_REF] Li | Pathophysiology of acute wound healing[END_REF]. Skin layer disorganization, involving stratum corneum, epidermis and dermis, observed in mice treated with B2R agonist, especially in diabetic mice, may thus reflect unbalance between hypo-proliferation of fibroblasts and hyper-proliferation of keratinocytes, impairing healing. Stimulation of keratinocyte proliferation and migration in vitro by B2R activation is in line with the thickening of epidermis observed in B2R agonist treated mice.

B2R activation effects on keratinocytes were associated with ERK-1/2 phosphorylation. This result is in agreement with previous observations showing that keratinocyte migration, proliferation and wound re-epithelialization involve ERK activation [START_REF] Gao | A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: activation of proteinase-activated receptor 1 and epidermal growth factor receptor[END_REF][START_REF] Li | Signals that initiate, augment, and provide directionality for human keratinocyte motility[END_REF][START_REF] Pastore | The epidermal growth factor receptor system in skin repair and inflammation[END_REF] and confirms the importance of this pathway in keratinocyte functionality.

The deleterious effect of B2R stimulation on wound healing was additive to the effect of diabetes. Abnormal keratinocyte and fibroblast migration, proliferation and differentiation, and decreased vascularisation contribute to delayed wound healing in diabetic patients [START_REF] Brem | Cellular and molecular basis of wound healing in diabetes[END_REF][START_REF] Albiero | Defective recruitment, survival and proliferation of bone marrow-derived progenitor cells at sites of delayed diabetic wound healing in mice[END_REF]. In these patients, keratinocytes in epidermis of chronic ulcers are highly proliferative [START_REF] Usui | Keratinocyte migration, proliferation, and differentiation in chronic ulcers from patients with diabetes and normal wounds[END_REF]. B2R agonist treatment probably worsened keratinocyte hyper-proliferation due to diabetes and stimulated migration, resulting in abnormal scar remodelling.

Interestingly, whereas B2R antagonist alone had no effect on the wounded skin in nondiabetic mice, it significantly accelerated wound repair in diabetic mice. Indeed, B2R blockade restored the normal proliferation and maturation pattern of the wounded epidermis in a murine model of type 1 diabetes (STZ). The beneficial effect on wound healing of B2R antagonist was extended to a second diabetic mouse model, closer to type 2 human diabetes (db/db mice). These observations indicate that endogenous kinins exert deleterious effects opposing wound repair in diabetic skin. The lack of effect of B2R antagonist in non-diabetic mice may be explained by low kinins bioavailability and/or reduced B2R synthesis in the skin in normoglycaemic condition. By contrast, in diabetes, B2R synthesis is upregulated in the skin and kinins production may also be enhanced.

The study highlights the organ specificity and duality of action of B2R. B2R activation simultaneously aggravates tissue damage in the wounded skin and reduces ischemic or hyperglycaemic tissue damage in heart and kidney [START_REF] Kayashima | The kallikrein-kinin system and oxidative stress[END_REF][START_REF] Bodin | Kallikrein protects against microalbuminuria in experimental type I diabetes[END_REF][START_REF] Griol-Charhbili | Role of tissue kallikrein in the cardioprotective effects of ischemic and pharmacological preconditioning in myocardial ischemia[END_REF]. Kinins are mainly inactivated in the circulation by the angiotensin I-converting enzyme (ACE/kininase II). ACE inhibitors increase kinin biodisponibility and are largely used clinically in a number of cardiovascular and renal indications, including in diabetes. No side effect related to impairment of skin wound healing or aggravation of diabetic ulcers has however been reported in clinical trials with ACE inhibitors. ACE inhibitors have not been studied experimentally in wound healing but angiotensin II type 1 receptor (AT1R) blockers and AT1R gene inactivated mice have been shown to display delayed skin wound healing in both non-diabetic and diabetic conditions [START_REF] Yahata | A novel function of angiotensin II in skin wound healing. Induction of fibroblast and keratinocyte migration by angiotensin II via heparin-binding epidermal growth factor (EGF)-like growth factor-mediated EGF receptor transactivation[END_REF]. As AT1R inhibition triggers angiotensin II type 2 receptor (AT2R) stimulation and subsequent kinin formation and B2R activation [START_REF] Siragy | Renin-angiotensin system modulates renal bradykinin production[END_REF][START_REF] Messadi-Laribi | Tissue kallikrein is involved in the cardioprotective effect of AT1-receptor blockade in acute myocardial ischemia[END_REF], kinins might be involved in the deleterious effect of AT1R blockade observed experimentally in the skin, although this remains speculative. Like for ACE inhibitors no side effect related to skin healing has been observed in clinical trials with AT1R blockers. In our study, the absence of effect of B2R antagonist in non-diabetic mice may be explained by low kinins bioavailability and/or reduced B2R synthesis in the skin in normoglycaemic condition. By contrast, in diabetes B2R synthesis is upregulated in the skin and kinin production may also be enhanced. Kinins are produced by tissue kallikrein and/or activated plasma prekallikrein. Tissue kallikrein is present in the skin [START_REF] Komatsu | Expression and localization of tissue kallikrein mRNAs in human epidermis and appendages[END_REF][START_REF] Schremmer-Danninger | Visualisation of tissue kallikrein, kininogen and kinin receptors in human skin following trauma and in dermal diseases[END_REF][START_REF] Poblete | Tissue kallikrein and kininogen in human sweat glands and psoriatic skin[END_REF]. However, in tissue kallikrein deficient mice [START_REF] Waeckel | Pathophysiology of genetic deficiency in tissue kallikrein activity in mouse and man[END_REF][START_REF] Meneton | Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice[END_REF], either rendered diabetic or not, we did not observe any alteration in wound repair, suggesting that tissue kallikrein is not importantly involved in kinin production in the wounded skin (unpublished results). On the other hand, plasma prekallikrein increases in diabetes [START_REF] Feener | Role of plasma kallikrein in diabetes and metabolism[END_REF] and this enzyme might be responsible for kinins production in the wounded skin, although this remains speculative.

In conclusion, B2R but not B1R activation exerts deleterious effects in the wounded mouse skin resulting in delayed healing, especially in diabetic animals. B1R and B2R are upregulated by diabetes in skin. Treatment with a B2R antagonist accelerates wound repair in two different mouse models of diabetes. Improving skin healing is still a therapeutic issue in diabetic patients, especially for treatment of foot ulcers. These results indicate that investigation of kinin B2 antagonism in human diabetic wound healing is warranted, and that kinin B2R antagonism could become a new treatment for cutaneous injury. It is worth noting that icatibant, a first generation pseudopeptide kinin B2R antagonist synthesized decades ago [START_REF] Wirth | Hoe 140 a new potent and long acting bradykinin-antagonist: in vivo studies[END_REF], has recently been approved for clinical use in an unrelated indication, angioedema [START_REF] Cicardi | Icatibant, a new bradykinin-receptor antagonist, in hereditary angioedema[END_REF].

Clinical perspectives

-Impairment of skin wound repair is a major complication of diabetes, favouring development of foot ulcers and contributing to their poor prognosis. Improving skin healing is a therapeutic issue in diabetic patients.

-Our study revealed that treatment with a kinin B2-receptor antagonist normalizes wound repair in two mouse models of diabetes, close to either type 1 or type 2 human diabetes.

-B2-receptor antagonism, already approved clinically in an unrelated indication, may become new treatment for cutaneous injury in diabetic patients.

Fig. 1 :Fig. 2 :

 12 Fig.1: B1R and B2R mRNA levels are increased in diabetic skin. Kinin receptor mRNA level in non-diabetic and diabetic mouse skin (wounded or not), measured by RT-qPCR. Data were normalized to 18S rRNA. Data are mean±SEM, n=7/group. **:p<0.01 Diab vs NonDiab

Fig. 3 :

 3 Fig.3: B2R but not B1R activation increases epidermal thickness and worsens skin disorganization in wounded skin in NonDiab and STZ-Diab mice. (A) Representative photographs of Massons's trichrome-stained sections of wounds at day 11 (2x; 10x; 25xmagnification) documenting change in the skin organization. Note thickness of epidermis and vascularisation and cellularisation of dermis. Bar = 1mm. Blue arrows show hypercellularisation, black arrows hypervascularisation, dashed blue arrow epidermal thickening and dashed black arrows skin layer disorganisation. (B) Quantification of epidermal thickness of the scar in NonDiab mice. (C) Quantification of epidermal thickness of the scar in STZ-Diab mice. Data are mean±SEM, n=7/group.

Fig. 4 :

 4 Fig.4: B2R-ant treatment increases mRNA level of MCP-1 and TNF-alpha. (A) MCP-1 and TNF-alpha mRNA level in scar of STZ-Diab mice treated with the B2R-ag or B2R-ant, measured by RT-qPCR. Data were normalized to 18S rRNA. (B) MCP-1 protein level in plasma of these mice. Data are mean±SEM, n=5-6/group.

Fig. 5 :

 5 Fig.5: B2R activation unbalances keratinocytes/fibroblasts proliferation. Cell proliferation after 48h of treatment with B1R-ag, B2R-ag and/or B2R-ant (A: keratinocytes; B: fibroblasts) at indicated doses. Data are mean±SEM, n=6 wells/group.

Fig. 6 :

 6 Fig.6: B2R but not B1R activation stimulates keratinocyte migration. (A) Representative images of keratinocytes migration (10×magnification) at 0, 15 and 30h post scratch. Scratch area is delimited by black box. (B) Scratch closure after 24h of treatment. Data are mean±SEM and expressed as percentage of scratch width measured at 0h. Each data point represents n=8 wells.

Fig. 7 : 1 )

 71 Fig.7: B2R activation induces ERK phosphorylation in keratinocytes. Phosphorylation of ERK-1/2 on keratinocyte lysates assessed by Western blotting after 0, 2, 5, 15 and 40 minutes of tretament with B2R-ag (1x10 -6 mol.L -1 ) and/or B2R-ant (1x10 -5 mol.L - 1 ). Results are representative of 3-5 independent experiments. *:p<0.05 vs t=0min.

TABLE 1 :

 1 Weight and blood glucose level of different experimental groups at time of wounding (beginning of the experiment) and 11 days after wounding (end of the experiment).

			Beginning of experiment	End of experiment
		n	Weight	Blood glucose	Weight	Blood glucose
			(g)	(mg/dL)	(g)	(mg/dL)
	NonDiab	30	26.2 ± 0.5	178 ± 6	25.7 ± 0.4	156 ± 12
	STZ-Diab
	PBS-treated	22	23.3 ± 0.2	471 ± 17 **	22.8 ± 0.3	464 ± 13 **
	B1R-ag-treated	21	23.5 ± 0.2	500 ± 17 **	23.9 ± 0.2	452 ± 21 **
	B2R-ag-treated	20	22.5 ± 0.2	456 ± 26 **	23.3 ± 0.3	438 ± 32 **
	B2R-ant-treated	7	23.0 ± 0.3	460 ± 34 **	24.0 ± 0.5	453 ± 26 **
	db/db mice
	PBS-treated	10	43.2 ± 0.9	386 ± 25 **	44.5 ± 1.2	379 ± 27 **
	B2R-ant-treated	10	43.5 ± 0.4	385 ± 34 **	45.8 ± 0.8	394 ± 29 **

** p<0.001: glucose level in all Diab groups vs NonDiab. NS among Diab groups.

Acknowledgments

The authors thank M. Moreau (Cordeliers Research Center, Paris) and the crew of the Functional Exploration Center (Cordeliers Research Center, Paris) for technical assistance and support in mice studies. We are very grateful to Pr P. Bruneval (anatomy and pathology department, European Hospital Georges Pompidou, Paris France) for his expert help in histological analysis. We also thank J.P. Girolami for his expertise.

Funding

This work was supported by Société Francophone du Diabète, Institut National de la Santé Et de la Recherche Médicale, Paris-Descartes University, Pierre and Marie Curie University.

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Summary statements

Kinin B2-receptor activation impairs skin wound healing in mice, in part through imbalance of keratinocyte/fibroblast proliferation. Such activation occurs in diabetes and contributes to delaying wound healing. B2-receptor blockade restores normal wound healing pattern in mouse models of diabetes.