The Nhan Luong
email: thenhan.luong@iutbayonne.univ-pau.fr

Sébastien Laborie
email: sebastien.laborie@iutbayonne.univ-pau.fr

Thierry Nodenot
email: thierry.nodenot@iutbayonne.univ-pau.fr

A Framework with Tools for Designing Web-based Geographic Applications

Keywords: D.2 [Software Engineering]: Design Tools and Techniques; I.7.2 [Document and Text Processing]: Document Preparation Design, Languages Document generation, Authoring tool, Mashups

Many Web-based geographic applications have been developed in various domains, such as tourism, education, surveillance and military. However, developing such applications is a cumbersome task because it requires several types of components (e.g., maps, contents, indexing services, databases) that have to be assembled together. Hence, developers have to deal with different technologies and application behavior models. In order to create Web-based geographic applications and overcome these design problems, we propose a framework composed of three complementary tasks: identifying some desired data, building the graphical layout organization and defining potential user interactions. According to this framework, we have specified a unified model and we have encoded it using Semantic Web technologies, such as RDF. Through a prototype named WINDMash, we have implemented some tools that instantiate our model and automatically generate concrete Internet geographic applications that can be executed on Web browsers.

INTRODUCTION

Currently, a fair amount of research and development has been conducted on Web-based application generation thanks to Web 2.0 technologies. Particularly in the domain of geographic information system (GIS), specific terms appeared in order to designate Internet Geographic Applications [START_REF] Haklay | Web Mapping 2.0: The Neogeography of the GeoWeb[END_REF]: "GeoWeb", "Geospatial Web" and "Web Mapping 2.0". Indeed, many Web-based geographic applications have been developed in different application domains (e.g., tourism, education, surveillance, military) and are using online mapping services (e.g., Google Maps1 , OpenLayers2 , Yahoo! Maps3 ).

However, developing such applications is a cumbersome task. The reasons are twofold: (1) they mix several components (e.g., maps, multimedia contents, indexing services, databases) which have to interact together and (2) developers have to deal with several technologies and different data structures as well as application models.

In this paper, we propose a framework for designing Webbased geographic applications. This framework is composed of three complementary tasks: [START_REF] Adida | RDFa in XHTML: Syntax and Processing[END_REF] identifying the desired data that have to be manipulated by the system, (2) specifying the graphical layout of the application, and (3) defining potential end-user interactions between components. According to this framework, we have specified a unified model with three facets and proposed to encode this model using RDF [START_REF] Manola | RDF Primer[END_REF]. We have chosen such a W3C standard because it enables to describe, to aggregate, to share and to reuse information embedded into each facet.

In order to experiment our proposal, we have developed some tools allowing designers to easily instantiate our unified model, i.e., create Web-based geographic applications. Moreover, from our proposed model, the designed descriptions can be computed into concrete Internet geographic applications and finally executed on a Web browser.

The remainder of the paper is structured as follows. In Section 2, we define the characteristics of a Web-based geographic application and illustrate an example. Moreover, we explain why it is currently difficult to design this kind of application. Thereafter, in Section 3, we describe some related existing systems. In Section 4, we specify our framework for designing Web-based geographic applications and detail in Section 5 its corresponding unified model. Section 6 demonstrates our framework through a prototype named WINDMash, especially some specific tools are presented in Section 7. Finally, we conclude in Section 8.

DESIGNING WEB-BASED GEOGRAPHIC APPLICATIONS

Designing interactive Web-based geographic applications that allow, for instance, completing and/or increasing the discovery of a territory, such as in tourism or in education, is currently a cumbersome task. Indeed, such applications may generally include several components that should interact with each other, such components are:

• Mapping components with, for instance, spatial annotations and various types of geovisualization (e.g., street map, satellite view);

• Content components, such as texts, images, audios and videos. In this paper, we will exclusively focus on textual components;

• Indexing components which automatically extract spatial entities from some given contents and identify spatial metadata, like geolocation information;

• Communication components in order to exchange data with database servers, e.g., GeoNames 4 .

In order to illustrate a typical Web-based geographic application, we present a use-case scenario in the following section ( §2.1). This example will be used all during the paper. Furthermore, we also explain why developing such applications is not straightforward ( §2.2).

A use-case scenario

Suppose a French course for foreigners where a teacher wants to explain the following concepts: Town, "Département" (a sort of province inside the country) and "Préfecture" (an administrative town inside a "Département"). More precisely, the teacher wants students to study a text written in French5 which contains several places names, e.g., Cannes (Town), Lyon ("Préfecture") and Savoie ("Département").

For pedagogical purposes, the teacher would like a Webbased geographic application, like the one illustrated in Figure 1. Indeed, it contains the text in French, a map containing spatial annotations and three lists of towns, "Préfectures" and "Départements". These lists should be automatically computed from the text. Furthermore, students are allowed to select a specific place in the lists. When clicking on a place, the corresponding term in the text is highlighted and the map is focused on this place.

Some requirements

Developing the interactive Web-based geographic application illustrated in Figure 1 with the behaviors described above is not a trivial task. It requires:

• Some programming skills, e.g., using JavaScript or AJAX, in order to create an interactive Web application;

• Knowledge about several databases schemas, and especially geographical databases in order to query and get spatial data, such as geolocations on a map;

• Manipulation of Web services (e.g., indexing services), particularly their inputs/outputs. Naturally, data structures have to be homogenized in order to confront and/or aggregate different services outputs.

Consequently, there is a need of a general framework which contains models and supports some tools for designing Webbased geographic applications. This framework is even more motivated in the next section.

RELATED WORK

End-user mashup programming environments are a new generation of online visual tools enabling users to quickly create, for example, Web-based applications [START_REF] Altinel | a data mashup fabric for intranet applications[END_REF]. They rely on metaphors that are easy to grasp by non-professional coders. They may bind together spreadsheets, the flow of linked processing blocks and the visual selection of GUI actions. In [START_REF] Beletski | End user mashup programming environments[END_REF] and [START_REF] Taivalsaari | The future of web applications[END_REF] several types of mashup environments have been summarized, some examples are: Yahoo! Pipes 6 , Microsoft Popfly 7 , Google Mashup Editor 8 , IBM Mashup Center 9 , MashMaker [START_REF] Ennals | mashups for the masses[END_REF], Marmite [START_REF] Wong | Making mashups with Marmite: towards end-user programming for the Web[END_REF], Vegemite [START_REF] Lin | End-user programming of mashups with Vegemite[END_REF], Exhibit [START_REF] Huynh | Exhibit: lightweight structured data publishing[END_REF] and Bill Organiser Portal [START_REF] Ro | Bill Organiser Portal: A Case Study on End-User Composition[END_REF]. 6 http://pipes.yahoo.com/pipes/ 7 http://www.deitel.com/Popfly/ 8 http://code.google.com/intl/en/gme/ 9 http://www-01.ibm.com/software/info/ mashup-center/ However, developing the geographic application example presented in Section 2 with these mashup environments is still difficult, while impossible with some of these systems. In fact, many of them do not take into account the specification of end-user interactions and especially the system reactions on the different application components. Furthermore, these systems are not designed to exclusively build geographic applications, hence they do not propose a framework for building such kind of applications.

Some recent works on Web Engineering, especially focusing on geographic applications, proposed architectures based on mapping services. For instance, Mashlight [START_REF] Albinola | A Lightweight Mashup Framework for Everyone[END_REF] is a Web framework for creating and executing mashup applications by building data processing chains. The generated applications contain by default one mapping component with geolocation information. Besides, DashMash [START_REF] Cappiello | Enabling End User Development through Mashups: Requirements, Abstractions and Innovation Toolkits[END_REF] offers to endusers more flexibility for creating geographic applications. Through a set of viewers, they can specify a graphical layout and visualize specific data inside them. Nevertheless, these systems do not allow to design end-user interactions.

SPARQLMotion10 is a visual scripting language and an engine for semantic data processing. Scripts implementing sophisticated data services and processing, such as queries, data transformations and mashups, can be quickly assembled with user-friendly graphical tools. Such a system is using Semantic Web technologies, like RDF [START_REF] Manola | RDF Primer[END_REF] and SPARQL [START_REF] Prud'hommeaux | SPARQL Query Language for RDF[END_REF]. However, as the previous systems, it does not provide solutions to specify end-user interactions and a general framework for constructing geographic application.

PhotoMap [START_REF] Viana | PhotoMap: From Location and Time to Context-Aware Photo Annotations[END_REF] offers graphical interfaces for navigation and query over some photo collections of users. Especially, with the map-based interface, itineraries followed by users are illustrated and photos with information are attached to specific places. Hence, end-users are able to retrieve where some photos have been taken either by clicking on specific places or by selecting a group of photos. This environment is another use-case for illustrating Web-based geographic applications. Nevertheless, this environment has been hardcoded by an expert and no design framework has been proposed by the authors.

In the next section, we propose a new framework for designing Web-based geographic applications. It should be considered to overcome the limits of the tools presented above.

A FRAMEWORK FOR DESIGNING WEB-BASED GEOGRAPHIC APPLICATIONS

To design a Web-based geographic application, we propose the framework depicted in Figure 2. This framework is composed of three complementary tasks:

1. Identifying the desired data that have to be manipulated by the geographic application. The data could refer to multimedia contents11 (e.g., the text written in French in Figure 1), some extracted information (e.g., in Figure 1, the list of towns automatically identified from the text) and some computed information (e.g., in Figure 1, the lists of "Départements" and "Préfectures" which correspond to the list of towns).

2. Specifying the graphical layout of the geographic application. This layout may be composed of several viewers, such as textual viewers, list viewers or map viewers. Thanks to the data that have been defined during the previous step, viewers might display some data sets. For instance, if some data about towns are determined, these towns will be displayed on a specific map viewer.

3. Defining potential end-user interactions on the data that are contained inside viewers. For instance, if a user clicks on a specific town listed in Figure 1, this town will be highlighted in the text written in French and the map will be focused on this town. As illustrated in Figure 2, even if we have ordered the three tasks, it is possible to go backward and forward during the design process. Actually, at any time the application designer may add, modify and/or remove some data, some viewers or some interactions. Furthermore, it is also possible to design a geographic application without interactions.

When all tasks have been completed, the Web-based geographic application can be generated and executed. Obviously, it is possible to update the generated application by repeating our proposed framework.

According to the framework presented in this section, for each task illustrated in Figure 2, we have specified a model and we have encoded it using RDF [START_REF] Manola | RDF Primer[END_REF]. Details and examples related to the use-case described in Section 2.1 are presented in the next section.

A UNIFIED MODEL FOR DESCRIBING AND COMPUTING WEB-BASED GEO-GRAPHIC APPLICATIONS

According to our framework proposed in Figure 2, we have defined a unified model for describing Web-based geographic applications. This model is structured into three facets:

• The data facet ( §5.1) describes the data that are manipulated by the application.

• The graphical interface facet ( §5.2) defines the organization of the application layout.

• The user interaction facet ( §5.3) specifies potential human-computer interactions.

Our unified model corresponds to the merge of all facet descriptions. We particularly show that these facets are complementary through the concept of Annotation. Furthermore, in order to encode, to aggregate, to share and to reuse each facet description, we have proposed an RDF/XML serialization [START_REF] Beckett | RDF/XML Syntax Specification (Revised)[END_REF]. Thanks to this standard, each facet can be described independently and may refer to other facet descriptions. Moreover, semantic queries can be executed on these descriptions. Each facet will be described and exemplified in the following subsections.

The data facet

As presented in Section 2, a Web-based geographic application manipulates data. In this paper, we consider two categories of data: Content and Annotation. The former refers to multimedia contents that might be composed of several segments, while the latter concerns the description of named entities (e.g., a person, a location).

As illustrated in Figure 3 (upper part), a content may hold several annotations referring to specific segments. Furthermore, an annotation may be connected with other annotations through different properties.

In this paper, we are considering exclusively textual contents inside a geographic application. Consequently, as presented in Figure 3 (lower part), a text may be segmented into paragraphs and/or tokens. Moreover, annotations will correspond to geographic information, e.g., places. [START_REF] Gaio | A global process to access documents' contents from a geographical point of view[END_REF] stated that geographic information is composed of three complementary features: temporal, spatial and thematic. We followed the same scheme, however the examples in the paper will focus only on the spatial feature.

Figure 4 presents an RDF/XML sample that corresponds to the data facet described in Figure 3. This description indicates that the file text.txt (i.e., the text written in French in Figure 1) contains an annotation (Annotation1) about an entity named Cannes. This geographic entity is located in the first paragraph of the text at the 9th token. Moreover, a geolocation and a geotype are associated to this entity.

Naturally, the description of Figure 4 could be enhanced with additional information. For instance, it may express that Nice, which is another spatial entity, is the "Préfecture" of Cannes. Since Nice does not appear in the file text.txt, this entity will not be related to a specific segment of the text. Furthermore, the description of Figure 4

The graphical interface facet

As illustrated in Figure 1, a Web-based geographic application contains a graphical user interface (GUI). This one may be composed of several viewers organized in the presentation layout. In this paper, we consider three categories of viewers: TextViewer, MapViewer and ListViewer. For instance, in Figure 1 there are a TextViewer which contains the text written in French, a MapViewer which includes Google Maps and three ListViewers enumerating some places.

Figure 5 presents our GUI facet. In this figure, each viewer may embed several annotations. More precisely, spatial entities may be highlighted in a text, displayed on a map or enumerated in a list. In fact, segments associated to annotations are used to locate entities, geolocations are useful for maps and geonames may be employed in lists. Figure 6 presents an RDF/XML sample that conforms to the GUI facet that we proposed in Figure 5. This description shows that the GUI contains a map viewer with a position specified by its top, left, height and width. This viewer embeds an annotation, i.e., Annotation1. Since the RDF merge operation has been clearly defined 12 , from the RDF/XML samples of Figure 4 and6, it is possible to display on the map the geolocation of Cannes.

< rdf : RDF > < rdf : Description rdf : about ="& ex ; gui . rdf " > < wm : title > French course </ wm : title > < wm : viewers > < rdf : Bag " > < rdf : li > < rdf : Description rdf : about ="& ex ; gui . rdf # Viewer1 " > < rdf : type rdf : resource ="& ex ; MapViewer "/ > < wm : width >400 </ wm : width > < wm : height >300 </ wm : height > < wm : left >300 </ wm : left > < wm : top >20 </ wm : top > < wm : hasData > < rdf : Seq " > < rdf : li rdf : resource ="& ex ; data . rdf # Annotation1 "/ > </ rdf : Seq > </ wm : hasData > </ rdf : Description > </ rdf : li > ... </ rdf : Bag > </ wm : viewers > </ rdf : Description > </ rdf : RDF >

The user interaction facet

A Web-based geographic application may be composed of several user interactions. For instance, in Figure 1 when a user selects a specific Town with a mouse click, this entity is highlighted in the text and the map zoom in on it. In [START_REF] Engels | Dynamic meta modeling: a graphical approach to the operational semantics of behavioral diagrams in UML[END_REF] and [START_REF] Stühmer | Lifting events in RDF from interactions with annotated web pages[END_REF], authors have formalized a vocabulary for defining user interactions, especially they are using the following concepts: Event and Action. An Event is generally characterized by two elements [START_REF] Stühmer | Lifting events in RDF from interactions with annotated web pages[END_REF]: the type of event (e.g., click, double-click, mouseover) and where the event occurred (e.g., on one entity in a viewer). An event may trigger several Actions, e.g., it might be a set of visual effects in the graphical user interface, such as highlight and zoom.

Figure 7 presents the user interaction facet which is inspired from [START_REF] Engels | Dynamic meta modeling: a graphical approach to the operational semantics of behavioral diagrams in UML[END_REF]. In this figure, events and actions occur on annotations. Consequently, thanks to the data and GUI facets proposed respectively in Figure 3 and 5, we could retrieve which viewer contains such annotation, its geotype, its geolocation and so on.

Figure 8 presents an RDF/XML sample that corresponds to the user interaction facet described in Figure 7.

In Figure 8, a potential end-user interaction I1 is defined. This interaction contains an event Evt1 and an action Ac-tion1. This description explains that if a user clicks on any annotations about Town in Viewer2, the Viewer1 will 12 http://www.w3.org/TR/rdf-mt/ zoom in on these annotations. Of course, several interactions could be defined and they may refer several times to the same annotations or the same viewers.

Note that the concept of Annotation has an important role, especially for designing a Web-based geographic application, because it allows to link the data, the GUI and the user interactions facets.

THE WINDMASH PROTOTYPE

We have implemented our proposal in a prototype named WINDMash 13 . This one allows designers to create and to automatically generate interactive Web-based applications handling geographic information. Figure 9 presents the architecture of our prototype. As illustrated in Figure 9, our prototype is composed of three modules (i.e., Data, Interface and User interaction) which correspond to our framework proposed in Figure 2. Each module manipulates some RDF/XML descriptions which comply with the model presented in Section 5. Naturally, the merge of all RDF/XML descriptions corresponds to an instance of our unified model which is used to compute and to generate the Web-based geographic application.

In this paper, we only focus on textual contents handled by our prototype. Hence, the data module may invoke several textual indexing services. For instance, the service 1 in Figure 9 could directly produce an RDF/XML output complying the data facet proposed in Section 5.1. Besides, other textual indexing services, like the services 2 and 3 , might be executed. For instance, we have used a service that automatically extracts places from a text and collects places' geolocations [START_REF] Sallaberry | Spatial Information Indexing Within Textual Documents Supported by a Dynamically Parameterized Web Service[END_REF]. However, the Web service implemented in [START_REF] Sallaberry | Spatial Information Indexing Within Textual Documents Supported by a Dynamically Parameterized Web Service[END_REF] produces XML outputs that do not correspond to the data facet proposed in Section 5.1. Consequently, we have 13 http://erozate.iutbayonne.univ-pau.fr/Nhan/ windmash3/ implemented an XSLT stylesheet in order to translate their XML outputs into some RDF/XML descriptions complying with our data facet.

In order to compute a Web-based geographic application from our unified model, we have implemented a code generator module written in PHP that produces XHTML Web pages containing JavaScript instructions. This module is using the RAP14 software package (RDF API for PHP) in order to analyze the RDF descriptions. Indeed, this package allows to query the descriptions with the SPARQL query language [START_REF] Prud'hommeaux | SPARQL Query Language for RDF[END_REF]. Moreover, we have used our WIND15 API (Web INteraction Design) which is fully described in [START_REF] Luong | an Interaction Lightweight Programming Model for Geographical Web Applications[END_REF]. This API enables to describe interactions between components (e.g., map, text) without having to know anything about the underlying Web Mapping Services (IGN Geoportail API, Google Maps API, OpenLayers API, etc.).

In the next section, several tools of our WINDMash prototype which allow designers to instantiate our unified model with its three facets, i.e., data, GUI and user interaction.

THE WINDMASH TOOLS FOR DESIGN-ING WEB-BASED GEOGRAPHIC APPLI-CATIONS

As illustrated in Figure 9, the WINDMash architecture is composed of three modules which concern, respectively, the data management, the GUI organization and the user interactions specification.

Consequently, three tools have been implemented in our WINDMash prototype for instantiating our unified model presented in Section 5. More precisely, these tools are: 1. A pipe editor which allows to combine different services and to filter the data manipulated by the application ( §7.1);

2. A graphical layout editor which is used to arrange, for instance, mapping components or multimedia contents ( §7.2);

3. A UML-like sequence diagram builder which allows to specify potential end-user interactions on the application ( §7.3).

In the remainder, each tool listed above is presented. Moreover, these tools are illustrated with screenshots based on the use-case scenario presented in Section 2.1.

Our pipe editor

In order to manage the data (i.e., contents and annotations) that should be manipulated by the Web-based geographic application, we have developed a pipes editor. This tool has been inspired from the Yahoo! Pipes editor 16 and enables designers to create a processing chain containing different services.

Figure 10 illustrates a possible processing chain which corresponds to the use-case presented in Section 2.1. Actually, from a given text (A), we would like to extract automatically some places (B). For that purpose, we invoke the Place-Extraction Web service which had been detailed in [START_REF] Sallaberry | Spatial Information Indexing Within Textual Documents Supported by a Dynamically Parameterized Web Service[END_REF]. Thanks to the identified places (B), we have defined two sets of annotations: towns (C) and "Départements" (D). Since the PlaceExtraction service classifies extracted entities, we have implemented some filters (i.e., Town and Department) in order to select the suitable entities. Finally, from the set of towns (C), we needed to get all corresponding "Préfectures" (E). Thanks to available geographical database (e.g., GeoNames, France IGN Database), the Prefecture service computes the appropriate list of "Préfectures" (E) from the list of towns (C). While constructing the pipes, it is possible to visualize at anytime the computed data by selecting with a double click the B, C, D and E items in Figure 10. Some services may also be customized according to specific needs, e.g., extract places that are situated in a given area. Furthermore, the given text may be updated for computing new datasets.

Each time a dataset is computed, such as the list of extracted places (B), it generates an RDF/XML description which corresponds to the data facet presented in Section 5.1. These descriptions are also accessible at the bottom left of the prototype in Figure 10.

We show in the next subsection, that these descriptions will be used in our graphical layout editor in order to display the data inside viewers.

Our graphical layout editor

Our graphical layout editor enables a designer to specify the graphical user interface of his/her Web-based geographic application. Indeed, the designer decides which type of viewer he/she wanted in his/her application (e.g., TextViewer, MapViewer, ListViewer) and how these viewers are organized inside the graphical layout.

Figure 11 illustrates how a designer may specify, with our tool, the graphical layout of the use-case presented in Figure 1. The menu on the left hand-side indicates the type of viewers that may be manipulated by the designer, the available dataset that have been computed with our pipes editor (Section 7.1) and the viewers that are currently used. In Figure 11, five viewers have been specified: a text viewer (1), a map viewer (2) and three list viewers (i.e., 3, 4 and 5).

Initially, when the designer drags and drops a viewer inside the graphical layout, this viewer is empty, except the map viewer which contains a map. If the designer wants to display some information inside viewers, from the menu, he/she has to drag the computed datasets and to drop them inside a specific viewer. For instance, if the designer wants to see inside the text viewer (1) the text that has been written in Figure 10, he/she has to drag from the menu the My Text element and to drop this element inside the My Text Viewer 1 viewer. Thereafter, if the designer wants to underline the places that have been extracted from this text, he/she has to drag the Places dataset from the menu and to drop it inside the My Text Viewer 1 viewer.

Similarly with the other types of viewers, if the designer wants to see the places on the map, he/she has to drag the Places dataset and to drop it inside the My Map Viewer 2 viewer. Finally, if the designer wants to see the lists of towns, "Départements" and "Préfectures" that have been defined in Figure 10, he/she has to drag the datasets and to drop them inside the list viewers, i.e., My List Viewer 3, My List Viewer 4 and My List Viewer 5.

Of course, it is possible to customize each viewer. For instance, the style of a text inside a text viewer may be modified, such as its font, its size, etc. Furthermore, the type of the lists may also be changed, e.g., with or without different kinds of bullets. Finally, different types of maps may be used, such as Google Maps, Yahoo! Maps, IGN Maps17 . . .

In the next subsection, we present how it is possible to specify potential end-user interactions using the layout components that have been designed in this section.

A "sequence diagram" builder for specifying end-user interactions

As required in the use-case scenario proposed in Section 2.1, when a user selects a specific place in the lists, the map should focus on this place and, if possible, the corresponding terms in the text should be highlighted. [START_REF] Hennicker | Modeling the user interface of web applications with UML[END_REF] proposed to design this kind of interaction through UML-like sequence diagrams.

Effectively, these diagrams are useful for designers because:

• they are based on few concepts to describe the interactions between a user and the system;

• they clearly distinguish events made by a user on the system (i.e., arrows from the user to the system) and the potential actions of the system to the user (i.e., arrows from the system to the user); • they can both describe the interactions between the user and the system, as well as the interaction between the components of the system.

Consequently, we have implemented a UML-like sequence diagram builder which is illustrated in Figure 12 18 . On the left hand-side of this figure, the menu is composed of the list of viewers that have been specified in Section 7.2 and the list of datasets that have been defined in Section 7.1.

The sequence diagram example illustrated in Figure 12 describes the following interaction: When a user selects, through a click (see the right arrow), a town contained in the viewer named My list Viewer 3, this town is highlighted (see the left arrow labeled with the term highlight) in the viewer named My Text Viewer 1. Moreover, the viewer My Map Viewer 2 also zooms in on this town (see the left arrow labeled with the term zoom).

When the designer has finished to build the sequence diagram, the WINDMash prototype handles a global RD-F/XML description which complies with the unified model presented in Section 5. Consequently, from this description and our code generator module, the designer could preview the Web-based geographic application by selecting the Preview button (see the buttons on top of Figure 12). If the generated application suits his/her needs, the designer may save the application and/or deploy it on a specific client. Otherwise, the designer may come back to the three WIND-Mash tools presented in this section (i.e., the figures 10, 11 18 Currently, this UML-like sequence diagram builder only allows to create lifelines and to specify messages between them via arrows. and 12) in order to add, to modify or to remove some application elements.

From the WINDMash tools presented in this section and, especially the examples illustrated in the figures 10, 11 and 12, the complete generated Web-based geographic application is available at http://erozate.iutbayonne.univ-pau. fr/Nhan/windmash3/demo/Frenchcourse/app.html. A video screencast of our WINDMash prototype is also available at http://erozate.iutbayonne.univ-pau.fr/Nhan/windmash3/ demo/Frenchcourse/video.html

CONCLUSION

In this paper, we have presented a framework dedicated to the design of Web-based geographic applications. This framework addresses three complementary tasks which concern the data manipulated by the application, the graphical layout and the user interactions. We have shown through our unified model for describing such kind of applications that annotations are central in the design process. Indeed, they can be used to describe entities, to display information inside viewers and to specify application behaviors. Furthermore, our proposed framework has been implemented in an online prototype named WINDMash. This prototype contains different tools that facilitate the instantiation of our unified model and automatically generates an executable Web-based geographic application.

Currently, our prototype only deals with textual contents. However, the data model presented in this paper is sufficiently generic to be extended in order to deal with multimedia contents, such as videos, audios and images. Furthermore, the geographic information manipulated by our WINDMash prototype can be imported from other repositories, for example from the LinkedGeoData 19 which exploits the spatial information collected by OpenStreetMap 20 . Hence, a future work would consist in developing a mediator between the imported LinkedGeoData datasets and our unified model. Moreover, we plan to extend our model in order to import non-geographic information, e.g., specific manual annotations.

Finally, we plan to export the generated Web-based geographic applications in XHTML+RDFa [START_REF] Adida | RDFa in XHTML: Syntax and Processing[END_REF] instead of XHTML. XHTML+RDFa export will enable WINDMash generated applications to take more advantage of the Semantic Web and, especially the Linking Open Data cloud [START_REF] Heath | Linked Data: Evolving the Web into a Global Data Space[END_REF].

ACKNOWLEDGMENT

Figure 1 :

 1 Figure 1: A Web-based geographic application for discovering French places.

Figure 2 :

 2 Figure 2: Our framework for designing Web-based geographic applications.

  may describe other spatial entities contained in the text, such as Biarritz or Lyon.

Figure 3 :Figure 4 :

 34 Figure 3: The data facet.

Figure 5 :

 5 Figure 5: The GUI facet.

Figure 6 :

 6 Figure 6: An RDF/XML sample of the GUI facet.

Figure 7 :

 7 Figure 7: The user interaction facet.

<Figure 8 :

 8 Figure 8: A user interaction encoded in RDF/XML.

Figure 9 :

 9 Figure 9: The WINDMash architecture.

Figure 10 :

 10 Figure 10: The WINDMash pipes editor.

Figure 11 :

 11 Figure 11: The WINDMash graphical layout editor.

Figure 12 :

 12 Figure 12: The WINDMash sequence diagram builder.

http://maps.google.com

http://openlayers.org

http://maps.yahoo.com

http://www.geonames.org

The English version of the text in Figure1is available at the following address: http://www.iutbayonne.univ-pau. fr/~slaborie/DocEng11/EnglishTranslation.txt

http://www.topquadrant.com/products/SPARQLMotion. html

In this paper, we only consider textual contents.

http://www4.wiwiss.fu-berlin.de/bizer/rdfapi/

http://erozate.iutbayonne.univ-pau.fr/Nhan/ windapi/wind.html

http://pipes.yahoo.com

http://www.geoportail.fr

This work has been supported by the French Aquitaine Region (project number 20071104037), the Département of Pyrénées-Atlantiques ("Pyrénées : Itinéraires Éducatifs" project) and the ANR MOANO project (http://moano.liuppa. univ-pau.fr).


MD Mogens Bernsdorf

Eva Balslev

Anne E Lykkesfeldt

Niels Kroman

Eva Harder

Hans Maase

Erik H Jakobsen

Dorthe Grabau

Bent Ejlertsen

Hans Von Der Maase

Value of post-operative reassessment of estrogen receptor α expression following neoadjuvant chemotherapy with or without gefitinib for estrogen receptor negative breast cancer

Keywords: breast cancer, neoadjuvant, estrogen receptor, phenotype changes, postoperative reassessment

Introduction

Expression of the estrogen receptor (ER) possesses a strong predictive value and constitutes a requisite for the efficacy of adjuvant endocrine therapy in breast cancer patients (1). Measurement of ER has for that reason become standard on all newly diagnosed invasive breast cancers (2;3) and accurate and standardized assessment of ER is therefore essential.

A negative association between ER expression and expression of receptors from the ErbB-family, like epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor II (HER2), has been demonstrated in a large number of clinical studies and overexpression of EGFR and HER2 are primarily observed in ER negative tumors (4;5). Both clinical and preclinical data demonstrate that progression of ER dependent breast cancer may occur via a shift from ER to EGFR/HER2 signalling (6-8), although the shift occurs with only modest upregulation of the ErbB receptor mediated signalling (8-10).

Absent ER expression is a complex multi-step and potentially reversible process and it has been suggested that absent ER expression, may be reverted by blocking of the ErbB system. Thus, several cell culture studies have shown that blocking EGFR and/or HER2 receptor signaling resulted in regain of endocrine sensitivity (10)(11)(12)(13)(14)(15)(16). Furthermore, in both clinical and preclinical studies, it has been demonstrated that treatment with trastuzumab and/or gefitinib restores ER expression in HER2 positive and ER negative breast cancer (16;17). In Munzone et al (17), three of ten patients changed ER negative phenotype to ER positive following treatment, indicating that re-evaluation of ER expression may be recommended in general.

Consequently, we sought to examine the effect of neoadjuvant treatment on ER expression in patients with early ER negative breast cancer and whether treatment with gefitinib, a specific EGFR tyrosine kinase inhibitor, would increase the possibility of endocrine responsiveness. The present study is an exploratory analysis but according to and defined by the study protocol. The study is based on collection of local pathology reports, followed by central immunohistochemistry (IHC) confirmation of tumors with ER phenotype change following definitive surgery. All patients received preoperative chemotherapy in the NICE trial and were randomized to gefitinib against placebo.

Patients and methods

Full details of the NICE study (clinicaltrials.gov identifier NCT00239343) have been described previously (18). In summary, NICE was a Nordic phase II, multicenter, two-armed double-blind randomized trial involving 12 centers in Denmark, Norway and Sweden, in which 181 patients were assigned to either four cycles of epirubicin (90 mg/m 2 ) and cyclophosphamide (600 mg/m 2 )(EC) plus 12 weeks of daily treatment with gefitinib (250 mg) or EC plus 12 weeks daily treatment with placebo.

Eligible patients were women with unilateral, primary operable, ER negative invasive breast cancer ≥ 2 cm.

Patients with inflammatory breast cancer, involvement of skin or muscle, involvement of supraclavicular lymph nodes or evidence of distant metastases were not eligible.

The primary clinical objective of the NICE trial was to compare the difference in pathologic complete response (pCR) rate in the two treatment arms and secondary objectives were complete and overall response. Tumor response was assessed by the response evaluation criteria in solid tumors (RECIST) criteria.

Written informed consent was obtained from all study participants prior to study entry and ethical committees with jurisdiction for the participating institutions approved the study protocol.

Patients who completed treatment in the NICE study and had available ER status, before and after neoadjuvant treatment, were eligible for this study.

Local pathology assessment

Local pathologists determined ER expression before random assignment. Immunohistochemistry was used in all participating institutions and ER was classified as negative for a proportion score of < 10% staining cells and ignoring intensity of staining.

Central pathology assessment

Formalin fixed paraffin-embedded tumor tissue was prospectively collected at baseline and following definite surgery (pre-and postoperative specimens, respectively). In all, complete specimens was obtained from 134 patients (74% of the randomized population). The Central DBCG Pathology Laboratory in Herlev, Denmark reviewed the material for residual tumor tissue and expression of tumor markers.

Assessment of ER and HER2

In tumors where local pathology reports indicated a change in ER phenotype, a central review of ER status was performed using IHC and scored with percentage of staining.

HER2 and EGFR copy number Fluorescence In Situ Hybridization (FISH) analysis was performed by use of DakoCytomation (HER2 pharm Dx and EGFR pharm Dx , Dako, Glostrup, Denmark). Tumors were scored as HER2 or EGFR positive/amplified when the gene to centromere ratio 2 and EGFR as deleted when the ratio was < 0.8.

Statistical methods

The percentage of ER staining cells were dichotomised as present (> 0%) or absent (0%), and as positive (≥ 10%) or negative (< 10%). Concordance denoted the proportion of tumors with the same classification by local and central assessment. Fisher's exact test was used to evaluate the difference in ER changes in the two groups.

Results

In all, 158/181 (87%) patients randomized into the NICE study completed treatment, of which 115 patients were eligible for this study; 59 patients in the gefitinib group and 56 patients in the placebo group. Reasons for excluding 43 patients were: a pCR was observed, leaving no tumor material for posttreatment ER evaluation (n=23); tumor was retrospectively identified as baseline ER positive (n=6); or no posttreatment ER evaluation was done locally (n=14). Baseline patient characteristics were well balanced in the two groups (table 1). 5.1% (3/59) of tumors in the gefitinib group and 3.5% (2/56) of tumors in the placebo group, with a total of 4.3% (5/115) and a difference of 1.51% (95% CI, -6.1 -9.1) changed from ER negative at baseline to ER positive after neoadjuvant treatment. All tumors with change in ER were revised and confirmed centrally.

Local and central ER evaluation and staining scores for tumors with ER phenotype change are listed in table 2. It is noteworthy, that in the tumors with ER reversion, between 15 to 90% of the tumor cells were ER

positive (fig 1).

Tumors with change in ER phenotype tended to be of ductal histology and HER2, EGFR and PgR normal/negative as this was randomly present in 4 tumors. Pathologic characteristics at baseline for the five tumors with change in ER are listed in table 3.

Other pathologic characteristics for tumors with a change in ER phenotype, such as HER2, EGFR and histology, were not different from the entire cohort (table 1 and2).

Of the 115 patients, 99 and 101 had both pre and post treatment HER2 and EGFR status performed, respectively. Changes in HER2 were observed in 3 tumors (3%, gefitinib, n=1; placebo, n=2) and in EGFR in 7 tumors (6.9%; gefitinib, n= 3; placebo, n=4). None of the five tumors with change in ER phenotype had any changes in HER2 or EGFR.

A partial response was observed in 3 tumors and stable disease in 2 tumors with change in ER phenotype (table 3). Because of the low numbers of tumors with change in ER status, no analysis of difference in response for tumors with and without ER phenotype change was done.

Discussion

This is the first report exploring ER-negative phenotype changes after a neoadjuvant gefitinib regime. All patients received EC with or without gefitinib and pathological tumor characteristics were collected at baseline and at surgery. Since central review revealed 1% of tumors to be a wrongly ER classified locally in the BIG 1-98 study (19), a central review of tumors with change in ER phenotype was conducted. The central review revealed no discrepancy with local ER classification (table2).

The primary goal of this study was to evaluate the effect of neoadjuvant treatment on ER expression and whether gefitinib, given neoadjuvant with EC, would have an increased impact on ER expression. Interestingly, 4.3% (5/115) of ER negative tumors changed ER phenotype, supporting that ER expression, is reversible, as also reported by others (6;7;12;17;20;21). The reversibility of ER expression challenges the clinical approach to patients with an ER resistent tumor, as patients with an initially ER negative tumor, that revert to ER positive, might benefit from endocrine therapy.

Together with our results, the significant shift in ER phenotype, from ER positive to negative, following neoadjuvant therapy observed in the trial by Taucher et al ( 21) and the variation in ER expression between primary and metastatic breast cancer reported by Arslan et al ( 22), further emphasizes the importance for renewed ER evaluation after neoadjuvant treatment and in the event of recurrence or metastases.

A higher turnover rate was anticipated in the gefitinib group, due to the inverse relation between expression of ER and EGFR/HER2 and due to the reversal of ER expression by suppression of EGFR/HER2 signaling, but no difference between the two treatment groups was observed. These results indicate that the inhibition of EGFR with gefitinib, may not be the only mechanism which restores ER expression and suggests that EGFR, may not alone drive cell growth in ER negative breast cancer. This is further supported by a recent review, describing different mechanisms of reversing ER expression and sensitization to endocrine therapy in ER negative breast cancer (15). In four of five tumors, ER reversion occurred in tumors with normal EGFR and HER2, and no conclusion on a change from ErbB to ER signalling can be made.

It remains unknown, whether the clinical activity of gefitinib can be equated to gefitinibs ability to revert ER expression, if such an ability excist. However, the clinical effect of gefitinib is neither depended on EGFR expression nor inhibition of EGFR phosphorylation (23;24) as well as gefitinib does not affect the expression of EGFR ( 23). In our study the majority of patients included were EGFR normal (table 1) and whether selecting patients based on EGFR amplification would have resulted in more tumors converting ER phenotype remains unknown, but no tumor that changed ER phenotype was EGFR amplified in the present study.

In the study by Baselga et al ( 23), the complete inhibition of EGFR phosphorylation, which did not translate into a clinical benefit, was achieved with gefitinib 500 mg/daily as monotherapy for treatment of advanced breast cancer. In the NICE study, a dose of gefitinib 250 mg/daily was chosen since no clinical benefit was observed in a comparison of gefitinib 500 mg/dailly to 250 mg/daily (25;26). Whether a dose of gefitinib 500 mg/daily could have increased the rate of ER reversions remain unknown.

The exact mechanism underlying the change in ER phenotype is unknown. As no discrepancy was observed between local and central review, the changes are likely due to a biological phenomenon, rather than inconsistent measurements. The intratumoral heterogeneity in breast cancer, may result in genetic subclones with different sensitivity to anti-neoplastic treatment (27). This could be a possible explanation for a change in ER phenotype, where, under the pressure of chemotherapy, a possible shift (clonal selection) from an ER negative dominant clone to an ER positive dominant clone could have occurred.

However, in the gefitinib treated group, two of the three tumors displayed a posttreatment ER level of 90%.

In one case, this was associated with stable disease, strongly supporting that gefitinib treatment may be responsible for the ER reversion. In the two patients with a partial response, selective survival and growth of a minor undetected population of ER positive cells cannot be excluded, although a posttreatment ER level of 90% indicate that reversion may have occurred.

Regardless of the underlying mechanism of restoring ER expression, in order to translate into a clinical benefit, the re-expressed ER must be functional in order to respond to endocrine therapy. In the study by Monzone et al (17), two of three patients with advanced disease, who reverted to ER positivity, were treated with endocrine therapy and one of these patients was without progression at three years. Whether or not reexpression of ER after neoadjuvant treatment indicate benefit from endocrine therapy remains unknown and requires further investigations.

Conclusion

Our data show that ER expression is reversible in a minor but significant fraction of ER negative tumors, emphasizing the importance for reliable and correct pathologic evaluation, in order to provide best treatment possible. Eventhough, gefitinib did not have an impact on tumors reverting ER phenotype, a possible role of gefitinib in the change of ER expression cannot be excluded.

The reversion of ER from negative to positive might indicate benefit from endocrine treatment in patients otherwise exempt from this option. Therefore, a reassessment of ER status and whenever possible, also markers associated with functional ER e.g. the progesterone receptor, should be performed following neoadjuvant treatment.

Reference List

(1) Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15year survival: an overview of the randomised trials. Lancet 2005 May 14;365(9472):1687-717.

(2) Goldhirsch A, Ingle JN, Gelber RD, Coates AS, Thurlimann B, Senn HJ.

Fig 1

 1 Fig 1 Immunohistochemical estrogen receptor staining in pre-and posttreatment tumor samples. (a) Baseline tumor sample from tumor 2 with 0% ER staining. (b) 40% ER staining in posttreatment sample from tumor 4 recieving EC + placebo treatment. Average ER staining throughout the sample was 20%. (c) 90% ER staining in

Table 1 .

 1 Pretreatment patient and tumor characteristics.

	Thresholds for

(6) Sainsbury JR, Farndon JR, Sherbet GV, Harris AL. Epidermal-growth-factor receptors and oestrogen receptors in human breast cancer. Lancet 1985 Feb 16;1(8425):364-6. (7) Sonne-Hansen K, Norrie IC, Emdal KB, Benjaminsen RV, Frogne T, Christiansen IJ, et al. (12) Xia W, Bacus S, Hegde P, Husain I, Strum J, Liu L, et al. A model of acquired autoresistance to a potent ErbB2 tyrosine kinase inhibitor and a therapeutic strategy to

Table 2 .

 2 Results of local and central ER evaluation.

			Local 1	Central 2
		Pretreatment	Posttreatment	Pretreatment	Posttreatment
	Gefitinib
	Tumor 1	negative	positive (25%)	0%	90%
	Tumor 2	negative	positive (30%)	0%	15%
	Tumor 3	negative	positive (NA)	0%	90%
	Placebo
	Tumor 4	negative	positive (100%)	0%	20%
	Tumor 5	negative	positive (80%)	0%	70%
	ER: Estrogen receptor. NA: Not available.
	1 Dichotomized data from local evaluation with 10% cut-off.

2

Central evaluation with percent of ER staining.

Table 3 .

 3 Baseline pathologic characteristics and response to NAC for ER changing tumors.

		HER2	PgR	Grade	Tumor	Histology	EGFR	Response
					size (mm)			(tumor reduction %)
	Gefitinib
	Tumor 1	normal	negative	2	21	ductal	normal	SD (29%)
	Tumor 2	normal	negative	2	21	ductal	normal	PR (52%)
	Tumor 3	normal	negative	2	70	lobular	normal	PR (57%)
	Placebo
	Tumor 4	amplified negative	3	40	ductal	deleted	SD (0%)
	Tumor 5	normal	positive	3	26	ductal	normal	PR (42%)

The NICE trial and this study was sponsered by Astra Zeneca. We would like to thank the following centers


Dr Selvan Govender
email: selvan.govender@daad-alumni.de

Biofilm productivity and concomitant cell autolysis in a membrane bioreactor

Keywords: Gradostat, manganese peroxidase, membrane bioreactor, nutrient manipulation, Phanerochaete chrysoporium, white rot fungi

Phanerochaete chrysoporium morphology and manganese peroxidase (MnP) productivity was characterised in a scalable, modularised 1145 cm 3 membrane gradostat reactor in response to switching between an enhanced production medium and a nutrient limited feed (50% C and N reduction).

Irrespective of the feed composition used nutrients permeating from the lumen of the ultrafiltration membrane matrix established nutrient gradients across the immobilised biofilm with distinct primary, stationary and decline growth phases observed. Severe nutrient C and N limitation did not change the cyclic nature of enzyme production (MnP max = 189.5 U l -1 ) but did reduce the overall bioreactor efficiency from 32 U l -1 d -1 to 22 U l -1 d -1 . Stress induced secondary metabolism resulted in concomitant cell autolysis causing biomass loss and increased operational flux after 20 days in the 33 day bioreactor operation cycle.

Introduction

Fungi produce a wide range of secondary metabolites which are of medical, agricultural and industrial relevance. Most fungi display filamentous growth. Their biosynthetic genes are clustered and conserved between organisms. This, together with the attendant physiological costs, suggests that these complex compounds could only be produced to benefit the organism even though many reports do not show any link to their survival [START_REF] Calvo | Relationship between secondary metabolism and fungal development[END_REF]. White rot fungi (WRF), such as the aerobic, shear sensitive basidiomycete Phanerochaete chrysosporium produce extracellular enzymes for lignin degradation based on an environmentally-induced physiological response to severe nutrient limitation (Mikiashvilli et al. 2005). These industrially and environmentally important ligninase enzymes (manganese peroxidases, lignin peroxidases and laccases) also degrade persistent xenobiotic chemicals. This has initiated significant research effort towards large scale enzyme production and recovery and or bioremediation. [START_REF] Cabuk | Biodegradation of cyanide by a white rot fungus Trametes versicolor[END_REF].

Commercial production of useful primary and secondary metabolites is often achieved by controlling the expression of genes involved in secondary metabolite production via manipulation of the growth nutrients or the specific environmental conditions. Nutrient limitation results in a lower growth rate and, in the case of WRF, this is a precursor to ligninase production where, in nature, it would be followed by further growth cycles once delignification makes more nutrients available. Based on the requirements for WRF, bioreactor design and consideration of the native state of the fungus, the gradostat bioreactor concept evolved as a viable option for continuous production of idiophasic enzymes and for bioremediation (Leukes et al. 1996;Ntwampe et al. 2008). The term gradostat applies because bi-directional contact occurs between the primary and secondary growth phases of the biomass, albeit that gas and liquid flow is uni-directional. The immobilised fungus mimicked the native growth and development of the basidiomycete in response to applied environmental stress, while the polymeric membrane provided a support matrix for physical retention of spores, space for subsequent germination and mechanical strength to accommodate the developing mycelium. Previous studies have followed the continuous fed bioreactor protocol of continuously supplying cells with substrate at growth limiting concentrations which maintain the cells in the exponential phase.

The membrane gradostat reactor (MGR) concept focuses on C, N and S limitation for high levels of MnP and lignin peroxidase (LiP) production, but the physiological and morhological effects are yet to be described and understood. An inherent feature of enzyme production in a gradostat reactor is the cyclic nature of extracellular MnP production. MGRs rely on biofilm development and the formation of nutrient gradients for idiophasic enzyme production. However excessive biofilm development which often sloughs off the membrane and blocks the reactor ports, coupled with cyclic enzyme production have hampered reactor efficiency and subsequently generated interest in nutrient manipulation and controlling the biofilm growth rate and morphology to improve enzyme productivity. Besides mimicking the native state of the WRF and complementing the biotransformation process, the MGR, coupled with a 'quality by design' approach to scale-up, also offers more operational freedom in addressing traditional bioreactor scale-up challenges such as maintaining homogeneity and adapting to gradients at a large scale. As such, the MGR has evolved via various bioreactor configurations from transverse-flow modules (Leukes, 1996) to capillary axial flow-modules [START_REF] Govender | Towards an optimised spore immobilisation strategy using P. chrysosporium, reverse filtration and UF membranes[END_REF]Ntwampe et al 2008).

This study investigates the nutrient supply regime (C and N ratios) compared with a recently developed production medium [START_REF] Govender | Nutrient manipulation as a basis for enzyme production in a gradostat bioreactor[END_REF]) and characterises the morphological and physiological response of immobilised P. chrysosporium, subjected to nutrient limitation in an axial-flow, 1145 cm 3 multi-capillary MGR. Manipulation of C, N, S and trace element nutrients for over production of MnP were investigated in relation to the spatial and temporal distribution of mycelial biomass within the membrane matrix. Oxygenation was provided in excess using both sterile air and 100% O 2 , while shear stress was kept to a minimum. The impacts on fungal growth and enzyme production are described using enzyme assays and ultrastructural analysis.

Materials and Methods

Microorganism, media and inoculum preparation

Phanerochaete chrysosporium ME446, maintained on 2% (w/v) malt extract/agar plates, was incubated at 39 C for 10 d, prior to spore harvesting while the resulting conidial spores were prepared by suspending the spores in sterile, deionised water followed by filtration through sterile glass wool to free it of mycelia. Spore concentration was determined by OD 650 . A production medium, supplemented with phospholipids and surfactants and characterised in a recent study [START_REF] Govender | Nutrient manipulation as a basis for enzyme production in a gradostat bioreactor[END_REF]) was used as a control in this study.

Membrane Preparation

Externally unskinned, anisotrophic polysulfone (PSU) capillary membranes were manufactured using the wet phase inversion process and the unique substructure morphology was designed to have low resistance to liquid transport and a large external surface area (Leukes et al. 1996). The well-defined ultrafiltration (UF) internal skin layers consisting entirely of closely packed and narrow-bore microvoids that extended from just below the internal skin layer of the membrane to the periphery, providing physical support for the growing fungal biofilm.

Gradostat bioreactor operation

Spores were immobilised in the membrane microvoid cavities by reverse filtration and was optimised [START_REF] Govender | Towards an optimised spore immobilisation strategy using P. chrysosporium, reverse filtration and UF membranes[END_REF]) to improve consistency in biofilm development. Membrane gradostat reactors (MGR), using externally unskinned UF capillary membranes as immobilisation matrices, were operated under the conditions described in Table 1. The MGR was dependent on aeration to generate a biofilm and to stimulate ligninase production. The air-flow regime consisted of pumping in sterile air via a peristaltic pump and supplementing with intermittent flushing of the reactor with 100% O 2 for 10 min every 12 h. The complete process flow diagram for continuous operation is illustrated in Fig. 2. The operational conditions selected for continuous operation were based on previous work with this gradostat system (Ntwampe et al. 2008;[START_REF] Govender | A scalable membrane gradostat reactor for enzyme production[END_REF] and were kept constant for the duration of continuous operation with changes made dependent on the nutrient switching scheme. MGR efficiency in this study was based on the average MnP production per day of operation.

Nutrient gradient manipulation

According to the Monod model, in the steady state the specific growth rate will equal the dilution rate.

Thus the specific growth rate may be controlled by altering the dilution rate or adjusting the rate of supply of fresh feed. This is normally used to investigate growth at submaximal rates [START_REF] Gow | The growing fungus[END_REF]. The advantage of controlling the specific growth rate in a MGR is that extended operation in the stationary phase could become possible. However, this is extremely difficult to achieve with the multicapillary MGR since it is a novel design and prior knowledge of optimal dilution rates would be required from the outset. The only 'known' criterion pertinent to biofilm growth available at the outset is the limiting substrate (C and N) concentration in the growth medium. Once MnP production was detected, it was assumed that a significant portion of the biofilm was in the stationary phase. It is hypothesised that by reducing the influx concentration of nutrient C and N it would be possible to maintain steady state production of MnP by controlling the growth phase (i.e. the stationary phase) of the immobilised biomass. The said nutrient stress was created by reducing the feed concentration of glucose [10% (w/v) to 5% (w/v)] and total nitrogen [1% (w/v) to 0.5% (w/v)] by an empirical value of 50% whilst leaving the dilution rate constant.

Enzyme activity and productivity

Mn (II) dependent peroxidase activity was determined by monitoring the oxidation of guiaicol spectroscopically. One unit of activity is defined as the initial increase in absorbance at 465 nm of 1 per min, and activities are reported in U l -1 while productivity was defined as the amount of product formed per unit reactor volume [START_REF] Govender | Towards an optimised spore immobilisation strategy using P. chrysosporium, reverse filtration and UF membranes[END_REF]. A glucose oxidase assay kit (Sigma) was used to determine residual glucose concentration, typically analysed for in the permeate flask. Residual nitrogen was measured at 400nm using Nessler's Reagent (Sigma). Unless otherwise stated all chemicals were purchased from Sigma, RSA.

Electron microscopy

For scanning electron microscopy (SEM), PSU membrane sections were fixed for 2 h in 5% (v/v) glutaraldehyde with 0.1 M phosphate buffer at 4 C and pH 7.5, after which the sections were dried in a water:ethanol gradient. Cross-sections of the membranes were prepared by freeze fracturing with liquid N 2 and examined using a Hitachi S-570 SEM (Japan). Fungal tissue to be analysed by transmission electron microscopy (TEM) were fixed in fresh glutaraldehyde, washed in two changes of 0.05M cacodylate buffer and post-fixed using OsO 4 . After at least two washes of 30 min each, the material was dehydrated in a graded alcohol series and then embedded in Spurrs resin, followed by sectioning, post fixation with uranyl acetate and Pb citrate and viewed with a JEOL 1210 TEM.

Results and discussion

Biomass morphology and productivity

Contrary to the practice of using separate germination and production media (Venkatadri and Irvine, 1993), this study used an enhanced production medium [START_REF] Govender | Nutrient manipulation as a basis for enzyme production in a gradostat bioreactor[END_REF]). The pump flow rate was kept constant so as to minimise the influence of hydrodynamic parameters on enzyme production and biofilm development. Different growth phases, i.e. primary, stationary and decline phases, were observed radially across the biofilm (Fig 3) after sectioning along the length of the capillary membrane. This observation was consistent with both the control and the nutrient limited feed and visual assessment of the membrane surface showed an initial dense, white biofilm which turned yellow/brown at the onset of ligninase enzyme production (Fig. 4). The colour change was attributed to the presence of excess trace elements in the feed solution. These observations were also characterised by both colour changes in the mycelium (Fig. 4b) and SEM observations of the fungal morphology indicating significant biofilm development which was further supported by trends in the permeation rate decrease in Fig. 4a, limiting the flux through the membrane.

Studies have shown that the morphology of the fungal mycelia can be related to their physiological state (Rittman et al. 1992). This morphological diversity; if regulated by controlling the operational regime could be an important parameter for optimal production of enzymes or other secondary metabolites in an MGR by selecting for a morphological growth phase that compliments the physiological secondary metabolite phase as correlated by other reports (Leukes et al. 1996;Rittman et al. 1992). From Fig 3, morphological differentiation occurs laterally as a function of the state of growth, which, in turn, is a function of the nutrient supply in response to spatial gradients radially across the biofilm.

SEM analysis shows different growth phases [primary (extension and production zone A), stationary (sporulation zone B) and decline (ageing zone C)] along the cross-sectional length of a single capillary membrane. The primary growth phase (zone A) was characterised by germinating spores and surface colonization with exploratory hyphae. Zone B consisted mostly of highly branched and fragmented hyphae, typical of the stationary phase. A high level of fragmentation is thought to occur due to increased vacuolation rather than just mechanical shear stress. Zone C (decline phase) shows a combination of copious conidiating mycelium and sloughing off of dead biomass followed by biofilm proliferation in the nutrient rich regions of the biofilm with the older biomass being pushed outwards towards the O 2 richer zone and finally necrotic spaces that were later observed indicating a loss of biofilm and concomitant hyphal autolysis

Nutrient gradient manipulation

The concentrations of glucose and total N in the feed system were reduced by 50 % (w/v) after 9 days as this was typically when one cycle of continuous production of MnP stopped and a few days before the onset of a new cycle. The subsequent cyclic MnP production could last considerably longer, due to the temporal and spatial domains generated radially within the immobilised fungal biomass by manipulating the concentration of glucose and or nitrogen in the inflowing medium which would then limit exponential growth as a consequence of continuous availability of the fresh medium. The advantage of controlling the specific growth rate in a MGR is that extended operation in the stationary phase for the idiophasic production of ligninases would become possible. However, this is extremely difficult to achieve with the multi-capillary MGR due to the as yet un-characterised design while a prior knowledge of optimal dilution rates would be required from the outset. The permeate aminonitrogen concentration decreased sharply during the initial germination and growth phase, but this rate of decline became less pronounced when culture age increased (Fig 5b). Glucose concentrations (Fig 5a) also declined rapidly as biofilm growth proliferated in zone A. This coincided with a rapid decrease in the permeation rate (Fig 4a). When fresh medium was introduced, growth proceeded as the feed permeated the immobilised biofilm, until the concentration of one particular nutrient component was reduced to a level at which it limited the specific growth rate [START_REF] Gow | The growing fungus[END_REF]. Increases in biomass lead to a decrease in substrate concentration and specific growth rate until balance is reached between biomass production and removal of nutrient through washout.

The reduction of the nutrient levels, however, resulted in another series of cyclic MnP production with peak MnP production of 190 U l -1 d -1 after 9 days compared to 250 U l -1 d -1 with the control feed (PM) after 7 days of continuous operation (Fig. 5c). This was exacerbated by the observation of 'bare' patches or necrotic zones on the biofilm. Here the level of mycelial 'removal' via hyphal autolysis and sloughing off was so intense that the native surface of the capillary membrane could be seen. MnP production was almost undetectable after 21 days and the increase in the permeation rate indicated a loss of immobilised biofilm (Fig. 4). The greater efficiency shown using the control or production medium (32 U l -1 d -1 compared to 22.5 U l -1 d -1 ) suggested that a thick biofilm and high flux range increased efficiency, most likely due to an adequate supply of nutrients and a residence time within the adsorptive capacity of the MGR. Addition of fresh nutrients (during the feed switch to nutrient limitation) led to a comparable second cycle of MnP production (Fig. 5c) in spite of the concomitant cell ageing (Fig. 6) which could also be due to the removal of additional metabolic wastes expected to back-diffuse into the lumen via re-circulation. The peak MnP production of 250 U l -1 d -1 with the 1145 cm 3 multi-capillary MGR is also a 45 x volumetric and a 12 x geometric scale-up of a 25 cm 3 single capillary MGR described by [START_REF] Govender | Nutrient manipulation as a basis for enzyme production in a gradostat bioreactor[END_REF] resulting in an 8.3 x increase in MnP productivity in contrast to a 6 x increase with the nutrient-manipulated, substrate-limiting feed system. This suggests that scale effects in the MGR were not as significant as manipulation of the nutrient gradient.

Fungal physiological response TEM analysis of sloughed off biofilm in the permeate line showed cell autolysis after 16 days of operation (Fig. 6a) while biofilm scraped of the capillary at the end of the 33 day cycle contained hyphae that had undergone severe autolysis. Glycogen deposits (another sign of cell aging) was also observed (Fig. 6). Hyphal autolysis was directly linked to loss of secondary metabolite production potential of Penicillium chrysogenum (Pusztahelyi et al. 1997). This could account for the absence of MnP activity during the latter stages of the run (days 25 -33) when hyphal autolysis was most severe.

Despite the restoration of the initial nutrient concentrations after 16 days hyphal degradation persisted until the end of the 33 day study period. An interesting observation was the formation of dolipore septa (Fig. 6b). Dolipore septa are elaborate septal complexes found in most fungi but their role in fungal physiology is still not very clear [START_REF] Gow | The growing fungus[END_REF]. Doplipore septa form in situations of extreme cell trauma or stress or even during ageing [START_REF] Alymore | Septal sealing in the basidiomycete Coriolus versicolor[END_REF]. The presence of paranthesomes (Fig 6b) supports this phenomenon because they are generally considered to provide a protective or selective function by regulating the cytoplasm immediately adjacent to the septal swellings and pores [START_REF] Alymore | Septal sealing in the basidiomycete Coriolus versicolor[END_REF][START_REF] Gow | The growing fungus[END_REF]. Under the severe nutrient limitation conditions imposed by extended operation of the membrane gradostat reactor, it seemed plausible that the formation of dolipore septa played a role in structural support, hyphal tip growth and maintaining rigidity of the hyphae.

Fig 6c depicts a SEM profile of a region of the capillary membrane where the biofilm was undergoing cell autolysis and formation of septa. The thick layer on the sloughed off biofilm surface was confirmed using IR spectroscopy as an extracellular polysaccharide sheath that built up around aging hyphae. Acid hydrolysis of these polysaccharides revealed predominantly glucose units linked by βbonds evident from the 890 cm -1 band in the IR spectra. Typically in nature, ligninolytic fungi use sheath polysaccharides to degrade lignin in wood by facilitating the joint action of enzymes providing a source of reducing power, and assisting in the diffusion of redox intermediates (Gutiérrez et al. 1995).

Conclusions

Switching from an enhanced production medium to an extremely C and N deficient feed early in the MGR operation cycle, did not change the general growth profile of the immobilised biofilm, with all three growth phases clearly documented. However, the nutrient limitation studies did not lead to an increase in MnP productivity (30% loss of bioreactor efficiency) or a deviation from cyclic MnP production. Moreover, the impact of nutrient starvation was extremely profound on the biofilm resulting in severe hyphal autolysis and visible loss of biofilm with predominance of the decline phase of growth. The formation of dolipore septa, the extensive polysaccharide build up on the capillary membranes and degenerating hyphae indicate the extent of stress and ageing of mycelia as a result of severe nutrient limitation. These results are useful for future work involving monitoring of glucose influx regime and the morphological growth state described could serve as an ultrastructural 'marker' for cells undergoing severe nutrient limitation and autolysis. Furthermore, this study shows that scale effects due to the transition from a 25 cm 3 to 1145 cm 3 MGR were not as significant as manipulation of the nutrient feed regime, which has a concomitant effect on biofilm morphology and physiology. A better understanding of the interplay between the growth limiting substrates and biofilm dynamics, coupled with knowledge of an optimal dilution rate could improve efficiency and induce the stationary phase to predominate across the biofilm for long term steady state enzyme production using the MGR.

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Fig. 6

 6 Fig. 1Illustration of the membrane gradostat concept with superimposed batch culture growth curves. (I) primary growth phase, (II) stationary phase, (III) decline phase. IB is the immobilised biomass, S is the ultrafiltration membrane skin layer, L is the lumen of the membrane capillary from which the growth medium is supplied, X is the radial distance from the lumen in the direction of nutrient flow and C is the concentration of the growth-limiting substrate

Table 1

 1 Dimensions and operating conditions of the gradostat bioreactor (See Fig.2)

	Parameter Parameter	Details Details
	Membrane surface area Membrane surface area	0.01357 m 2 0.01357 m 2
	Extra-capillary reactor volume Extra-capillary reactor volume	0.001145 m 3 0.001145 m 3
	Growth medium supply regime Constant flux Growth medium supply regime Constant flux
	Permeate flow-rate Permeate flow-rate	0.0100 to 0.017 l.d -1 0.0100 to 0.017 l.d -1
	Oxygen supply rate Oxygen supply rate	1 vol. (vol ECS) -1 .min -1 1 vol. (vol ECS) -1 .min -1
	Air flow regime Air flow regime	Transverse to capillaries Transverse to capillaries
	Temperature control Temperature control	Lagged shell (37ºC) Lagged shell (37ºC)

Ackowledgements

This study was supported by the National Research Foundation and the Deutscher Akademischer Austausch Dienst.