Shahram Nourizadeh

C Deroussent
email: claude.deroussent@medetic.com

Y Q Song

J P Thomesse
email: thomesse@loria.frshahram.nourizadeh

Medical and Home automation Sensor Networks for Senior Citizens Telehomecare

Keywords: Telemedicine, Tele-Healthcare, Elderly Healthcare, Smart Home, Distributed health record, Actimetry, Fuzzy logic, Home automation I

This paper presents a home Tele-healthcare system for the senior citizens. The system uses home automation sensors to detect activity level of the persons. It is equipped also with other technologies to monitor them and detect any abnormal state in their health situation, like as bed and chair sensors, a mini PC connected to a TV, medical sensors, a wireless camera network. The system detects health abnormalities at an early stage through the frequent monitoring of physiological data. The system is designed for the elderly patients who wish to spend their old age in their own home, because of its potentials to increase independence and quality of life for seniors who prefer to live in their own homes and to realize cost savings for the health care system. By using this system MEDeTIC, a non-profit organization proposes a new concept of building smart homes named "Vill'Âge" at France.

INTRODUCTION

Nowadays a big rate of dependent elderly requiring healthcare services must leave their home and move to special healthcare centers and this is often not acceptable for them. Advanced communication networks and Internet technologies have a key role in the development of smart homes. Healthcare integrated smart homes can be used to allow the seniors and their family members to benefit from a safe home environment with activities and services accompaniment for their daily steps. These technologies have a key role in the development of smart homes.

To reach these benefits, most countries are now facing an urgent requirement to provide appropriate retired home environments solutions for senior citizens and allow them to rest in their own homes. For same reasons, elderly telehomecare, videoconferencing and tele-consultation systems have been attempted by many researchers too.

Many telemedicine and e-health systems are being developed and many innovations and ICT-based emerging solutions are close to be operational, nevertheless the expected take-up did not occur yet, since proposed systems and their targeted medical benefits are certainly too segmented and disconnected the ones from the others. In addition, most of existing products and services in this field rely on proprietary technologies, which is a substantial obstacle to the development of complementary services or applications, and thus of the market.

Consequently, population like elderly, disabled people or chronically ill people, who are often suffering from coexisting troubles, are not encouraged to dare benefiting from ICT and innovations, as available solutions are not complete, not compatible the ones with the others, not scalable, neither end-to-end nor comprehensive, and remain then unaffordable for someone who would try to subscribe to each relevant service covering a more or less significant part of its medical and social needs, without ensuring a global management of the daily problems.

The fragmentation of experimentation, projects and products did not yet give birth to any efficient and affordable holistic solution that could fulfill the various targeted beneficiaries and users' expectations. The development costs and R&D expenses required for a proper adaptation to each community of users and beneficiaries' needs make investment very high, as time-to-market delays are uncertain and no business model has been successfully assessed yet in this domain. The accompanying measures, at institutional, ethical, legal, financial and organizational levels remain insufficient, which prevent the development and the sustainability of such assistance and at-home monitoring services.

Finally, the population is neither confident nor familiar enough with these innovation-based solutions, especially in such a medical or health-related purpose, although their development requires that they become a natural component of the medical and social environment and infrastructure. For instant, in this section we will name just some of the existence systems.

The remainder of this paper is structured as follows: section II discusses state of the art. Our system, its uses and functionalities are described in section III, while section IV describes challenges and some discussion notes. Our studies and future works are resented in section V and section VI provides concluding remarks.

II. STATE OF THE ART

A telehealth application is a complex system that requires the integration of various sub-systems and applications. As we said, nowadays, many telehealth and e-Home applications are available; however, it is difficult to find the system that matches exactly the end-user requirements. The current developments and innovations are limited in terms of commercial and operational perspectives and suffer from certain essential phenomena.

In [START_REF] Newman | A High Performance Web-based Teleconsultation System for Island Telemedicine[END_REF] the authors presented a teleconsultation system. This system is based on web technologies and the user accesses the system over the Internet or an intranet. The system therefore is designed to do a teleconsultation, and it is not integrated with the other healthcare modules or an automatic analysis of patient's health situation. TOPCARE [3] is a telehealth system for mobile care and homecare of high risk patients, elderly and care needing people. The system is an open telecare platform solutions for telemedicine services. The overall objective of TOPCARE is to develop technical devices and telecommunication structures and to lay the organizational groundwork for bringing co-operative health care services into the home of patients. System comprises a telematic homecare platform (THP) backbone, the development of telematic home stations (THS) and health professional stations (HPS) and a communication server that will manage the network administration, the health professional registration at the THS, the device communication, and the Internet access. Therefore, in TOPCARE, interaction is limited to only the clients having TeleHealth Client (THC) module.

The Interactive and cooperative tele-monitoring of the dialyzing at home (DIATELIC) is a system of telemonitoring of dialyzing at home by the technology of DPCA (Dialyze Peritoneal Continue Ambulant) [4]. It was developed by LORIA in cooperation with the doctors of the ALTIR (Lorraine Association of Treatment of Renal Insufficiency) and a doctor advice of the Health insurance. Research works drove to the Diatelic system. This system is only designed especially for the dialyze patients.

The AFIRM team of the laboratory TIMC-IMAG in Grenoble developed a project called System of Information and Communication of the Inteligent Home for Health (SIC -HITCH) [START_REF] Vincent Rialle | Modélisation d'un support de communication pour des personnes âgées à domicile Abir Ghorayeb1[END_REF]. Objective is to monitoring the patient in his home with sensors installed in his home, by triggering off alarms in the appropriate urgent centers, in case of feelings of faintness, in falls or in abnormal situations. This system is an experimentation and simulation platform.

III. PROPOSED SYSTEM

The requirements presented in the previous sections and remaining obstacles or brakes make the design of monitoring and assistance tools quite complex, as each pathology or disability generates its own set of requirements and constraints. Such context increases the need for the development of innovative global ICT-based solution allowing to implement personalized and person-centric care process and tools, which can evolve with the users' medical and social and physical evolution, and which can compile the various collection and analysis of data to ensure a reliable and sound diagnosis, medication and evaluation of the medical and social state of the person. To reach these advantages, we developed a patient oriented system.

A. System architecture

By a user requirement analysis, we propose a Multi-tiered architecture. Fig. 1 shows the system's general architecture. The elements of this architecture are: B. Software architecture Fig. 3 shows the software architecture of the system. As we can see, we used a modular multilayer architecture in this system. The architecture has 4 layers: a) Command receiver layer: This layer receives the commands from remote controller and transfers them to user interface layer. This system can work with different types of commands, as like as sound, a normal IR remote controller and also PDA. We developed a remote controller application for PDA that uses Wife to communicate with PC. This layer receives also the data from different medical and home automation sensors.

b) User interface layer:

This layer presents a simplified user friendly and dynamic graphical user interface. This layer is a modular and has 5 different modules:

Video conferencing, Home automation, medical, services and multimedia (TV, Video and Music). Fig. 4 shows the medical interface of the application.

c) Application layer:

This layer has also 5 main modules: Video conferencing that consists of Flash player, media player for multimedia, service, Home automation, Medical, data analyzer, data integration and local database. The data received from Home automation and medical sensors will be integrated in Data integration and then Data analyzer will analyze them to detect and anomalies.

d) Data base layer:

In each mini PC we have a local data base which save all the information received from different sensors. This data base is synchronized with the main data base in the web server. The pre-process in the mini PC will be search to detect most urgent problems in health state of the person. And in the central server application will process the data to do activity monitoring and to detect problems like diabetes risks and...

e) Communication layer:

This layer facilitates the communication between PC and servers via Internet and also between PC and Home automation devices.

C. Distributed patient record and web services technology:

As shown in Fig. 3, the patient record has a distributed architecture, and we have a local database in each client computer. We used a distributed patient record because in distributed database architecture the data is not stored entirely at a single physical location. Instead, it is spread across a network of computers that are geographically dispersed and connected via communications links. We have therefore a large database capacity and incremental growth, and like all distributed databases, we have a reliable, available and flexible database. The most important advantage of a distributed database for our system is that a distributed database allows faster local queries and can reduce network traffic. In this system, in each client computer we have a pre-treatment of patient's data to detect the eventual emergency problems. This distributed database architecture helps to early detection of emergency problems, that means by checking the local database, if the data from healthcare devices are outside the ranges defined in their care plan, or if the data have not been received when expected, the system will trigger an alarm that may result in an automated connection with medical call centre.

This system uses web services to interact between the client and the server and it has intention to address the need to standardize the transmission of physiological data, by using the platform independence of web services and the structural independence of XML in the development of a web service for data transmission, processing and storage of physiological data for the service of tele-monitoring. By using web services, the system requires minimal additional technological elements and minimal technical requirements support.

D. Home automation system and Activity monitoring

The system gives not only to the patients a full control of Home automation devices, by using system's simplified interface but also generates the alerts by using home automation sensors, which allow an indoor localization of the beneficiary and can, thus, propose a fall detection service generating alerts. For instance, the system allows doctors or healthcare providers to measure the physical activities of elderly. The system uses a more passive method. It senses a person's presence in a room by home automation sensors installed throughout the living areas, such as motion and presence detectors, light, magnetic and temperature sensors.

Activity patterns are then analyzed in the central computer for unusual behavior, which use an intelligent method by using Fuzzy Logic. On the other hand, by using the data from home automation sensor and medical devices, the application stored in the web server, will analyze the medical record by using intelligent methods like fuzzy logic or other more complex cognitive systems, to find the potential health problem. By detecting a problem, the system will trigger an alarm that may result in an automated data transmission to a Doctor.

E. Video conferencing and Tele-consulting

Telecare is related to offer health care services over Internet or a wide area network, and in this domain, videoconferencing is one of the important solutions, allowing real-time interaction between patients and the Doctors. The offered system is integrated with a simplified and userfriendly videoconference system which is adapted to elderly needs. This sub system is used to have a video meeting by the families and also to request an online consulting. The teleconsulting steps are:

1. Patient connects to videoconferencing system 2. System sends a message to Doctor 3. System loads patient's medical record to Doctors computer 4. Doctor joins the conference 5. Consulting 6. Doctor fills the consulting form and saves it to medical record 7. End of consulting 8. System generates a billing and sends it to patient 9. Patient can view the billing in his interface and can pay it online

IV. CHALLENGES

In this type of systems, several challenges need to be addressed. The technological challenges regard the generic features of the dynamic database, of the wireless sensor networks, of the different supporting platforms, of the video call centre, and web services, to allow a flexible and smooth interaction among those items and to anticipate further additional functionalities, interacting devices, and so on. In this respect, the methodology of specifications, development and integration of the technological features must be realized with a major concern for some important concepts, like: Interoperability, Scalability, Evolution and customization according to the users' medical and social profiles and needs.

In addition, as the healthcare industry is turning to information technology to help solve its business issues, specially provide to quality patient care services, it is important to develop QoS (Quality of Service) specification in distributed health information systems [START_REF] Ray; Weerakkody | Quality of service management in health care organizations: a casestudy‖ Computer-Based Medical Systems[END_REF].

Unlike traditional systems, many non-end-to-end mission-critical applications envisioned for healthcare systems, which are complex systems, because in these systems, we can find different subsystems, such as, wireless sensor networks, LANs, software platforms, home automation systems, mobile systems, Internet… that require different QoS requirements on the system, and these requirements pose unprecedented challenges in the area of QoS support in healthcare systems. Any failure or lack of performance on the system which could not be tackled in a reasonable delay may have some damageable consequences on the solutions' acceptance and development potential: the confidence is a basic and elementary factor of acceptance or reject, such incident could also generate a psychological defiance towards ICT's in general and towards such innovative assistance and monitoring services.

V. EVALUATION

For the technical evaluation of the system, a flat is entirely equipped to act as demonstrator and as laboratory. We did some studies also to evaluate the system between the senior citizens, as like as [START_REF] Boulanger | Preliminary Based Service Evaluation for Elderly People and Healthcare Professionals in Residential Home Care Units‖ Digital Society[END_REF]. Our first study shows that all the healthcare professionals have a positive attitude toward dedicated means for ICT practical application (50% -totally agree‖, 33.3% -strongly agree‖ and 16.6% -lightly agree‖). Majority of interviewed elderly residents, in this study, declare not knowing what would be their family attitude toward ICT use (45.83%); 37.5% think that their family would be favorable to ICT use and 16.67% think that the family would be unfavorable to ICT use, but this study with the projected family attitudes of elderly people shows that the limitations experienced in everyday life do not lead to a favorable or unfavorable category of ICT user [START_REF] Boulanger | Preliminary Based Service Evaluation for Elderly People and Healthcare Professionals in Residential Home Care Units‖ Digital Society[END_REF].

Due to these results, definition and revision of planned applications (scenarios improvement), behavioral intention and acceptance of designed implemented solutions and finally actual usage and acceptance evaluation are important parameters in system development and design.

For this reasons design and integration effort for the technical and R&D activities of our project focus on the design of services and interfaces ensuring a maximum level of natural understanding on the way to use that is to say with an enhanced effort on the prerequisite and experimentation feedback's of the users who are suffering from chronic conditions and not familiar-and sometimes reluctant-with ICT and innovation-based devices and systems. Ergonomic interface, accessibility, scalability and evolutionary solution according to the evolution of the users' needs are the important aspect of development.

VI. CURRENT WORKS

This section will present come of our current works and projects to complete the system. The first part will present our project to integrate ZigBee environmental sensors with home automation sensors. This is a common project with Schneider Electrics Company -France section. (See: http://www.schneider-electric.fr/). Second project presented in this section, is a Fall Detection project by many French companies.

A. Integration ZigBee with Home automation protocols

As we said in III.A and the Fig. 3 shows, the system is integrated with vital sign monitoring devices and sensors. The system has possibility of integrating the system with the wireless devices and sensors with different communication methods such as Bluetooth, ZigBee, USB and the other technologies. The medical data are store in the database aromatically; without any manual action. We aim in this project to integrate ZigBee protocol with home automation protocols, such as KNX, IHC. Among the many wireless technologies, ZigBee is one of the most promising ones for home automation; a wireless home networking system can be configured using ZigBee alone [START_REF] Woo | Seung Ho Hong -KNX -ZigBee gateway for home automation‖[END_REF].

B. Fall detection with wireless camera networks

Nowadays, the best choice for elderly fall detection is to use a bracelet with a push button around the neck. Unfortunately, if the person forgets to carry the system, it is possible that the help does not arrive early. The idea of this project is to resolve this problem by using a noninvasive and automatic system to accomplish a system of surveillance at home for the seniors living alone. By using a camera installed in every room and home automation sensors, we will be able to follow the person and detect potentially dangerous situations.

In addition, by detecting such event, it will be possible to alert a medical call centre, and if needed, to urgent services. The cameras installed in the home send the images to a mini PC which is equipped with an application will use group of home automation sensors and cameras and an intelligent algorithm to detect a fall. Functionnal architecture of fall detection system Fig. 5 shows the fonctionnal architecture of this fall detection system. As transmission is made via wireless or wired network which is also shared by other applications like home automations, we use a feedbacking system (Flux control) in order to manage the load of the network. In case of a big load in the network, images will be transmitted with a bigger delay and thus the transmission rate will change dynamically. In image transmission, we also use a filtering algorithm (m,k)-filter [START_REF] Koubâa | Graceful Degradation of Loss-Tolerant QoS using (m,k)-Firm Constraints in Guaranteed Rate Networks[END_REF] proposed in TRIO team of Loria. The application analyzes the images to detect if the person is in a critical situation or not using the information of home automation sensors, the system already knows the exact position of person and can therefore determine if a help is necessary.

Fig. 1 .

 1 Fig. 1.System's general architecture

Fig. 2

 2 Fig.2shows the hardware architecture. It presents the different hardware that must be installed in each home; it also shows the different platforms of the system.

Fig. 5 .

 5 Fig. 5.Functionnal architecture of fall detection system

VII. CONCLUSION

In this paper, we have presented a distributed system to support remote medical consultations and elderly management and homecare across global wide area networks and heterogeneous platforms. By using this system, MEDeTIC (www.medetic.com) , a non-profit organization, offers a new concept of building smart homes by using telemedicine and home automation, named in French -Maisons Vill'Âge®‖. The first housing schemes are in building with implements of the system's components.


Philippe Nird

Danielle Derocq

Marie Maynadier

Monique Chambon

Ilaria Basile

Magali Gary-Bobo

Marcel Garcia

Heat shock cognate 70 protein secretion as a new growth arrest signal for cancer cells

Keywords: Animals, Blotting, Western, Breast Neoplasms, metabolism, pathology, physiopathology, Cathepsin D, genetics, metabolism, Cell Count, Cell Line, Cell Line, Tumor, Cell Proliferation, Chromatography, High Pressure Liquid, Culture Media, Serum-Free, pharmacology, Electrophoresis, Polyacrylamide Gel, Female, HSC70 Heat-Shock Proteins, genetics, metabolism, secretion, Humans, Microscopy, Electron, Scanning, Neoplasms, metabolism, pathology, physiopathology, RNA Interference, Recombinant Proteins, pharmacology, Signal Transduction, drug effects, physiology cancer, growth inhibition, Hsc70, secretion, extracellular, cathepsin D

Previous studies indicated that density-arrested cancer cells released an unidentified growth inhibitor whose secretion was prevented by overexpression of the lysosomal protease cathepsin D. Here, this growth inhibitor was purified by affinity chromatography and identified as the heat shock cognate 70 protein (Hsc70) based on its peptide microsequencing and specific antibody recognition.

Among intracellular proteins, including other heat shock proteins, only constitutive

Hsc70 was secreted in response to the high cell density. Moreover, Hsc70 secretion from cancer cells was generated by serum deprivation, whereas its cellular concentration did not change. Prevention of Hsc70 secretion by cathepsin D overexpression was associated with the formation of multilayer cell cultures, thus indicating a loss of contact inhibition. In addition, we demonstrated that supplementing the culture medium with purified Hsc70 inhibited cell proliferation in the nanomolar range. Conversely, removal of this extracellular Hsc70 from the medium by either retention on ADP-agarose or competition at the Hsc70 binding site restored cell proliferation. Hsc70 appears active in human breast cancer cells and hypersecreted by direct cathepsin D inhibition. These results suggest a new role of this secreted Hsc70 chaperone in cell proliferation that might account for the higher tumor growth of cancer cells overexpressing cathepsin D.

Introduction

Stress-related proteins are known as heat-shock proteins (Hsp) and are divided into families according to their average molecular weight. The 70-kDa Hsp family is composed of heat-inducible proteins (Hsp70), which are expressed under cellular stress conditions, and heat shock cognate proteins (Hsc70), which are constitutively expressed without any stress stimulation (

). [START_REF] Dworniczak | Structure and expression of a human gene coding for a 71 kd heat shock cognate protein[END_REF] These ATP-dependent proteins are involved in important cellular functions. Hsc70 is most efficient when associated with heat shock factor cochaperones (

), and could also bind to nascent polypeptides ( ) and to polypeptides containing [START_REF] Morimoto | of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators[END_REF][START_REF] Beckmann | Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly[END_REF] abnormal amino acids, dissociate clathrin from clathrin coats ( ), and promote lysosomal degradation of cytosol Rothman & Schmid, 1986 proteins ( ). Hsc70 contains signal peptides which allow its nucleolar or its cytoplasmic localization [START_REF] Chiang | A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins[END_REF] Tsukahara et al.

). Hsc70 is expressed in non-malignant mammary cells as well as breast cancer cells (Soulier et al. 1993;;2004[START_REF] Vargas-Roig | Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy[END_REF] ) and the overexpression of Hsp/hsc70 in chemoresistant cancer cells ( , ; ; [START_REF] Kao | Application of differential display, with in situ hybridization verification, to microscopic samples of breast cancer tissue[END_REF][START_REF] Ciocca | Response of human breast cancer cells to heat shock and chemotherapeutic drugs[END_REF]2003[START_REF] Lazaris | Proliferating cell nuclear antigen and heat shock protein 70 immunolocalization in invasive ductal breast cancer not otherwise specified[END_REF] has prompted studies about possible clinical markers of these proteins ( ; [START_REF] Vargas-Roig | Heat shock proteins and cell proliferation in human breast cancer biopsy samples[END_REF][START_REF] Morimoto | of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators[END_REF]Ellegde et al. 1994 Nylandsted et ; ; ;

)). While the heat shock protein 70 family is al., 2002[START_REF] Thanner | Heat-shock protein 70 as a prognostic marker in node-negative breast cancer[END_REF][START_REF] Ciocca | Heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications[END_REF][START_REF] Torronteguy | Inducible heat shock protein 70 expression as a potential predictive marker of metastasis in breast tumors[END_REF] primarily implicated in several intracellular functions, the presence of extracellular Hsp70 was evidenced on tumor cell surfaces (Multhoff ; ), suggesting an immunological role through, for instance, the stimulation of effector cells of the innate et al., 1995[START_REF] Melendez | Heat shock protein 70 and glycoprotein 96 are differentially expressed on the surface of malignant and nonmalignant breast cells[END_REF] immune system ( ; ). Stressed rat cells ( ) and human cells like [START_REF] Udono | Heat shock protein 70-associated peptides elicit specific cancer immunity[END_REF]Srivastava, 2005 Hightower et ) have shown that contact inhibition of cell growth, i.e. the [START_REF] Abercrombie | Observations on the social behaviour of cells in tissue culture. II. Monolayering of fibroblasts[END_REF][START_REF] Eagle | Growth regulatory effects of cellular interaction[END_REF] mechanism by which cell-cell contacts mediate antiproliferative signals, plays a fundamental role in regulating homeostasis in normal tissues. In contrast, the loss of contact inhibition is one trait of cancer cells, which can also grow to higher saturation densities and form multilayered foci (

). This cellular behavior contributes to disruption of natural tissue barriers, and favors tumor [START_REF] Abercrombie | Contact inhibition and malignancy[END_REF] spreading.

We previously demonstrated that the growth of cancer cells to higher density was promoted when these cells overexpressed cathepsin D (cath D) ( ; ). Furthermore, we have shown that these effects are due to modulation of extracellular [START_REF] Garcia | Overexpression of transfected cathepsin D in transformed cells increases their malignant phenotype and metastatic potency[END_REF]Glondu et al. 2002 growth inhibitor(s) produced by high density cells, but the nature of the inhibitor(s) is still unknown (

). In the present [START_REF] Liaudet | Transfected cathepsin D stimulates high density cancer cell growth by inactivating secreted growth inhibitors[END_REF] report, we demonstrate that Hsc70 is the main protein secreted by density-arrested 3Y1-Ad12 cancer cells and is associated with the antiproliferative activity of conditioned medium. The extracellular release of Hsc70 is not the result of cell lysis but appears positively regulated by either serum deprivation or cell density, or negatively regulated by cath D overexpression. In addition, human breast cancer cells also secreted Hsc70, and this secretion is enhanced by adding the cath D inhibitor pepstatin A or by cath D silencing. Finally, the growth inhibition obtained at nano molar concentrations of purified secreted Hsc70 in 3Y1-Ad12 cancer cells or recombinant Hsc70 in human cell lines strongly supports the hypothesis that decreased Hsc70 secretion may be involved in the loss of contact inhibition and high density growth of cancer cells.

Results

Purification and identification of an extracellular protein associated with growth inhibition

Conditioned culture media from 3Y1-Ad12 cells arrested at confluency were used to purify factor(s) associated with growth inhibition.

Protein components were separated by HPLC and the elution profile was recorded with an UV detector (

). Each fraction was Figure 1A evaluated for antiproliferative activity in rat mammary adenocarcinoma RBA cells (Cohen et al., 1974) test which was previously shown to be a better antiproliferative test than that with 3Y1-Ad12 cells (

). The antiproliferative activity appeared to be [START_REF] Liaudet | Transfected cathepsin D stimulates high density cancer cell growth by inactivating secreted growth inhibitors[END_REF] concentrated as a single peak around fraction 48, which corresponded to an apparent molecular weight of 240 kDa in a gel filtration column (arrows in

). This activity was also found as a single peak in another HPLC condition performed on a Mono Q Figure 1A anion-exchange column with an elution carried out with a salt concentration of 0.2 M NaCl (data not shown).

In order to identify the proteins associated with this antiproliferative activity, each active fraction was analyzed by sodium duodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE), and at least 15 proteins were detected by silver staining (

). Some of Figure 1B these were serum contaminants since they were not detectable in the medium after 24 h of metabolic cell labeling with S-methionine.

35

This serum contamination was illustrated by the presence of the most intense band at 66 kDa, which corresponded to bovine serum albumin. Two major neo-synthesized proteins with an apparent molecular weight of 30 and 73 kDa appeared to be correlated with the antiproliferative activity. More attention was given to the 73 kDa protein since its secretion was previously found to be decreased by cancer cells overexpressing cath D ( ). To further characterize the 73 kDa proteins, this band was extracted from the gel [START_REF] Liaudet | Transfected cathepsin D stimulates high density cancer cell growth by inactivating secreted growth inhibitors[END_REF] after conditioned medium separation, and N-terminal sequencing was then performed on three trypsin digestion peptides using an Automatic Peptide Sequencer system (

). The sequences were compared to protein databases and their analysis clearly revealed Figure 1C that these peptides corresponded to the 70 kDa heat shock protein sequence ( ; ). Moreover, O [START_REF] Malley | Constitutively expressed rat mRNA encoding a 70-kilodalton heat-shock-like protein[END_REF] ' [START_REF] Sorger | Cloning and expression of a gene encoding hsc73, the major hsp70-like protein in unstressed rat cells[END_REF] peptide 570-583 was able to differentiate the constitutive Hsc70 among other members of the highly conserved heat shock protein family owing to the presence of residues I570, N575, and I577. In addition, the sequence of this fragment also indicated that the secreted Hsc70 was the isoform 1 protein since isoform 2 did not contain residues encompassing the 570 583 sequence. Western blots with specific Hsp70 and Hsc70 antibodies confirmed that Hsc70 was the secreted protein (

). Figure 1D Hsc70 is secreted by high density cells but not shed by lysed cells

In order to determine whether Hsc70 was the result of secretion by cancer cells and not a cell lysis product, we compared the patterns of both the secreted and intracellular proteins of confluent cells on SDS-PAGE after metabolic labeling with S-methionine (

).

35

Figure 2A

The relative protein patterns strongly differed between the extracellular and intracellular compartments. As examples, the 256 kDa protein was abundantly secreted but barely present within the cells, whereas the 135 kDa protein was poorly secreted. The amount of secreted 70 kDa band corresponded to 19 of the total amount of secreted proteins, whereas the intracellular amount of this band represented only 2 % % of the total labeled intracellular protein content in these cancer cells. In contrast, in cath D overexpressing cells, the secreted 70 kDa band is reduced to 0.1 of the total secreted proteins. From a macroscopic standpoint, we quantified the percentage of cell death in a trypan % blue exclusion assay. Over the same assay period, only 1.6 of the total number of control and cath D overexpressing cells died. This % suggests that release of the protein content by passive mechanisms is limited and identical in both situations ( ). In addition, we Figure 2B used the property of the heat shock protein family that binds ATP to subject the conditioned medium to ATP-agarose chromatography. The bound proteins were eluted prior to SDS-PAGE analysis and silver staining revelation (

). The elution profile showed that the Figure 2C detected Hsc70 protein was the only excreted Hsc protein present in the medium. Fractions 4 to 6 contained the majority of the secreted Hsc70. Overall, these results indicated that the extracellular Hsc70 was the result of protein secretion in culture medium rather than being part of the general release of intracellular proteins following cell lysis.

Contact inhibition and relationship with hsc70

We next examined the morphological changes between high density 3Y1-Ad12 controls and cath D overexpressing cells. Scanning electron microscopic studies revealed morphological alterations in confluent cells when comparing controls and clones stably transfected with cath D cDNA. Control cells exhibited a flattened morphology of monolayer cells at confluency with rare disseminated foci (Figure ). In contrast, transfected cells continued to proliferate with overlapping cell layers and achieved a higher density multilayer ( 3A Figure 3B ) containing aggregated colonies. This loss of contact inhibition was even more pronounced when cultures were prolonged for over 10 days, as revealed by cellular DNA quantitation to monitor cell growth ( ). Control cells reached the plateau phase at around 18 Figure 3C μ g DNA/well on day 6 and onwards, whereas transfected cells did not reach the plateau phase even on day 10 of the experimentation. The maximal density for the transfected cells reached 38 g DNA/well. We secretion from control cells and from clones expressing cath D ( μ). This ability of the transfected cells to proliferate and form multilayered colonies was linked to a marked decrease in Hsc70 Figure 3D secretion (

). Cath D expressing cells secreted 60 less Hsc70 at confluency than the control cells. We have also tried to Figure 3D % decrease Hsc70 secretion by RNA interference silencing in order to further demonstrate its implication in contact inhibition. However, we did not obtain a such genetic proof since cells became detached during the experiment and died (data not shown). This result was in accordance with data of Florin et al. (Florin et al. 2004) indicating that Hsc70 knockout cells were nonviable.

Regulation of Hsc70 secretion by cell density, serum deprivation and lysosomotrophic bases

The effects of different growth regulating parameters were explored on Hsc70 expression. Hsc70 levels were evaluated in both the intracellular and extracellular compartments of two cath D transfected clones and their corresponding controls after 24 h standard culture with increasing amounts of serum in the culture medium (

). No significant differences were observed with respect to the amount Figure 4 of intracellular Hsc70 in the controls and the cath D clones (

). However, an increase in Hsc70 occurred within both cell types Figure 4A with serum removal from the culture medium. In contrast, secreted Hsc70 was markedly different (p 0.0014) in clones expressing cath D = as compared to control cells (

). Control cells secreted large amounts of Hsc70, with a mean ranging from 24 to 36 ng/10 cells Figure 4B 6 depending upon the percentage of serum in the culture medium, whereas cath D clones produced about 10-fold less Hsc70 than the controls (2.5 vs 24 ng/10 cells). For serum withdrawal, a slight increase in secreted Hsc70 was observed, and the transfected clones 6 produced about 5-fold less Hsc 70 than the control cells. Hsc70 secretion was also influenced by the cell density, as shown in , Figure 4C and different effects were observed in the controls and the cath D expressing clones. In control cells, an increase in the cell density from 0.4 to 1.5 10 cells/cm is associated with enhancement of extracellular Hsc70 from 33 to 60 ng/10 cells. When clones expressed Cath D 6 2 6

and formed multilayer cells, scarce amounts of extracellular Hsc70 could be recorded and no significant modulations of Hsc70 were observed throughout the tested density range. In the same culture conditions, we used the trypan blue assay to evaluate the percentage of cell death. For control cells, as well as for the transfected clones, the mean cell death did not exceed 2.4 of total cells (data not shown), as % expected for these cultured cells.

To further confirm that the cath D enzyme was involved in Hsc70 secretion, we neutralized cath D maturation in acidic compartments by using weak bases such as chloroquine or ammonium chloride, as previously described by Liaudet ( ). As shown by [START_REF] Liaudet | Transfected cathepsin D stimulates high density cancer cell growth by inactivating secreted growth inhibitors[END_REF] Western blotting, when such bases were added to the medium, secreted Hsc70 was significantly increased from 7.7 ng/10 cells in cath D 6 cells to 47 ng and 38 ng/10 cells in chloroquine and ammonium chloride treated cells, respectively (

). These results suggest that 6 Figure 4D the acidity of cellular organelles, which is necessary for cath D maturation and activity, is required to prevent Hsc70 secretion. It is known that a pH increase induces a conformational change in cath D since the N terminal becomes positioned in the active site cleft at neutral pH, masking both active D33 and D231 residues to the substrate, as revealed by crystal structure ( ; [START_REF] Baldwin | Crystal structures of native and inhibited forms of human cathepsin D: implications for lysosomal targeting and drug design[END_REF][START_REF] Metcalf | Two crystal structures for cathepsin D: the lysosomal targeting signal and active site[END_REF] ).

Cath D expression did not affect the responsiveness to extracellular Hsc70

To verify the hypothesis that Hsc70 secretion and Hsc70-mediated growth inhibition are independent mechanisms, we complemented the culture medium with S-methionine labeled conditioned media from control and cath D cells (

). As expected, the cellular 35 Figure 5A binding of the 70 kDa protein was observed only in the presence of a conditioned medium (CM) obtained from control cells which secrete Hsc70. Interestingly, the amount of 70 kDa protein bound to cath D cells was strictly identical to that retained by the control cells. In addition, conditioned medium from control cells inhibited proliferation of cath D recipient cells to the same extent as for control cells ( ). These data indicated that cath D expression did not affect neither the Hsc70 cell uptake nor the Hsc70-induced growth Figure 5B inhibition

Involvement of extracellular Hsc70 in antiproliferative cell activity

To assess the antiproliferative activity of this protein, we supplemented the culture medium with an ATP-agarose purified Hsc70 from serum-free conditioned medium, as described in . Increasing nanomolar amounts of Hsc70 were then added to log phase Figure 2C proliferating cells. As shown in , up to 2 nM purified Hsc70 markedly decreased cell proliferation in a dose-dependent manner. Figure 6A Thereafter, higher amounts of Hsc70 did not significantly influence cell proliferation. Hsc70 decreased cell proliferation to 24 (i.e. 76 % % inhibition) as compared to an inactive fraction used as control. To determine whether or not the binding site of extracellular Hsc70 was required for its antiproliferative activity, we used an RNase A protein bounding Hsc70 at position 450 462 through its KFERQ sequence ( -) (residues 7-11 from RNase A). The RNAse A from up to 1 g/ml totally reversed the inhibitory effect of [START_REF] Agarraberes | An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation[END_REF] μ conditioned medium from confluent cells (CM) (

). This indicated that the binding of Hsc70 to proteins containing KFERQ was Figure 6B probably required to avoid proliferation. Moreover, the addition of the S-peptide ( ) (corresponding to the 1 20 [START_REF] Chiang | A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins[END_REF] -N-terminal amino acids of RNAse A) prevented much of the activity of the conditioned medium thereby suggesting that extracellular Hsc70 played a major role in the growth inhibition (

). On the contrary, the additional growth inhibition observed after serum Figure 6C withdrawal was not totally prevented by S-peptide, indicating that serum may contain an additional factor. Moreover, retention of Hsc70 by ADP-agarose partially prevented the antiproliferative effect of CM (

). Altogether, these data indicated an antiproliferative Figure 6D activity of extracellular Hsc70 requiring the binding to the substrate containing the KFERQ sequence.

Effect of Hsc70 on human breast cancer cell lines and cath D modulation

Extracellular Hsc70 also modulated the growth of human cancer cells (Figure S1). Recombinant Hsc70 but not Hsp70 decreased by 25 and 20 the proliferation of MCF7 (Figure S1-A

Discussion

This study provides evidences of the contribution of Hsc70 in cancer cell growth regulation through secretion of this protein in the extracellular environment. We isolated this extracellular protein for its antiproliferative activity and identified it as the heat shock cognate 70 protein through microsequencing of trypsin fragments. The sequence of the fragment at the amino-terminal end of the protein is characteristic of the Hsp70/Hsc70 family within the ATP-binding site of the protein ( ; ). [START_REF] Munro | An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein[END_REF][START_REF] Sorger | Cloning and expression of a gene encoding hsc73, the major hsp70-like protein in unstressed rat cells[END_REF] Retention of the protein on an ATP-affinity chromatography column suggests that a functional ATP binding site is present on the molecule. In addition, the carboxy-terminal end of the protein fragment allowed us to clearly identify this protein as the rat Hsc70 isoform 1, since this Cterm sequence is specific to the Hsc70 isoform 1 and is missing within isoform 2 (

), in addition to the [START_REF] Tsukahara | Molecular and functional characterization of HSC54, a novel variant of human heat-shock cognate protein 70[END_REF] monoclonal antibody recognition which discriminates an antigenic determinant localized only within the specific isoform 1 protein. This 646 residue protein was highly conserved during mammalian evolution and differs only by the N579S amino-acid change between human and mouse or rat (

) whereas the hsp70-gene family is characterized by a large diversity ( ). In O [START_REF] Malley | Constitutively expressed rat mRNA encoding a 70-kilodalton heat-shock-like protein[END_REF] ' [START_REF] Brocchieri | Translocation of Constitutively Expressed Heat Shock Protein Hsc70 to Synapse-Enriched Areas of the Cerebral Cortex After Hyperthermic Stress[END_REF] addition, the identification of this protein as being Hsc70 was confirmed by the fact that an Hsp70 antibody was not able to recognize this protein. This protein was eluted in a gel filtration system as a single active peak corresponding to approximately 290 kDa, indicating that the protein may exist as a multimeric (possibly tetrameric) species. Such self-association has been described via size-exclusion chromatography when an Hsc70 concentration of over 4 M was loaded onto a fast pressure liquid chromatography column ( μ

Fouchaq et

). al., 1999 Hsc70, like many other heat shock proteins, remains intracellular and the detection of a such protein in the extracellular compartment might be the consequence of either cell lysis or cell death, which is always possible in cell cultures, or in contrast due to active release by the cells. According to the first hypothesis, the protein pattern between the two compartments would be qualitatively similar, considering the extremely higher abundance of some major intracellular proteins. This hypothesis was not confirmed by our data, since the absence of major intracellular proteins in the extracellular compartment suggests that the existence of Hsc70 out of cells is due to an active secretion. Hsc70 does not present the sequence leader to be secreted through the classical pathway. However, the use of a general lysosomal inhibitor, such as ammonium chloride which alters the lysosomal pH, functions as an enhancer of Hsc70 secretion. It should be stressed that the opposite effect has been reported for Hsp70 from prostate cancer cells, where an increase in the intralysosomal pH inhibited Hsp70

secretion (

). Taken together, these results suggest that the routes of Hsp70 and Hsc70 secretion differ. [START_REF] Mambula | Heat shock protein 70 is secreted from tumor cells by a nonclassical pathway involving lysosomal endosomes[END_REF] These observations could be linked to the fact that, although they have very similar sequences, an increasing body of data indicate that these two proteins have different physiological functions ( ; ; ; [START_REF] Dressel | Differential effect of acute and permanent heat shock protein 70 overexpression in tumor cells on lysability by cytotoxic T lymphocytes[END_REF][START_REF] Rohde | Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms[END_REF]Goldfarb et al., 2006 Tutar et al., ). Another possible alternative route for Hsc70 secretion is via exosomes ( ; ; 2006 [START_REF] Agarraberes | An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation[END_REF]Thery et al., 1999 Geminard et ; ). In our model, however, we were unable to demonstrate the presence of Hsc70 within a 100 000 g pellet al., 2001[START_REF] Clayton | Induction of heat shock proteins in B-cell exosomes[END_REF] × isolated from conditioned medium (data not shown).

Moreover, we also found that Hsc70 secretion was increased by two events that negatively regulated cell growth, i.e. serum starvation and cell confluence. These events trigger numerous cellular modifications in cancer cells, including intracellular regulator overexpression ( ). The modulation of Hsc70 secretion by cell confluence was abolished when cells [START_REF] Marchesini | Role for mammalian neutral sphingomyelinase 2 in confluence-induced growth arrest of MCF7 cells[END_REF][START_REF] Anagnostopoulou | Differential effects of Stat3 inhibition in sparse vs confluent normal and breast cancer cells[END_REF] overexpressed moderate levels of cath D. Previous data have shown that overexpression of cath D in 3Y1-Ad12 rat cancer cells was sufficient to increase both the metastatic phenotype of the cells and their capacity to proliferate to a higher cell density and form colonies in soft agar. In breast cancer patients, cath D overexpression can be measured in tumor tissues, and several clinical meta-analyses have correlated the cytosolic concentration of cath D in primary tumors with an increased risk of metastasis (

). It should be [START_REF] Bossard | Keeping data continuous when analyzing the prognostic impact of a tumor marker: an example with cathepsin D in breast cancer[END_REF] noted that the present results were obtained for cath D levels below the median concentration noted for primary breast cancers (70 pmol/mg cytosol protein) (

). An attractive hypothesis would be that Hsc70 serves as a substrate for cath D [START_REF] Garcia | Biological and clinical significance of cathepsin D in breast cancer metastasis[END_REF] overexpressing cells within acidic endolysosomes, and thus the pH of this organelle would be neutralized by lysosomotropic agents inhibiting cath D activity and increasing Hsc70 secretion. The antagonistic effect of cath D overexpression on Hsc70 secretion indicates that these two proteins might be localized in the same organelle. In this regard, Mambula et al. ( ) suggested that [START_REF] Mambula | Mechanisms for Hsp70 secretion: Crossing membranes without a leader[END_REF] endolysosomes contain both Hsc70 and cath D proteins. Although the mechanism of action of cath D is not known, the reversal of cath D action by lysosomotropic compounds in 3Y1-Ad12 cells, by pharmacological inhibitor pepstatin A in the human breast cancer MCF7 and MDA-MB-231 cell lines, or the siRNA silencing, suggests that the enzyme might proteolyse Hsc70, or another protein that is required for Hsc70 secretion. Our unsuccessful attempts to hydrolyse Hsc70 by cath D has advocate for a cath D indirect effect on Hsc70 secretion.

However, an interaction between cath D and Hsc70 cannot be excluded since a sequence equivalent to the classical KFERQ motif (Chiang ;

) for Hsc70 binding was found at position 109 (KvERQ) in the N-termimus of cath D, & Dice, 1988 [START_REF] Terlecky | Polypeptide import and degradation by isolated lysosomes[END_REF] corresponding to an external domain of this protein, as revealed by crystal structure analysis ( ; [START_REF] Baldwin | Crystal structures of native and inhibited forms of human cathepsin D: implications for lysosomal targeting and drug design[END_REF][START_REF] Metcalf | Two crystal structures for cathepsin D: the lysosomal targeting signal and active site[END_REF] ).

Finally, we demonstrated that secreted Hsc70 inhibited cell proliferation in the nanomolar concentration range in rat cells. This effect corresponded to most of the antiproliferative activity of the conditioned medium. Nanomolar concentrations of recombinant Hsc70 also modulated the breast cancer cell proliferation, however, to a lesser extent, indicating that Hsc70 could be part of a more complex structure to fully reduce growth. The prevention of the Hsc70 antiproliferative activity due to competitors at the Hsc70 binding site also suggests that this binding site is involved in the antiproliferative response. Moreover, we also showed that cellular binding to Hsc 70 and its subsequent antiproliferative response were clearly unchanged within all clones overexpressing cath D. This indicates that the Hsc70 inhibition of cell proliferation is independent of cath D overexpression, whereas its secretion is not. These findings suggest that the routing pathways for Hsc70 secretion and uptake are quite distinct. In conclusion, we provide the first evidence that the heat shock cognate protein 70 is secreted by confluent cells, and that this extracellular protein plays an active role in the contact inhibition of cancer cells. Moreover, the decrease of this secreted chaperone might account for the higher growth of cancer cells overexpressing cath D.

Materials and Methods

Cell line and culture

Rat embryo cell lines transformed with adenovirus type 12 (3Y1-Ad12), RBA rat mammary adenocarcinoma cells (Cohen et al., 1974) were maintained in RPMI supplemented with 5 FCS.

%

Stable transfectants of 3Y1-Ad12 expressing human cath D, or controls transfected with the vector alone, were cultured with 400 μ g/ml geneticin as previously described (

). [START_REF] Liaudet | Transfected cathepsin D stimulates high density cancer cell growth by inactivating secreted growth inhibitors[END_REF]

Protein labeling

For S-methionine labeling, cells were incubated for 24 h at 37 C in 1 ml methionine-free DMEM supplemented with 200 Ci/ml 35 °μ 35 S-methionine (>1000 Ci/mol; Amersham). The secreted proteins were analyzed by SDS PAGE, followed by fluorography.

Growth inhibition

The growth inhibitory activity of conditioned media, HPLC fractions or purified Hsc70 protein, was measured on recipient low-density cells (RBA rat mammary cancer cells or 3Y1-Ad12 rat embryo cancer cells) growing for 5 days in the log phase in the presence of 2 FCS. Controls were performed with similar volumes of medium, or buffer used in the same conditions. The DNA content % was determined in triplicate wells as previously described (

). [START_REF] Liaudet | Transfected cathepsin D stimulates high density cancer cell growth by inactivating secreted growth inhibitors[END_REF]

Purification of 70 kDa protein and microsequencing

Confluent 3Y1-Ad12 cells were washed and incubated for a further 24 h in serum-free medium. The media were dialyzed against distilled water, adjusted with 20 mM Tris (pH 7.5) and 1 mM CHAPS prior to concentration by lyophilization. The proteins were purified in a procedure involving ion-exchange chromatography with an elution performed with a 0 0.5 M NaCl gradient. The antiproliferative activity of each fraction was then evaluated in an RBA-mammary cell test which has previously shown to be a better antiproliferative test than that involving 3Y1-Ad12 cells. Fractions containing the inhibitory activity were pooled, then applied onto a gel filtration column using HPLC (Amersham-Pharmacia). The inhibitory activity of each HPLC fraction was determined as previously described. The proteins from positive fractions were pooled, lyophilized and separated on SDS-PAGE with low ammonium persulfate concentration, without heating or the addition of 2-mercaptoethanol. The major 70 73 Kda protein associated with growth inhibition was isolated from a non-reducing Coomassie blue stained SDS-PAGE, solubilized, then digested with trypsin for peptide isolation and sequencing in a Beckman automatic peptide sequencer.

High pressure liquid chromatography (HPLC)

HPLC was performed on a normal phase column using phosphate buffer as solvent. A linear gradient (1 ml/min, 30 min) was used and absorbance was measured at 280 nm. Fractions of 30 sec were collected, dried under nitrogen flow, then dissolved in 0.1 M phosphate buffer, and analyzed for the cell proliferation procedure.

ATP-agarose chromatography

Conditioned medium were prepared from high density control 3Y1-Ad12 cells after 24 h secretion in serum-free conditions. The conditioned medium was dialyzed, concentrated by lyophilisation and equilibrated in buffer D (20 mM NaCl, 15 mM 2-mercaptoethanol, 3 mM MgCl2, 20 mM Tris, pH 7.5) through a PD10 column. The concentrated medium was then loaded onto a 5 ml ATP-agarose gel *

  ) and MDA-MB-231 (Figure S1-B) cells, respectively. The treatment with pepstatin A % % significantly increased Hsc70 secretion (Figure S2-A) in both cell lines. Moreover, by silencing cath D, an increase in Hsc70 secretion was observed in both cell lines. (Figure S2-B).

Figure 1

 1 Figure 1Identification of the 70 kDa-protein as the heat shock cognate protein Hsc70 ( ) Conditioned medium obtained from confluent 3Y1-Ad12 cells labelled with S-methionine was dialyzed, concentrated and purified on A 35 an HPLC system as described in the Materials and Methods. The UV elution profile was recorded at 280 nm (dashed lines). The antiproliferative activity of the collected fractions was measured by adding 75 l of each odd fraction to triplicate culture wells containing μ 100,000 RBA cells growing in the log phase. The DNA content was evaluated after 5 days incubation and the values are expressed as a percentage of DNA recovered in control wells treated with buffer alone (solid lines). ( ) HPLC fractions from the above experiment B containing the antiproliferative activity were subjected to 12 SDS-PAGE separation without any reducing agent, and silver stained (left) or % processed for fluorography of S -labelled proteins (Brighty & Jassal). ( ) Identification of rat Hsc70 by peptide microsequencing. The 35

Figure 2 C

 2 Figure 2 Hsc70 from culture medium is secreted and is the only heat shock protein detectable ( ) 35-S-Met protein profile after SDS-PAGE separation and fluorography analysis for the intracellular proteins (I) or the secreted proteins A (S) from control cells and secreted proteins from cath D overexpressing clones (Cath D S). Arrows represent examples of proteins present in only one compartment. ( ) The cell death was evaluated using trypan blue staining for control cells and Cath D transfected clones. ( ) B %

Figure 3 Figure 4

 34 Figure 3 Loss of contact inhibition and repression of Hsc70 secretion induced by cath D Control and cath D expressing cells were cultured for 6 days on glass coverslips and analyzed under a scanning electron microscope. Bars represent 90 nm. Inserts represent a 5-fold magnification. ( ) A characteristic monolayer cell culture depicts confluent control cells. The high A confluency did not allow observation of the cell contours. ( ) Overlapping cell layers characterized the cath D transfected cells. Abundant B foci were seen as spheroids. ( ) Cell quantitation was performed by DNA content measurement using the DABA method. Two control cell C lines (open circles) and two cath D transfected cells (filled circles) were monitored for a 10 day period. Results are means for two clones run in triplicate. ( ) In the same experiments, Hsc70 content released for a 24 h period in the medium at day 6 was quantitated by Western blot D and expressed as ng for 10 cells for 24 h ( p 0.0148) 6

Figure 5

 5 Figure 5 Cath D expression and responsiveness to secreted Hsc70 ( ) Confluent control cells or Cath D expressing clones were cultured in the presence of S-methionine for 24 h. The corresponding A 35 conditioned media were then added in the presence of 1 mM non radioactive methionine, to confluent control (C) or cath D recipient cells. After a 24 h incubation and two washes, the cell proteins were subjected to SDS-PAGE and fluorography. ( ) Proliferation of control or cath B D cells cultured in the presence of the indicated concentration of proteins recovered from conditioned media of control density-arrested cells.

  Figure 6

[START_REF] Fouchaq | Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70[END_REF]

[START_REF] Fouchaq | Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70[END_REF]

[START_REF] Fouchaq | Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70[END_REF]

[START_REF] Fouchaq | Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70[END_REF]

[START_REF] Fouchaq | Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70[END_REF]

[START_REF] Fouchaq | Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70[END_REF]

[START_REF] Fouchaq | Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70[END_REF]

Data analysis

Statistical significance was analysed using student s test from GraphPad Prism, version 3.00 for Windows (GraphPad Software Inc., ' t San Diego, CA, USA). Values of p < 0.05 were considered significant.

column (Sigma). The column was washed successively with three volumes of i) buffer D, ii) buffer D supplemented with 0.5 M NaCl, and iii) buffer D. Finally, the retained proteins were eluted with 1 ml fractions of buffer D 3 mM ATP. To test the antiproliferative activity, + the fractions were washed three times with culture medium and concentrated twice with Centricon Biomax 5.

Scanning electron microscopy

Cells were grown on glass coverslips in phenol red-free DMEM with 5 DCC/FCS for 5 days. Cells were fixed with glutaraldehyde % (3.75 ) for 2 h and post-fixed with 1 osmium tetroxide (1 for 1 h in Millonig buffer at pH 7.3. Coverslips were rinsed in Millonig % % % buffer and dehydrated through a graded series of alcohol and isoamyl acetate before critical-point drying. After mounting on an aluminum support, samples were coated with gold, and then examined with a JEOL Model JSM-35 scanning electron microscope at 15 kV.

Secreted Hsc70 quantitation

SDS-PAGE was performed with known amounts of commercial Hsc70 as standard tracks. After Western blot, the tracks were scanned and quantified using scion image for Windows (Scion Corp. Frederick, MA, USA).

Ackowledgements:

We thank Jean Derancourt, (Centre de Recherches de Biochimie Macromoleculaire du CNRS, 1919 Route de Mende, 34293 Montpellier Cedex 5, France) for peptide fragments analysis. The authors also thank the Centre R gional d Imagerie Cellulaire (Montpellier France) for Footnotes:

Conflict of Interest

The authors declare no conflict of interest.

Supplementary information is available at http://www.nature.com/onc/


Bilel Nefzi
email: bilel.nefzi@loria.fr

Hugo Cruz-Sanchez
email: hugo.cruzsanchez@loria.fr

Ye-Qiong Song
email: ye-qiong.song@loria.fr

SCSP : an Energy Efficient Network-MAC Cross-layer design for wireless Sensor Networks

This article presents SCSP (for "Sleep Collect and Send Protocol") for wireless sensor networks, a network-MAC cross layer design that resolves the inherent conflict between energy efficiency and throughput. The protocol uses in its MAC layer a new paradigm that we call "sleep, collect and send". The idea of SCSP is that a router sleeps for a given amount of time, wakes up and collects data from its children and other routers and then send them into a burst during a period of time that we call transmission period. In its network layer, the protocol uses a hierarchical tree structure as network architecture and a tree routing protocol. SCSP does not require synchronization between routers and dynamically calculates the sleep and collect periods according to the amount of incoming traffic. The protocol is implemented and simulated in OPNET simulator.

I. INTRODUCTION

Many wireless sensor networks (WSN) applications such as forest surveillance are deployed for long periods with only batteries as main power supply. In order to take account of energy efficiency, MAC protocol designs use duty cycling mechanisms in which nodes periodically switch between sleep (low-power) and active modes. However, adding sleep periods degrade network throughput which is an essential parameter for some applications and situations. For example, when a fire is detected in the forest, a big number of messages will be generated thus stressing the high throughput functionality of the MAC protocol. Therefore, nodes in WSN must be capable of achieving high throughput rates while minimizing energy consumption. When an urgent event occurs, there is no need for energy saving anymore. Hence, we need a dynamic adaptation mechanism of sleep and active periods.

To address this challenge, we propose the SCSP protocol, a network-MAC cross-layer design that dynamically adapts the active and sleep periods according to the amount of received traffic. SCSP consists of a MAC protocol that works according to the paradigm "sleep, collect and send", similar to the "store and forward" in wired switches except that we add a sleep state. Besides, it does not require synchronization between routers. Thus, it is scalable. The network layer uses a modified version of the ZigBee [1] tree routing that is more tolerant to node failures. MAC layer provides the list of neighbor routers to the network layer which in its turn provides multiple forwarding choices to it. Hence, MAC protocol has the possibility to change the next-hop router during transmission if the message is not successfully transmitted after some tries.

The rest of the paper is organized as follows. Section II presents the related work. In section III we describe the SCSP design. In section IV, we evaluate its performance through the simulation of a tunnel supervision scenario. Section V concludes the paper.

II. RELATED WORK

S-MAC [START_REF] Ye | Medium access control with coordinated adaptive sleeping for wireless sensor networks[END_REF] and B-MAC [START_REF] Polastre | Versatile low power media access for wireless sensor networks[END_REF] are the most popular contention based MAC protocols in WSN. S-MAC is designed with energy consumption minimization and self-configuration support in mind. S-MAC periodically switches between sleep and active states. It provides a synchronization mechanism that maintains a common sleep schedules between neighbor nodes which enables low-duty-cycle operation in multi-hop networks while minimizing energy consumption. The protocol uses an RTS/CTS transmission scheme and a virtual carrier sense technique similar to those in IEEE 802. 11 [3] which are used to provide adaptive listening and avoid overhearing unnecessary traffic. Finally, it presents a message passing technique which fragments a long message into many small fragments and transmit them in a burst.

B-MAC provides power management via low power listening. Nodes periodically switch between sleep and active states (sleep period is called check interval). When a node wakes up, it checks channel activity. If activity is detected, the node stays awake and receives the incoming packet. However, if the medium is clear, the node goes back to sleep. Each transmission is preceded by a preamble as long as the check interval so the intended receiver is definitely aware of the transmission and receives the incoming packet. SCSP has the advantages of both protocols and provides further enhancements. It uses the sleep and wake up periods as S-MAC does. However, during the sleep periods, a node periodically wakes up, checks channel activity and stays active if the channel is not free as B-MAC does. This combination avoids S-MAC's synchronization overhead thus improving scalability. In addition, by bursting data, a preamble is sent once for the first message to transmit thus improving throughput. Finally, SCSP dynamically adapts active and sleep periods while they have to be set during network deployment or through reconfiguration mechanisms for S-MAC and B-MAC. IEEE 802.15.4 [START_REF]Wireless Medium Access Control (MAC) and Physical Layer (PHY) specifications for low-rate Wireless Personal Area Networks (LR-WPANs). IEEE standard for information technology[END_REF] standard specifies the physical layer (PHY) and MAC sublayer for low data rate, low-cost wireless personal area networks (WPANs) of fixed, portable or moving devices with no battery or very limited energy consumption requirements. In its beacon enabled mode, the MAC sublayer uses a superframe structure, defined as the time between two successive beacon transmissions, that is composed of an active period and a sleep period. The superframe structure is adapted to a star topology. [START_REF] Koubaa | A Time Division Beacon Scheduling Mechanism for IEEE 802.15.4/Zigbee Cluster-Tree Wireless Sensor Networks[END_REF] proposed a solution to enable the use of superframe structures in multi-hop networks. However, it is synchronized and centralized thus non scalable.

Reactive routing protocols like AODV [START_REF] Perkins | Ad Hoc On Demand Distance Vector (AODV) Routing[END_REF] and DSR [START_REF] Johnson | The Dynamic Source Routing Protocol for Mobile Ad Hoc Networks for IPv4[END_REF] and pro-active routing protocols like OLSR [START_REF] Jacquet | Optimized Link State Routing Protocol (OLSR)[END_REF] and DSDV [START_REF] Perkins | Bhagwat Highly Dynamic Destination-Sequenced Distance Vector (DSDV) for Mobile Computers Proc[END_REF] are not suitable for duty cycled MAC protocols. Path discovery in reactive protocols has a high delay and energy costs while in pro-active protocols, performing frequent route updates and taking sleep and wake up schedules into account is expensive. In our protocol we used a modified ZigBee tree routing (ZTR and m-ZTR) scheme which does not require any message exchange or route discovery. The modification tackles some of its weakness, namely its tolerance to node failure.

III. SCSP DESIGN

A. General parameters and network architecture

We consider a wireless network composed of simple nodes and router nodes (named router in the rest of the document). A simple node is a device that generally has measurement sensors built in and has limited routing decisions capabilities. A router is a device that implements full routing and network management protocols. However, it can act like a simple device when its routing and management capabilities are disabled.

The network is organized into a tree and uses the tree addressing scheme presented in ZigBee standard. The network formation procedure will be developed in Sect. III-C.

All nodes (simple nodes and routers) use CSMA/CA protocol to access the medium but with different maximum backoff intervals in order to give routers higher chance to access the medium; we used the unslotted version of CSMA/CA proposed by IEEE 802.15.4 (used in the non beacon enabled mode). SCSP operates on the top of the "2450 MHz DSSS IEEE 805.15.4" physical layer; the data rate is equal to 250 kbps.

B. Channel access protocol

1) General description: When a node starts, it executes the association procedure to integrate the network. For a router, once the association is successfully completed, it starts sending beacon frames to provide useful information to its neighbors (including its children) and synchronization to its children. Routers use a superframe structure, defined as the time between two successive beacon frames. The superframe is divided into three periods; Sleep Period (SP), Waiting Period (WP) and Transmission Period (TP). We call the sum of SP and WP a subframe. During the SP, the router enters into sleep mode. When the SP expires, the router becomes active. However it does not transmit packets one by one upon their arrival. Instead, it goes to reception state and collects data from its neighbors (simple nodes or routers) during the WP. After the end of the WP, the router starts transmitting all packets queued in its buffer in a single burst. This is done in the TP. Finally, at the end of the TP, the router sends the next beacon and starts another cycle. The superframe structure is illustrated in Fig. 1(a). Simple nodes are always in sleep mode unless they receive a packet to transmit from the higher layer. Full details of the WP, TP, SP and the operating mechanism of simple devices are given next.

2) Waiting period: The W P depends on the amount of incoming traffic; the higher the traffic volume, the longer the WP duration. The W P duration for a router r is given by (1).

W P = N max • d S , if N r c = 0 N max • d R , otherwise (1)
N r c is the number of children associated to the router r. d S and d R are the maximum amount of time needed by a simple node and a router, respectively, to perform the collision avoidance rocedure (d BP ), sense the channel (t CCA ) and detect that it is Idle (at the first attempt), change the antenna state from receiver to sender (t tat ), send the packet (d pkt ), wait for an acknowledgment (t ack ), and receive it (d ack ). See Fig. 2. d S or d R define one slot time.

N max is an integer that has to be estimated using the incoming traffic (it defines the number of slots). Its value gives the duration of the W P . It has a minimum value of 1. The estimation algorithm is described next.

The estimation algorithm. Let's define first the set Ω ǫ IN as the set of W P s during which the router receives data and W P k , k ǫ Ω these W P s. We define also, U k , k ǫ Ω as the ratio giving the sum of the service times of received packets ( iǫE k 1 µi , E k is the set of received packets during W P k ) divided by W P k duration. 1 µi is the service time of packet i (see Fig. 2). U k is shown in [START_REF] Ye | Medium access control with coordinated adaptive sleeping for wireless sensor networks[END_REF]. We note that U k can be greater than 1 in the case where the router is still receiving a packet from a neighbor router while the W P ends (recall that the router is not preemptive).

U k = i ǫ E k 1 µi W P k (2)
Let us denote by S k the exponential moving average of

U k , k ǫ Ω S k = (1 -α)S k-1 + αU k-1 (3)
α is the smoothing factor. We used a non linear filter where α is bigger when U k-1 ≥ S k-1 allowing S k to adapt more swiftly to traffic increase. After a number of trials, we found that values of α, α 1 = 0.008 and α 2 = 0.01, gave the best of all-around performance. The computation of S k resembles that of the average queue length in RED [START_REF] Floyd | Random Early Detection (RED) gateways for Congestion Avoidance[END_REF] and the round trip time in TCP [START_REF] Postel | Transport Control Protocol[END_REF].

S k is evaluated before sending the k th beacon frame and is used in the Algorithm 1 to determine the value of N max for the k th W P (thus its duration). In the algorithm, T hr max , T hr min and N M AX designate the maximum threshold of S k , the minimum threshold of S k and the maximum value of N max , respectively. T h max allows the increase of the WP's duration. When set too high, the WP will not respond rapidly to the network load growth. When set too low, bandwidth will be wasted since the W P will increase too fast. T h min is the minimum utilization ratio under which the protocol performance will degrade significantly. T h min is used to decrease the WP's duration : set too high, WP decreases fast which diminish the benefits of our algorithm. When set too low, WP's duration decreases too slowly compared with the network load growth. N M AX bounds the value of N max to prevent a high increase in the W P duration. It is set to 15 in all simulations.

Intuitively, S k calculates the average utilization ratio of the W P . If S k exceeds T hr max , the value of the N max is increased which augmentates the duration of the W P . If S k decreases below T hr min , the value of N max is decreased. We can see that changing the W P duration, affects the value of U k (2) and so S k which in its turn affects the value of N max . Besides, S k depends also on the traffic load. Hence, our algorithm appears like an iterative process where the objective is to find the good value of N max that reflects the incoming traffic load while satisfying an utilization ratio between T hr min and T hr max .

3) Transmission period: The collected data during the waiting period are transmitted in a burst. At the beginning of the TP, the router sends a preamble frame; its duration is superior to the wake up period of routers 1 . This way, all neighbor routers will wake up. However, if the network is configured to stay active all the time, preamble transmission is disabled. After that it sends the queued packets. CSMA/CA protocol is used to transmit the preamble packet. Then, the remaining 1 The wake up period is the same for all routers Algorithm 1: Any router has at least one message to send during the TP which is the beacon frame. If the router has buffered messages to send, it transmits the beacon after finishing the transmission of these buffered messages. Otherwise, it must contend to access the channel using CSMA/CA before sending the beacon frame. In this case, the preamble frame is not sent. We shall notice that a beacon frame transmission is always considered successful since beacons are not acknowledged.

Estimation algorithm / * Update S k* / α = α 1 if U k-1 ≥ S k-1 then α = α 2 end S k = (1 -α)S k-1 + αU k-1 / * Update N max* / if (S k ≥ T hr max ) then N max = N max + 1 else if S k ≤ T hr min then N max = N max -1 end end N max = max(1, min(N max , N M AX))
After the end of the WP, the transmission procedure described above begins only if the channel is idle. Otherwise, the WP is extended until it becomes idle. At each detection of a busy state, the WP is extended by d S seconds (d S is defined in Sect. III-B2) .

4) Sleep period: During the SP, the router goes to sleep. However it periodically wakes up and senses the channel. If the channel is free it goes back again to sleep, otherwise it remains active as far as it receives data from the medium. The router goes back to sleep if the SP did not expire yet (in that case, it starts the WP) and if the channel remains idle for a period of time equal to the sum of d S and the maximum backoff duration of a router. This amount of time guarantees that no other router is starting its transmission procedure before it goes to sleep.

The duration of the subframe is a constant defined by the network administrator and the duration of the WP depends on the traffic load. Thus, the duration of the SP depends also on the traffic. When the traffic load increases, the WP increases while the SP decreases and vice-versa. This is the dynamic adaptation of the sleep and active periods which is illustrated in Fig. 1(b) and Fig. 1(c) for a heavy and a light traffic, respectively.

5) Simple nodes operating mechanism:

A simple node is always in sleep mode unless it receives a packet to transmit from the higher layer. In that case, it wakes up and listens to the channel. It keeps listening to the channel until it receives a beacon frame from a neighbor router. Then, it extracts the SP and WP durations and the address of the router which sends this beacon and goes back to sleep during the SP period specified in this beacon frame. After the expiration of SP, it wakes up again. Now the simple node is synchronized with the WP of the beacon frame sender. Finally, it sets the next hop address of the packet to the address extracted from the beacon frame and uses CSMA/CA to send it.

If a node has another packet to transmit or if the transmission of the packet being transmitted fails (due to a CSMA/ CA failure or an non reception of an acknowledgment frame), the simple node must check first if it can transmit it in the remaining time of the WP. If it is the case, it sets the next hop address of the packet to the address extracted from the beacon frame and uses CSMA/CA to send it as the previous packet. Otherwise, it must restart the whole procedure; locate a beacon frame, sleep during the SP period of the beacon frame of the sender and send the packet.

Any other packet received during the transmission procedure is queued. At the end of transmission of all the queued packets, the simple node goes back to sleep. Note that the next hop can be a router that is not the father of the simple node.

C. Network formation 1) Association:

We denote by the root, the router that starts the network and specifies all its parameters (like the subframe length and parameters needed for address attribution). The root is equivalent to the the ZigBee coordinator in the ZigBee standard.

The association mechanism works as follows. When the root becomes active, it starts sending beacon frames periodically; the root starts using the superframe structure immediately. If a router or a simple device wants to join the network, it starts collecting beacons during a period of time that we call "collection period" (CP). During this period, for any beacon received, the node records all the information provided and stores the record into a list. The list is sorted according to the depth of the sender which is the minimum number of hops to reach the root using only parent child links and the number of children associated to it; the head of the list contains the record with the minimum depth and the number of children. When the CP expires, if the list is empty, the CP restarts. Otherwise, the node selects the head of the list and tries to locate the next beacon of the "selected node", in order to be synchronized with it. Finally, it sends an association request during WP of the selected sender. Upon the reception of an association request, a router sends immediately a reception response containing the assigned address and the network parameters. This is the only case where a given router sends a frame during the WP. The association response frame acts like an acknowledgment; if a node sends an association request and does not receive an association response, it considers the frame lost and moves to the next record in the list if any and repeats the synchronization process. Otherwise, it starts again the CP. The association procedure is executed once for all routers, during network establishment, unless the root decides to reset the network.

2) Address attribution: The network addresses are assigned using a distributed address allocation scheme that is designed to provide every potential parent with a finite sub-block of network addresses to distribute to its children. During network establishment, the root determines the maximum number of children per parent (Cm), the maximum number of routers (Rm) between these children as well as the maximum depth of the network (Lm) (the root has a depth of 0). A function called Cskip(d) (equation 4) is used after that to calculate the size of the address sub-bloc being distributed by each parent located at depth d.

Cskip(d) = 1 + Cm • (Lm -d -1), if Rm=1 1+Cm-Rm-Cm•Rm Lm-d-1 1-Rm , otherwise (4)
The network address distribution is as follows. The root has always the address 0. For routers, the address assignment uses the value of Cskip(d) as an offset: if the node is the first served, its address is one greater than its parent address. Otherwise, the addresses are separated from each other by Cskip(d). For simple nodes, network addresses are assigned in a sequential manner using the following rule :

A n = A parent + Cskip(d) • Rm + n (5)
Here 1 ≤ n ≤ (Cm -Rm) and A parent represents the address of the parent.

D. Routing protocol

1) ZigBee tree routing: End devices (simple nodes in our case) always send their data to their father. For routers, ZigBee tree routing (ZTR) is based on some comparison rules: for a ZigBee Router with address A at depth d, if the following logical expression is true, then a destination device with address D is a descendant. If it is the case, the Next Hop is given by the following rule:

A < D < A + Cskip(d -1) (6)
• N = D for ZigBee end devices where

D > A + Rm • Cskip(d) • N = A + 1 + ⌊ D-(A+1) Cskip(d) ⌋ × Cskip(d) , otherwise (7)
If the expression is not true, the destination is not a descendant and the message should be routed through A's parent.

2) Modifications: First, simple devices does not always send their data to their father. Instead, they send it to the first received beacon sender. Finally, in prior work, Nefzi [START_REF] Nefzi | Performance Analysis and improvement of ZigBee routing protocol[END_REF] proposed an improvement of ZTR using neighborhood table in routing decision in order to improve the worse-case delay. Our modification follows the same logic since neighborhood table can be efficiently built using beacon information. The modification works as follows. MAC layer provides the list of neighbor routers to the network layer. This table is updated whenever a router receives a beacon frame. First, by default, the next-hop address is the one provided by ZTR with only one exception; if the destination is a descendant of a neighbor node, the next-hop is set to this neighbor. Finally, if the nexthop as calculated by ZTR is not a descendant, the network layer sorts the neighbor table according routers depth; the neighbor router with the minimum depth is placed first.

During TP, if a transmission fails after some tries and if the next hop is the father and the destination is not the father (MAC layer knows its father address), it picks up the head of the list and sends the message again to this router. If the neighbor table is empty or the destination is a descendant, the message is destroyed.

The modification works when the next-hop as given by ZTR is the father. This is because the sink node is generally the root. Besides, first level nodes tend to die first because most of the traffic crosses them to reach the destination.

IV. SIMULATION ANALYSIS

We present in this section a simulation study of SCSP which we implemented in OPNET [START_REF] Opnet | [END_REF] (Modeler v14.5). We analyze the energy consumption and end to end delay with different network conditions and protocol parameters. We study its dynamic throughput adaptation behavior. We compare m-ZTR with the basic ZTR. For energy consumption calculation, we use the micaz mote [START_REF]Micaz datasheet[END_REF]

A. Scenario description

We consider a multi-hop network, which simulates a tunnel supervision system, in a surface of 250m x 50 m (Fig. 3). It is composed of seven routers (blue circles in the figure) and 16 simple nodes (gray squares). Simple nodes hear only the two nearest routers and the transmission range of routers is equal to the distance between R3 and R2 (86m). The sink node is the root node. Cm, Rm and Lm are equal to eight, four, and four, respectively. Simple nodes generate a traffic with a constant inter arrival time and a constant packet length equal to 450 bits 4 .The root node is always active and has unlimited energy.

B. Network lifetime

We define the network lifetime as the time of the first router failure. In this set of simulations, we varied the sub-frame The network lifetime increases almost linearly as the subframe length increases because routers sleep much more time. For light traffic load (2min and 1min message generation rates), the network lifetime increases as the subframe duration increases. The improvement in comparison with the "always active" scenario is 2 to 20 times depending on the duration of the subframe. For heavier traffic load, network lifetime is almost constant; on one hand, routers wake up more often during their SP to get data and on the other hand, the WP duration is longer since it is adapted according to amount of incoming traffic. SCSP obtains better results except when routers are always active. This is because SCSP has higher MAC overhead than IEEE 802.15.4 non beacon enabled mode; beacon transmissions for instance.

C. End to end delay

We run the previous set of simulations again and measured the average end to end delay as a function of the supeframe length. The results illustrated in Fig. 5 show that IEEE 802.15.4 MAC always outperforms SCSP. This is because of SP and beacon transmissions overhead. However, if we take into account the inter arrival time characteristic of the traffic, we can establish a trade off between energy saving and end to end transmission delay. For example, for an inter arrival time equal to one minute and a subframe length equal to 100 slots, the average end to end delay is equal to 1560ms which is considered reasonable in comparison with the 60 seconds of inter arrival time. Hence, considering the traffic characteristics, the end to end delay and the energy consumption, the network administrator can efficiently calculate the optimal subframe length that maximizes the network lifetime.

D. WP adaptation

In this set of simulations, simple nodes 11 to 18 transmit data starting at 1500s and ending at 2100s with a generation rate of two messages per second. The rest of simple nodes start transmitting at 10s until the end of the simulation with a message generation rate of five seconds. The subframe length is equal to 20 slots. We used the m-ZTR. The energy is set to maximum to avoid nodes death. We plot the WP duration as a function of the simulated time for all routers. Results obtained by routers R2 and R3 are shown in Fig. 6 along with the total traffic sent (in bits/sec) and results of the other routers are illustrated in Fig. 7.

For all routers except R1, the WP duration increases after 10s. This is because some simple nodes start transmitting data at the same time (10s). Thus, the WP is adapted quickly to support this increase in the traffic volume. After that, the WP of all routers goes again to its minimum value, because the generated traffic volume is not big and is smoothed according to the active and sleep router schedules. Similarly, between 1500s and 2100s, eight nodes start transmitting data. Again, R2, R3 and R4 adapt their WP to this increase in traffic volume because most of simple nodes that start transmitting at 1500s send traffic to R3 or R4 which have R2 as father. Note that the WP duration of R2 remains equal to 9.632ms (two slots) between 1500s and 2100s because the traffic volume corresponds to a WP duration of two slots. However, the WP duration of R3 and R4 oscillate between one and two time slots. This is because, simple nodes are free to choose the router to which they send data. So, R3 adapts its WP when it is chosen and vice-versa. Since, the traffic generation rate is constant, simple nodes generally wake up at the same time and tend to select the same router. WP of R1 and R5 remains at minimum since most of the traffic is routed through R7 and R2.

SCSP efficiently adapts its WP duration to the incoming traffic volume. This is an important feature of our protocol since it solves the problem of throughput degradation introduced by forcing nodes to periodically enter sleep mode.

E. Routing

In this set of simulations, we fixed the subframe length to 10 slots and data generation rate to one message every five seconds. Then we measured the traffic received by the sink node and the total energy consumption during the simulated time.

Figure 8 shows the performance of ZTR and m-ZTR. Before first router's death, m-ZTR performs slightly better than ZTR due to the additional transmission attempt to a neighbor node when the maximum retransmission limit is reached (it is fixed to five in all simulations). After the first router's death (R1 which is the same for both protocols) and compared to ZTR, m-ZTR maintains the whole network connectivity for three additional minutes before the received traffic falls due to the death of R2. This is due to the flexibility of selection of nexthop routers by simple nodes and routers.

Hence, m-ZTR is more tolerant to node failures than ZTR.

V. CONCLUSION This paper presents SCSP, a network-MAC cross-layer design which ameliorates energy consumption by switching between active and sleep periods and dynamically adapting them according to the amount of received traffic. The protocol uses a simple but efficient routing protocol that does not need route maintenance or discovery and works jointly with the MAC layer to enhance its fault tolerance properties. Simulation results show that SCSP, extends the network lifetime and connectivity in comparison with IEEE 802.15.4 MAC.

Future work consists of implementing the protocol within micaz motes.

  Fig. 1. Subframe structure

Fig. 2 .

 2 Fig. 2. d S , d R and 1 µ variables

Fig. 3 .

 3 Fig. 3. Simulation scenario

Fig. 4 .

 4 Fig. 4. Energy consumption with different message generation rates

4 Fig. 5 .

 45 Fig. 5. Comparison of end to end delays between SCSP and IEEE 802.15.4

Fig. 6 .

 6 Fig. 6. Total traffic sent in bits/s and WP duration in seconds of R2 and R3

Fig. 8 .

 8 Fig. 8. Comparison between basic and modified ZTR

  current and voltage parameters which

	Receive mode current (mA)	19.7
	TX current (mA)	17.4
	Active mode current(mA)	8
	Sleep mode current (µA)	15
	Voltage (V)	3
	TABLE I
	MICAZ MOTE PARAMETERS
	Data rate (kb/s)	250
	CCA duration (µs)	0.128
	Initial energy (J)	100
	Wake up duration (ms)	10
	T hrmax	0.75
	T hr min	0.28
	d S (ms)	4.816
	d R (ms)	3.536
	TABLE II
	SIMULATION PARAMETERS
	are given in table I. Table II summarizes general simulation
	parameters 3 .

Acknowledgments are enabled in SCSP

We set the initial energy to 100J. However, a typical Alcaline AA battery which is used by micaz motes has a theoretical energy of 15390J[START_REF]Alcaline Technical Information[END_REF]

We supposed that the deployed application generates periodic messages. However, SCSP is not specifically designed for such applications.