Christian Cote
email: chrsitian.cote@univ-lyon3.fr

ACTIVITY ANALYSIS, SEMANTICS AND METADATA BUILDING

Keywords: metadata, activity analysis, semantics

We propose a methodology for building consistent metadata for the description of scientific working documents. Our methodology is supported by distributed cognitive theoretical principles and implemented through a combination of anthropological, semantic, and linguistic approaches. Our analysis is applied to a particular pharmacist activity, namely the adaptation of posology. Issues focus on the representation of situations that describe individuals, tools and artifacts. Regular relations between these situations characterize types that allow the description of conceptual contents associated to these empirical objects. Because these situations and types are expressed by a set of metadata and then associated to current metadata, our proposal extends the nature of entities described by metadata to useful internal activity artifacts.

Introduction.

We propose a new set of metadata that precisely describes some contents of documents which were created and used in the course professional activities. Big data and e-science entail the description of heterogeneous documents in research infrastructures [START_REF] Rousidis | Metadata for Big Data: A preliminary investigation of metadata quality issues in research data repositories[END_REF] . If we extend the purpose to the frontiers of e-science, for example professional setting in which electronic health record in medical care is used, questions of data description, discovery and use become crucial. Metadata are a guarantee for data discovery and reuse in a big data context [START_REF] Qin | How portable are the metadata standards for scientific data? a proposal for a metadata infrastructure[END_REF] . However, we need to specify the relevant set that precisely and universally describes these data.

We enhance the precision of the content representation through the characterization of universal data features that connects heterogeneous documents between them. The creation of a new metadata set for the description of specific sorts of documents is unnecessary [START_REF] Qin | Functional and architectural requirements for metadata: supporting discovery and management of scientific data[END_REF] . Therefore, we do not offer an alternative to existing metadata sets but, rather, we propose some modules that will specify the intrinsic contents of some documents which cannot easily be described by usual metadata.

Any metadata set is based on a presupposed document function in a library or an infrastructure activity. This role entails the selection of some features: for example, Dublin Core 4 and the other metadata sets like MODS a are based on bibliographic description for librarian activity. By contrast, a http://www.loc.gov/bibframe/pdf/marcld-report-11-21-2012.pdf. http://www.loc.gov/standards/mads/rdf/. http://www.loc.gov/standards/mdc/index.html. namely bearer and content, for any information artifact [START_REF] Smith | The Information Artifact Ontology (tutorial), Semantic Technology for Intelligence, Defense, and Security[END_REF] . The bearer description emphasizes the characterization of the document function in context. However, the pragmatic dimension of the bearer can be more precisely expressed by considering meaning and reference. The content dimension is characterized by the notion of "aboutness". We enhance this dimension by the characterization of the individuals the document refers and, more precisely, by showing how the documents work on these individuals. [START_REF] Borgo | Technical Artifact: An Integrated Perspective[END_REF] . By contrast with the B. Smith & al. 11 , for us, meaning refers not only to objects, but also to the different steps of the information processing from individuals in the world to symbolic information.

Methodology.

We propose an innovative methodology based on the complementarity between an analysis that describes these documents according to their context, their role, and their object and another that specifies the linguistic entities and discourse structures that realize these functions. The linguistic dimension has a special interest for the automatic extraction of metadata values and the creation of collections based on individual types, events, etc. [START_REF] Stvilia | Research project tasks, data, and perceptions of data quality in a condensed matter physics community[END_REF] The cognitive anthropology framework allows a characterization of the context (creation and use), constraints on meaning and document functional structure. It represents some relations between documents, actor reasoning, and material environment. We do not propose a general and procedural model of the activity g but a representation of information building, conveyance, and interpretation.

However, the conceptual framework of cognitive anthropology has not been designed for information conveyance and interpretation analysis: it cannot represent the linguistic content of the artifacts. Its range is limited to the cognitive activity associated to material objects and spatial configurations. On the other hand, semantic situation theory [START_REF] Barwise | Situation and attitudes[END_REF] preserves the distributed characterization of the cognition and explains the expressions and discourses meaning in context. Our study combines the two perspectives in order to promote a stable definition of the information artifacts, tools, and individuals in the world. Analysis issues will be externalized to form metadata.

Creating this set requires a complete setting composed of a small set of qualitative data. This setting is an exemplar type of any professional activity which implies documents. In this analysis, each proposal of document description refers to an identified phenomenon in the world and in documents. The adaptation of posology for antibiotherapy in hospital pharmacy presents a double advantage:

1. The whole process of the activity matching is explicit and 2. It requires documents that can be considered both for practice and research.

(b) Information content entity (ICE) -an entity which is about something and which can potentially exist in multiple (for example digital or printed) copies -a jpg file or a pdf file g This sort of model is too abstract and general and cannot represent the daily arrangement and circumstantial variations.

Distributed and situated cognitive anthropology: information artifacts as foundations for activity.

As presented by A. Fjuk and S. Ludvigsen, "activity theory constitutes a rich framework for studying different forms of practices as developmental processes, with individual and social issues interlinked at the same time. In other words, activity theory affords analyses of social phenomena on different levels; activity -at the level of social systems; action -at the level of the individual learner acting intentionally; and operation -at the level of the concrete operation, procedure or behaviour" [START_REF] Fjuk | The complexity of distributed collaborative learning: Unit of analysis[END_REF] .

More recently, theories of situated and distributed cognition proposed a more precise characterization of the activity process. Considering that cognition is not only a human internal capability, they have proposed a characterization of objects, tools, and documents as externalized memories. These analyses allow a definition of documents as representational artifacts.

E. Hutchins [START_REF] Hutchins | Cognition in the Wild[END_REF] proposals have a specific interest. Following Hutchins, culture organizes the natural world in a functional way: natural world entities are cultural markers in the realization a particular operation while humans externalize complex reasoning by making tools and artifacts. For example, Hutchins explores how plane speeds may be memorized by cockpits. One essential principle of anthropology is that preexisting cultural solutions for a specific problem can be shared in other contexts in order to solve some similarly structured problems h .

The adaptation of posology is a particular activity of hospital pharmacy that controls patient individual elimination -thereby drug toxicity risks-and calculate dosage on the basis of patient behavior. Adaptation can be described by pharmaceutical concepts such as drug elimination and therapeutic goal. This descriptive level is fairly efficient for characterizing pharmaceutic reasoning but the characterization of how and why the documents are relevant in the course of the action requires a description of this activity at a more abstract level. Activities indeed rely on core specific documents properties such as how a document allows working about an individual in the world, how it contributes to inference, or how it represents a complete situation in the world integrating some changes.

The adaptation of posology is a particular task in which the natural world is only accessible with samples wich are analyzed by tools and operation issues are reported into artifacts. Pharmacists have developed a large network of information both with a view of working on patients without any access to them, and of recording the individual information in memories and knowledge of the previous patients' behavior.

In this study, we do not address the different aspects of the cognitive activity during process matching but rather, in the material recording of information and knowledge. We limit activity analysis to information acquisition, conveyance, and duplication through different activity artifacts and tools.

The following figure represents the information trajectory from the real world to the workspace of the pharmacist: It characterizes the context of the artifact and the essential concepts we will use for our analysis and the metadata elements:

h The first application domain for this cognitive model was the Micronesian navigation and it has been extended to the "Western navigation" and airplanes cockpits. Now, we successively describe the different parts of this trajectory, considering individuals and universes, tools and artefacts.

Patient bedroom Locations of operations in hospital

Hypothesis of the three universes.

Natural language theories usually, consider that there exist three components for sign: things in the world, conceptual knowledge, and words [START_REF] Ogden | The Meaning of Meaning: A Study of the Influence of Language Upon Thought and of the Science of Symbolism[END_REF] . Knowledge externalization (in tools, representational artifacts and protocols) changes our conception of meaning: the world is structured by culture. The three universes translate the three sign constituents into observable entities of the activity. These observable entities will be described by metadata.

Entities of the natural world designate every individual in the world before being integrated into a cultural framework. An individual [START_REF] Strawson | Individuals[END_REF] is a continuant 18i . Although individuals j may be culturally processed into objects of enquiry, they remain individuals. The natural world objects can be represented only by an indexical or a proper name. Symbolization entails a classification and a status: the individual becomes an object for information and knowledge investigations. We can work on an individual only if we use tools and representational artifacts. One paradox that may explain the development of information and information networks in pharmacy is that while pharmacists have many tools and processes both for symbolizing the patients' states thanks to various measures and analyses and for associating these states to populations recorded in databases, the whole processing remains unable to exactly predict patients' behavior.

Each individual can be represented by some specific features in the framework of the activity:

-Patient: agentive (e.g. resistance, elimination) and spatial features i "Entities that continue or persist through time, such as objects, qualities, and functions" in J. Arp Then, artifacts and any information about a fact are indexed by individuals. Any proper name is classified in the type <individual> and has some properties associated with it.

Artifacts and tools are material expansions or externalizations of human cognition. They are characterized by constraints, parameters, and issues and have representational properties. The constrained issues of tool operations are the integers that are conveyed to the representational artifacts but these integers cannot predict the quantity and nature of the observable facts: human reasoning on these representations boils down to inferences from these issues to the individuals. Contrary to natural language, artifacts integrate different dimensions of the world such as measurement analysis [START_REF] Ruttenberg | IntroductiontoOntology Introductions, International Conference on Biomedical Ontology[END_REF] , analogic representations, and annotations.

Artifacts and tools have three dimensions:

-Functional description. Artifacts and tools are represented by their function that indicates the frame of interpretation of the reported issues. -Composition and structure. This description characterizes what things mean in the artifact or the tool. Messages that convey issues have syntactic structures and rules of composition. -Conveyance and classification. Any tool has an issue symbolically -realized or purely mentalthat is reported to required representational artifacts. This knowledge is partly represented by type interpretation that characterizes the regular classification of anchored k facts into predefined files or boards.

User world defines the framework of the actor, considering both personal and community skills. The user world is composed of a social and collective knowledge about tasks and goals. The specific user knowledge is represented by its professional skills such as its ability to manipulate tools and resources in conformity to specific goals. This knowledge makes relations between the two previously presented universes: it connects what can be interpreted in the workspace about the individual in the world and what is expressed by the tools and artifacts. It can be segmented into proper human knowledge about the social activity organization and descriptive knowledge that concerns what we can learn about the individuals from the represented information l .

Tools as externalized cognitive ability.

Tools are defined in cognitive anthropology by externalizing a part of a collective cognitive ability. A tool is represented by its operative abilities, together with its goals, its foundational knowledge, and its provisional issues. The operation is an implemented inference, that is, meaning production (or symbolization) is devoted to tools. If the definition of an artifact can be applied to any serialized k Anchoring characterizes the function from a symbolic entity to a material situation in the world. l This knowledge can be captured only by analyzing the interactions between users and the relation to technical and scientific publications.

support of information, then a tool is associated to any service that produces information from something in the world.

At descriptive level, we represent a tool through a simple operation which requires a parameter on some object in the world. The operation issue is a symbol which represents this object by considering the parameter. Hutchins' representation of a tool is slightly more precise than Ruttenberg's because it associates knowledge (via the parameter) to the operation of symbolization. The Hutchins representation addresses knowledge distribution in the workspace.

A simple externalized operation is schematized by Hutchins as follows in the navigation context: At a symbolic level of description, we identify an information structure that can be communicated and interpreted after it was represented in an artifact.

The notion of artifact.

The notion of artifact is essential in Information Science, ontology, and cognitive anthropology. In his definition of information artifact, Barry Smith [START_REF] Smith | Document acts[END_REF] introduces a pragmatic dimension in reference to J. Searle [START_REF] Searle | The Construction of Social Reality[END_REF] . But the speech act framework seems too limited regarding the role of the artifacts in the activity. The Hutchins proposition directly associates an information artifact to a cognitive structure. From a functional viewpoint, memories play an essential role in the coordination and cooperation in long-term therapy design. They concern what has happened during the therapy. The memorial and mediation role of the artifact in the activity introduces some precisions into the characterization of the whole artifact. The heading of the artifact is more a reference to the activity and to the processes that this activity is based on than a title that characterizes the identity of the document.

Each course of the activity depends on context and circumstances. Tools and artifacts present intangible regularities in the activity. Regarding memories, artifacts have the property to be continuant n (or endurant). If we now consider the whole activity, the artifact is the space that reports and memorizes the operation issues. Then it is used as a resource that presents a structured collection of information for any use. Information artifacts have both analogic and prescriptive dimensions. They record scientific knowledge about the activity and then regulate the individual interpretation of the described facts.

Examples of sheets from the pharmacy workspace are used for extracting some expressions from these artifacts. The associated attributes are presented below.

m An information bearer should not be confused with a document: the bearer characterizes only the material frame of a content representation. A sheet of paper can contain some different bearers. It's an artifact per se, but each information collection structured in the artifact by a spatial or visual bearer is an artifact.

n "An entity [bfo:Entity] that exists in full at any time in which it exists at all, persists through time while maintaining its identity and has no temporal parts." [] comment: Definition: An entity [bfo:Entity] that exists in full at any time in which it exists at all, persists through time while maintaining its identity and has no temporal parts. http://www.berkeleybop.org/ontologies/obo-all/bfo/bfo.obo Identified expressions Attributes "Posology adjustment for dose regimen by…" "Adaptive control for antibiotherapy" "Adaptive control for antibiotherapy -filereference population".

Artifact function

"clinical information" "information about treatment" "kinetic issue" "General information" "Bacteriology" "Associated therapy" "related pathologies" "Therapy" "Further information" "kinetic" Represented event Name of the patient "Vanco" "Bile duct Infection + streptococcus" Individual "Kinetic after one day therapy" "Measured plasmatic concentrations (μg/ml)" Ct0 = 7,26 μg/ml Cpic = 12,77 μg/ml » Tool Parameter Issues

Spatial and temporal dimensions of the artifacts: a functional bearer for classification.

Because of its material dimension, an artifact may be considered as an area, that is to say, a spatially distributed access to information in the workspace. Hutchins [START_REF] Hutchins | The role of cultural practices in the emergence of modern human intelligence[END_REF] demonstrates how some spatial natural structures like stars in the sky are used as temporal measures in navigation. This operation of transformation of a spatial dimension into a temporal measure is explained by the human cognition innate ability. This property is characterized in our artifacts by the articulation of temporal-token information into a spatial-type representation: information in time is incorporated into a spatial artifact o . Now, we can precise the different functions of the representational artifacts in the activity p :

The first function is the memory of the achieved operations, then an individual trajectory within the framework of shared knowledge. Artifacts represent the memory of the therapeutic actions on the individuals and then, by inference, what we have learned about individuals.

The second function is a symbolic and partial representation of the individuals. The issue of an operation is about a singular individual state and is published in a file or a pre-printed sheet that classifies this issue into a common framework. This artifact classification of the information into a typical frame is the graphic representation of a common view of the individual and of the way the activity works with it. Symbols are used for anchoring the artifact to the world and for referring to the information. Since pharmacy presents several types of individuals, the artifacts represent the interaction between a patient, a molecule and bacteria. This function allows us to reason about how we interact in the world.

The third function is a classification of a succession of representations.

The artifact is a media of representation of a common and a priory scientific construction of the reality and is realized by the bearer structure. This function is fulfilled by an event q classification [START_REF] Ferrario | Towards an Ontological Foundation for Services Science[END_REF] . It associates predefined categories of accepted events or facts to the reported information about the individuals. This dimension correlates a regular form reasoning (about elimination or therapy) to specific individuals.

User situation and interpretation: semantic characterization of the artifact contents.

Now, we explore the discourse and activity articulation, especially the complementarity between formal semantic theories and distributed cognition principles. In many professional activities such as pharmacological analysis or research, we reason about individuals in the world only with data and without access to this individual. Then the organization of these data for the user interpretation is a condition for the success of the activity and the relevance of decisions. This characterization is founded on the interaction between information, the natural world and user goals.

o Everybody has an individual history but the same anatomy. p These artifacts are supported by the claim that cognition has a dual representation: internal in the human mind and external in tools and mediation artifacts.

q We define an event as follows: "Although the term "event" has often a dynamic connotation, we use such term in the more general sense of entity which occurs in time (also called perdurant in the DOLCE ontology). In this understanding, states and processes are considered as special event kinds". (Ferrario & Guarino, p.3 We start by presenting situation theory and the three foundational situations which characterize the interpretation of the identified objects. We proceed by presenting the types which are generated by the regular relations. Finally, we show how these types may be modelled.

a. Situation theory.

Situation theory [START_REF] Devlin | Logic and information[END_REF] proposes a formal representation of meaning in a distributed context. We first present the foundations of the proposed model, concerning what is meant by meaning and situation.

Considering the user interpretative positions, meaning is defined from the user position in the course of his activity to situations in the world. Situation theory postulates that information interpretation is defined by relations between situations. This situation is structured under the principle of distribution and complementarity between heterogeneous r and symbolic entities. The situation structure is based on but represents some primitives of human reasoning with information [START_REF] Kratzer | Situations in Natural Language Semantics[END_REF] .

A situation accepts, or supports, information on the condition that a word having a conceptual principles or definite descriptions. This simple predicative structure is characterized as "infon" s and this "infon" is supported by a situation. Given a situation, s, and an infon σ, meaning has relations to strings of parametric values. These values are defined by direct reference:

s ⊨ σ, and if the infon is "John saw Mary running", we write:

s ⊨ see (j, s',t) ⋀ s'⊨ run (m,t').

Any situation must be parametric to accept a collection of infons t . Relations between concepts are characterized by properties.

In situation theory, the interpretation is defined by the inference of true propositions from expressed information to situations:

-intensional interpretation reasons about type or generalization, -extensional interpretation reasons about token or specification.

This interpretation implies that each individual, tool operation, and artifact has its proper type-token interpretation. This interpretation requires some prior knowledge about the symbolization operations, the information conveyance and the individuals. This prior knowledge is externalized in the representational artifacts by the bearer structure. This externalization is mediated between information world (i.e., individuals and tools) and the pharmacist's goals and skills.

r Heterogeneity is defined by constraints on meaning: information and situations associate terms that have different ways to mean and refer: conceptual meaning, direct reference, etc.

s "I adopt the word "infon" to denote an object of the form ≪P, a 1 ,…,a n ,i≫, where P is an n-place relation (for some n), a 1 ,…,a n , are objects appropriate for the respective argument place of P, and i is equal to 0 or 1. The notation and the name are intended to emphasize the fact that "infons" are semantic objects, not syntactic representation." (K. Devlin, op. cit., pp. 22-23). t This position is opposed to the principle of ontology theories. According to these theories, meaning is based on the semiotic triangle that postulates a relation between a concept, a lexical entity and an object in the world.

Three foundational situations.

Initially, J. Barwise & J. Perry distinguish three sorts of situations (op. cit., p.6) to characterize the meaning of an utterance. An utterance is defined as an action that conveys information and describes a statement. Utterance generally but not necessarily refers to a sentence. We define utterance as any description of the individuals, obtained by the operations issues. An individual occurs in a described situation by an indexical and a time markers. An "infon" is a predicative structure interpreted in a situation. Then, a situation is a propositional framework in which the information can be verified or not.

For one informational structure, we identify three situation types:

- It represents the common knowledge which is necessary for uttering and interpreting information. This situation is represented by the artifacts.

These situations explain why the representation provides a partial view of the world and why we can make some inferences about this world. These interpretations overlap each "infon" interpretation.

We now present the application of this conceptual framework on the informational dimension of the adaptation of posology. We characterize each situation by S and each situation supports a relation between heterogeneous entities. These entities which are characterized at a type level means that each entity classifies some tokens.

The described situation characterizes the fact that there exists an individual (associated to time and location markers) in the world:

The utterance situation indicates that an operation represents a part of the individuals with a required parameter: The resource situation indicates that the parameters associated to the individual representation are issued from a conceptual structure u . This structure is externalized by the artifacts composition:

These three situations are generic because they define the foundational entities and operations of the informational dimension.

Linguistic features cannot be considered alone. A word becomes a concept if it satisfies two criteria:

-The ability of this word to have an intensional meaning -Its ability to convey and classify some specific information from a location in the activity to another.

Therefore, a predicate is characterized by the generic property of a word to convey and classify information in a distributed context. It classifies a succession of information in that it represents an event and in that each individual and operation issue is an argument. The characterization of the document properties by linguistic categories entails an artifact content description based on the classes of information accepted and shared by the artifact. When the issue of an operation is reported into a sheet or a file, it becomes a proposition since it can be interpreted at any time by everyone in the same way.

The interpretation takes place in the frame of the activity and then requires each identified situation. Interpretation in S 2 requires the interpretation of S 1 that requires S 0 .

b. Type relations between situations.

We now characterize the relations between the different situations. The regularity of these relations allows a type representation that surrounds one instance of activity matching. This type representation accounts for learning in the activity, knowledge accumulation, and discovery. Types correspond to materially attested relations in the activity and these relations stem from interpretations and their related knowledge.

Distributed cognitive anthropology characterizes how knowledge is manifested through material organization whereas situation theory represents how the uttered information is interpreted in this context. Distinctively to navigation (or other technical activities analyzed by Hutchins), adaptation is based on a knowledge acquisition about the portions of the world, especially individuals v . The individual behavior cannot be predicted and a knowledge acquisition about this individual is a u This representation overlaps the distinction between an « artifact function » that designates an independent bearer (like a sheet or a file) and an « event representation » in which the bearer accepts a collection of issues from defined operations. v Adaptation of posology is both a routine activity in hospitals and a research activity.

Resource situation: representational artifact S 2 ⊨ th. [(S 1 ⊨ par. (S 0 ⊨ ind. (t,l)t,l)), (t,l)] (S 2 ⇒ S 1 and S 1 ⇒ S 0 ) → (S 2 ⇒ S 0 ) th. : theoretical constraint (associated to the artifact structure)

S 2 ⇒ (S 1 ⇒ S 0 )
process that overlaps a succession of dosing proposals. A mental representation of the individual properties overlaps the synthesis of the operation issues. That is why the adaptation activity is characterized as a control expertise: dose regimens can be modified regarding to the patient behavior.

Situation theory defines situation types for characterizing abstract situations. We propose a type representation of the relations between individuals, tools and artifacts in conformity to the information conveyance. Knowledge is represented by a classification [START_REF] Dretske | Information and the Flow of the Information[END_REF] of any token in a type and by the recognition of information by its classificatory type. Situation theory postulates that abstraction is a particular human quality that accounts for intensional dimension of knowledge. Human internal knowledge is associated to a type characterization of any specific situation w . Following the principle of externalization, an artifact materializes some abstract knowledge. The report of the patient's last name on these artifacts anchors the knowledge associated to the support. Any type is realized at a token level by an "infon". The predicate has a conceptual meaning and bases the information conveyance from a situation to another in the activity frame x .

The three foundational situations are populated by concepts when information is integrated into regular pre-printed sheets and electronic files. These regularities are represented by types. We now explore their specific properties both at the level of the information and the artifact model.

First we present the type mechanism. After the representation, we introduce the range of this type in the interpretation context. The denomination of the type interpretation follows.

Individual interpretation and artifacts.

When we consider medicine and pharmacy, the real world includes patient, molecules and bacteria. These individuals are articulated to the different properties of the artifact: the spatial property for the patient body, the temporal property for the molecule processes (essentially elimination) and both spatial and temporal properties for the bacteria. The analog property of the artifacts anchors the space and time dimensions of the individuals [START_REF]Logical reasoning with diagrams[END_REF] .

We now represent how a situation in the world is represented by an artifact considering the classificatory rule:

The type (T) is the relation that indicates the classification of the individuals in the artifact function. This classification has an important consequence: since each individual is classified into a regular artifact, it can be compared to any other and primarily to the individuals of the previous therapies.

w Following F. Dretske (op. cit. p.86), we adopt this definition of knowledge: "K knows that s is F = K's belief that s is F is caused (or causally sustained) by the information that s is F. (…) Knowledge is identified with information-produced (or sustained) belief, but the information a person receives is relative to what he or she already knows about the possibilities at the source. Since there is a covert reference to knowledge on the righthand side of the equation, the equation does not tell us, as it purports tell us, what knowledge is."

x This proposition is supported by the solidarity between artifact visual properties and predicative linguistic entities.

T 0 ⊨ S 0 | S 2
We categorize this type as <INDIVIDUAL CATEGORY> y . For example, this type is linguistically realized by the predicate "patient name".

Operation issue interpretation in artifacts.

The whole information about an operation is conveyed to the artifact and represented by an information structure z . An information structure is a grammatical composition integrating words referring distinctively: last names have a direct reference and by contrast, and event names have a mental significance. The classification requires an event name that means that this conveyed issue represents a state in the course of the event about these individuals. Then, an information structure classifies the whole components of the tool/operation situation in the artifact.

The conveyance of the issue and its classification into an event representation can be written as follows:

This type classifies any operation issue into the event representation. Considering that operations select a specific dimension of the individuals through the parameter, we obtain a representation of the individuals constrained by an operation aa . This type can be categorized as <CONSTRAINT>. It is linguistically realized by the predicate "parameter".

Event interpretation in artifacts.

We now further define the type relation that holds between event representation and artifact function. Since symbolization is defined by a classification, discourse (i.e. information representation in artifacts) is characterized by a relation between event structures and activity goals. The theoretical constraints presented into the foundational situation (S 3 ) introduce a type/token distinction between the temporal-linear course of the events and the type-spatial structure of the artifact bearer. In this way, at type level, an event about the patient is spatially classified considering the artifact function in the course of the activity. The dimension of situations changes because the artifact is not only considered as a singular frame for interpreting one instance of information but also as a spatial and temporal representation of some goal-oriented externalized knowledge.

The interpretation about the whole individuals requires relations between the different events. These relations are called theories and represent inferences between events at an intensional level:

Every event, obtained by the report of devoted operation issues, is classified into the artifact function denomination. An event has no pragmatic dimension. The classification of some pre-defined events into the artifact function allows the interpretation of an individual <BEHAVIOR> during the therapy. This type is linguistically realized by the predicate "event name".

Synthesis of interpretation requirement.

We now propose a representation of the different interpretation positions integrating the associated knowledge and structural dependencies. We distinguish a partial interpretation that concerns represented events about the individuals and a global interpretation that concerns the individuals and the activity management. Context and content situations distinguish individual and tool situations from artifact situation. Event and social knowledge are externalized by artifact bearers while individual and tool knowledge are externalized in the world. They characterize the content of the reported information.

The activity process management is based on social knowledge which is distributed and shared among the different pharmacists. Social knowledge presupposes knowledge about individuals and how these individuals are represented (by tools and events). Tool knowledge is an a priori structure that constrains social interpretations as well as event interpretations. Knowledge about the individual behaviors during the therapy associates the different individuals and is both anchored on the event representation and the knowledge of individuals. The correlation between the different event proceedings is a basis for a behavior characterization that is interpreted in terms of action and activity management. It follows that social knowledge is supported both by individual behavior and by individual category: their complementarity is a condition for a sound decision.

Document description founded on activity information use.

Up to this section, we have considered activity and information independently from document description. Now we provide some metadata principles. Document description is a communication tool that facilitates resource retrieval considering the diversity of user interests: in e-science and big data context, a collection of resources can be of interest because it presents information about world entities, tools, events and the matching of an activity. Because we can represent the content and context of the information both at the type and token levels, we can generalize the methodology to the description of any activity anchored artifact, tool and individual bb .

As with the situation and type characterization, we distinguish two sets of metadata: content environmental metadata for situations, and bearer internal metadata for types. Before the characterization of each schema, we present the modular structure of the situations and types: This representation is centered on artifacts. For a tool-centered description, S 1 is classified into the different artifacts of the activity. This remark can be extended to the represented events and the individuals.

We summarize the previous issues and their representation by metadata as follows: Reference characterizes situations describing something in the world (S 0 and S 1 ). Mediation characterizes S 2 structures. Abstraction is supported by types: it concerns knowledge associated to the relations between situations and complex interpretation.

We propose the following strategy to build metadata based on the following idea: representation languages 28

Relations between situations and types.

Now we can present the relational properties that allow inference between situations and types. We These relations represent reasoning between metadata modules. They present direct inferences from situations to types and then facilitate reasoning with metadata modules.

Conclusion

With the increasing amount of metadata sets today, one may wonder whether it is really necessary to introduce a new one. But we can extend the descriptive properties of metadata to content and document context if we adopt a theoretical framework. This methodology appears as a response to the B. Smith's criticisms 30 about metadata foundation and a contribution to extension of the metadata coverage. Document description becomes more structured and new relations between external and internal artifact components can be specified in such a way that they present inferences from conceptual representations to anchored descriptions. This property is a great advantage for the modular description which allows an independent processing of each module. We have presented only one inference between modules but some other properties may be identified.

We have also developed a methodology based on the solidarity between linguistic phenomena and material processes. This methodology has entailed a coherent set of metadata based on both the distribution of the described entities and information conveyance. Our perspective is more empirical and based on attested terms than usual ontological processing. Then the model can be extended to relations between artifacts, information exchanges and inferences in the course of the activity. This perspective allows the description of entities like tools and individuals that are not actually recorded by library metadata. We will extend this analysis to the activity description, considering the workflow of artifacts, communication and workplace. In this way, a whole activity will be described.

Our metadata set is designed to be complementary to usual sets like Dublin Core for example, or ontology likes IAO. We do not propose any other KOS but artifacts and tools externalized knowledge is connected to external resources (such as controlled vocabularies or terminologies). Our perspective allows primarily an intrinsic characterization of the used metadata, distinct from external controlled vocabularies, and further, the possibility to extract automatically the linguistic entities that will be used as values in the description.

Our metadata set is based on the adaptation of posology but we have preserved a generic representation in order to enable a large spectrum of applications.

Figure 1 .

 1 Figure 1. Overview of the information building, conveyance and interpretation in the frame of the adaptation of posology.

Figure 2 .

 2 Figure 2. Schematizations of the externalized operations.

Figure 2 . 1 .Figure 2 . 3 .

 2123 Figure 2.1. Operation in navigation Figure 2.2. Generic representation

Image 1 .

 1 Initial adaptative control sheet. Image 2. Adaptative control board. Image 3. Posology adaptation recommandation.

  Described situation characterizes what sort of fact of the world is described by the reference. -Utterance situation characterizes what sort of representation choices has been selected for these facts. The utterance or expression framework characterizes the situation. It concerns the tool operations and issues. -Resource situation characterizes what sort of theoretical framework supports these choices.

1 ⊨

 1 Described situation: world individual S 0 ⊨ ind. (t,l) Utterance situation: operations about this individual S [par. (S 0 ⊨ ind. (t,l)), (t,l)] S 1 ⇒ S 0 Par : operation parameter

Figure 3 .

 3 Figure 3. Schematization of the knowledge distribution and relations in the activity.

Figure 4 .

 4 Figure 4. Structure of the metadata modules.

Figure 5 .

 5 Figure 5. Articulation between cognitive dimensions, semantic model and metadata.

  use the range and domain properties to characterize what a situation or a type means by abstraction and anchoring. These properties are realized in a situation or among two situations (in the case of types). Range and domain properties represent classifications: this relation construction represents what a situation means in a conceptual domain and the impact of a concept in a situation.This representation classifies facts into concepts and the realization of concepts in facts. More technically, situations are considered as containers and these containers have a domain characterized by a concept. Conversely, a concept has an individual as range (except constraints that have an event name as range).

Figure 6 .

 6 Figure 6. Schematic representation of the inferences between situations and types.

Table 1 . Categorization of the linguistic expressions extracted from the activity artifacts.

 1

Table 7 . Behavior metadata and predicative representation.

 7 allow different descriptions which are based on their predicative structure and identical categories of symbolic entities 29 :

	Bearer	{	T 0 : < individual category> ⊨	S 2 <artifact function> T 2 : <behavior> ⊨ T 1 : <constraint> ⊨	}
	Content	{		S 0 : <individual>	S 1 : <tool>	S 2 : <represented event>	}
	Activity cognitive functions	Represented by	Semantic model	Described by
	Abstraction			Types	External controlled languages
							Content environemental
	Reference			S	0 & S	1 situations	metadata
							Metadata set
							}
	Mediation			S 2 situations	Bearer context metadata

bb Metadata description is limited to entities identified as externalized knowledge. Knowledge architecture requires ontology.

y This classification is emphasized by the database (used for estimation calculus) USC*PACK, which is available in the pharmacists' workspace. z An information structure is the linguistic and, more particularly, the grammatical representation of the "infon".aa The event defines concept realized by the operation issue and the constraints characterizes how this realization is obtained.T 1 ⊨ S 1 | S 2 T 2 ⊨ S 2 | S 2

-Every situation can be represented by an attribute-value pair, where each attribute is the predicate of the" infon". The token component of the situation is represented by a string of values, and each situation exemplifies one of these values. -Every relation between components in a situation generates an RDFS triple. Then a situation is represented by a RDFS schema. -Every regular situation builds a structure of triples and this schema describes the resource.

-Types are schemas based on relations between schemas.

Resources identify the entities of the activity we will describe. The predicates are linguistic entities defined as properties. These properties are empirically attested: they are extracted from artifacts. They associate the resource to individuals and operations components (characterized by nonpredicative symbolic entities). These properties are associated to memories (of the different individual names, parameters and events). The extracted values have the same meaning in the pharmacist community and then, they are considered as a common professional vocabulary. In the case of types, predicates are informed by external controlled languages which require a manual indexer decision.

Content environmental metadata.

Content environmental metadata modules characterize the artifact content. This content refers to external individuals and operations. Each module contains an informational predicative structure in which each situation is represented by a resource or subject, a predicate and a limited set of values or objects.

INDIVIDUAL: an individual represents a resource in the world identified by an entity name, a last name which may be categorized and a time marker. TOOLS: the resource is a simple denoting operation requiring a parameter on something in the world. The predicate is represented by the parameter name and the issue by one entity in a string. The issue is in time indexed but this issue is not necessarily of interest for metadata.

Metadata module Predicative representation

Metadata module Predicative representation

Bearer internal metadata.

ARTIFACT: an artifact is a functional bearer that represents individuals and tools issues in the course of the activity. Each artifact contains different identified sequences of issues for a same operation and individuals: each sequence represents an event. The artifact function is marked by the title and the formal markers (spatial representations) associated to the artefact structure. Events are characterized by the symbolic entities associated to visual markers of solidarity between information expressions.

Metadata module Predicative representation

Artifact situation: Artifact function: Represented event: Time:

Artifact situation:

Represented event:

operation name:

Metadata module Predicative representation

Individual category: Patient name: Artifact name: Category name:

Subject: patient name Predicate: artifact name Object: Category name Exemple Individual category :

Patient name: M. x Artifact name: "Adaptation posologique de traitement par…"

Category name: patient âgé Table 5. Individual category metadata and predicative representation.

T 1 : CONSTRAINT: each artifact function is associated to the report of tool operations. Every tool name is interpreted at a type level as the name of an event (measured by the tool). The constraint represents exactly how the event is represented and why this representation is both partial and in adequacy to the event. T 2 : BEHAVIOR: Behavior represents the structure of the different events in the artifact. Each event is classified into the artifact structure and owns a meaning in relation to the other events. They figure out what the relations between events mean about the whole individuals. This reasoning is a condition for the action.

Metadata module Predicative representation

Metadata module Predicative representation

Behavior


Nicole Andenmatten

Saskia Egarter

Allison J Jackson

Nicolas Jullien

Jean-Paul Herman

Markus Meissner
email: markus.meissner@glasgow.ac.uk

Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

cannot be observed upon induction with high doses of the rapamycin analogue Shield-1, the inducer of the ddFKBP-system 1 (Fig. 1c). We found that one-hour incubation with rapamycin is sufficient for efficient lacZ excision (Fig. 1d). Next we designed a gene-swap strategy, where loxP sites flank the cDNA of a GOI. Removal of the cDNA by Cremediated recombination positions the YFP under control of the constitutive promoter, resulting in green fluorescent parasites (Fig. 1e). We tested this strategy by transfecting RH DiCre parasites with a vector containing floxed KillerRed 10. Parasites incubated in the presence of rapamycin displayed a highly efficient switch from red-to green-fluorescent parasites (96 %), as demonstrated by microscopy and FACS analysis (Fig. 1f,g). We have successfully employed this strategy to generate an arsenal of KO mutants. Importantly, this strategy allows visual identification of YFP positive KO parasites after successful GOI removal. Furthermore, we generated a parental strain, ku80::diCre, where homologous recombination is favoured (Supplementary Fig. 1).

Next we applied this technology to functionally dissect critical components of the parasites' invasion machinery. We generated conditional KO mutants for the gliding motor, myosin A (MyoA), the micronemal protein MIC2, and parasite actin (Act1; Fig. 2a and Supplementary Figs. 2345). These invasion factors are considered essential for host-cell invasion based on the interpretation of phenotypes obtained in knockdown (MyoA 3 and MIC2 8 ) or overexpression mutants (Act1 11 ). To produce the conditional myoA KO parasites, firstly we introduced a second floxed and ty-tagged copy of myoA (regulated by the chimeric promoter p5RT70) 12 , randomly into the genome of the RH DiCre parasites. The endogenous myoA was then replaced with the cat gene, producing parasites containing only one copy of myoA (Supplementary Fig. 2). For mic2 and act1 we directly employed the gene-swap strategy in ku80::diCre and exchanged the endogenous GOI for the gene-swap construct, where expression of the respective cDNA is under the endogenous promoter's control (Supplementary Figs. 4 and5). We verified all genomic modifications using analytical PCR on genomic DNA (Supplementary Figs. 2345). The parasite strains containing floxed versions of the GOI (loxPMyoA, loxPMic2 and loxPAct1) did not show any detectable phenotype. (Fig. 2a,b and Supplementary Figs. 2345). In contrast, Cre-activation with 50 nM rapamycin for 4 -8 hours resulted in a mixed population consisting of uninduced and knockout parasites for all three lines (Fig. 2a and Supplementary Figs. 2345). The excision efficiency was dependent on the strain; loxPMyoA displayed the most efficient recombination of ~ 75 %, loxPMic2 approximately 40 % and the recombination rate in the loxPAct1parasites was about 20 %, when analysed 24 -36 hours after induction. Given that KO parasites are expressing YFP, it is straightforward to perform phenotypic characterisations of the respective conditional KO, or to enrich this population using FACS sorting (Supplementary Fig. 5 and data not shown).

Surprisingly, although we verified that the conditional myoA KO has similar egress, gliding motility, and host cell invasion phenotypes as previously described for a myoA knockdown mutant (Supplementary Fig. 2) 3 , we successfully isolated a clonal myoA KO parasite line that can be permanently maintained in culture. Similarly, we achieved isolation of a clonal mic2 KO parasite line, demonstrating for the first time that neither myoA nor mic2 are essential for parasite survival. This suggests the existence of a MIC2-and MyoAindependent invasion mechanism (Fig. 2a,b). We verified the absence of the respective genes using analytical PCR on genomic DNA and absence of the proteins in Western blots and IFA for both clonal KO lines (Fig. 2a and Supplementary Figs. 3 and4). In a growth assay of myoA KO and mic2 KO parasites we could see that both KO strains displayed a significant growth defect that was more pronounced for myoA KO parasites when compared to control parasites (Fig. 2a,b). Importantly, the reintroduction of myoA led to full complementation of myoA KO parasites, demonstrating a specific phenotype (Supplementary Fig. 3). Interestingly, MyoA deletion did not affect secretory organelles or localisation of other glideosome components, such as the myosin light chain 1 (MLC1) or the Gliding Associated Protein 45 (GAP45; Fig. 2c). Immunofluorescence analysis of mic2 KO parasites showed the absence of MIC2 (Fig. 2a), while other micronemal proteins AMA1 and MIC3 remained unaffected and displayed normal localisations (Fig. 2d and Supplementary Fig. 4). As described previously for a MIC2 knockdown, the localisation of MIC2's interaction partner, M2AP, and its unprocessed form was severely disrupted 8 , and could be found in punctate spots within the parasite in addition to the parasitophorous vacuole (Fig. 2d and Supplementary Fig. 4).

In contrast to MyoA and MIC2, we were unable to isolate a clonal act1 KO parasite line, demonstrating that actin is essential for parasite survival. We never observed any plaques formed by YFP positive act1 KO parasites upon induction of loxPAct1 (Fig. 2a and Supplementary Fig. 5). An immunofluorescence time-course of YFP parasites in the induced loxPAct1 population established that Act1 protein levels are significantly decreased as early as 24 hours after induction and are undetectable after 72 hours (Fig. 2e). Although a role of Act1 in parasite replication has been questioned previously 13 , we found that Act1 depletion resulted in a strong apicoplast segregation defect -a plastid-like organelle (Fig. 2e). No other gross morphological consequences were obvious (Fig. 2e and Supplementary Fig. 5). Intriguingly, we found that green-fluorescent parasites can be easily identified in the induced loxPAct1 population up to 10 days after induction, indicating host-cell invasion in the absence of Act1. Indeed, when parasites are released from the host cell 72 hours after induction, the act1 KO parasites remain capable of infecting host cells (Fig. 2e). However, these parasites died within the host cell due to apicoplast loss, resulting in a typical delayed death phenotype 14 .

In summary, the DiCre system enables the efficient generation of conditional KO mutants with a clear and interpretable phenotype, as background expression of the GOI can be excluded. We present here three KO examples of genes previously described as essential for host-cell invasion and demonstrate that the parasite is capable of employing alternative invasion mechanism(s). MyoA and MIC2 are shown to be dispensable for host-cell invasion. A few remaining actin molecules could enable invasion, but since a striking effect on apicoplast replication can be observed as early as 24 hours after act1 excision, we consider this possibility unlikely. Instead, we favour the interpretation that T. gondii is capable of employing an Actin/MyoA/MIC2-independent mechanism to invade the host cell. Future studies will elucidate this novel finding in detail. This conditional knockout technology provides researchers with a novel tool that produces complete KO for the analysis of essential genes. Our understanding of T. gondii invasion has been remodelled as a result of this technology. Consequently our study necessitates reanalysis of previously described "essential" invasion factors, to define the minimal invasion machinery. We have successfully adapted the DiCre system for the malarial parasite Plasmodium falciparum (Collins et al., unpub. data), demonstrating the potential of this technology to enhance all apicomplexan biological research.

Methods

Cloning of DNA constructs

All primers used in this study are listed in Supplementary Table 1. The FKBP-Cre59 open reading frame (ORF) consists of NLS, FKBP12, linker and Cre1-59; FRB-Cre60 ORF consists of NLS, FRB T2098L, linker and Cre60-343 9 . The respective ORFs were amplified using the primers FRB-Cre60/ FKBP-Cre59 fw and FRB-Cre60 rv or FRB-Cre60/ FKBP-59 fw and FKBP-Cre59 rv and cloned into the plasmid p5RT70mycGFPPfMyoAtailTy-HX 15 using EcoRI and PacI as restriction sites (p5RT70FKBPCre59-HX; p5RT70FKBPCre60-HX). In case of p5RT70FKBPCre59-HX, hxgprt was exchanged for dhfr derived from pDHFR-Tsc3 16 via SacII (p5RT70FKBPCre59-DHFR). To generate a plasmid for integration of both Cre subunits into the same genomic locus, FKBP-Cre59 was amplified with the primers p5RT70FKBPCre59 fw and rv using p5RT70FKBPCre59-DHFR as template. The resulting p5RT70FKBPCre59 fragment was cloned into p5RT70FFRBCre60-HX using KpnI (p5RT70DiCre-HX). Finally the hxgprt resistance gene was exchanged for dhfr via SacII. The resulting vector is referred to as p5RT70DiCre-DHFR.

To generate ploxPTyMyoAloxP-DHFR, the expression cassette p5RT70TyMyoA, which allows expression of N-terminal Ty-tagged MyoA, has been placed between the two LoxP sites of pLox-P30/11LacZ-Lox-CAT 17 using ClaI and PacI. The CAT selection cassette on this vector was exchanged for dhfr from pDHFR-Tsc3 using NotI and XbaI as restriction enzymes resulting in the construct p5RT70loxPTyMyoAloxP-DHFR.

A ku805′UTR-DiCre-ku80 3′UTR cassette was constructed in order to replace the hx ORF by diCre in ku80::hx strain 18 . For this purpose, the 5′UTR and the 3′UTR of ku80 locus were amplified using ku80 5′UTR fw/rv or ku80 3′UTR fw/rv primer pairs, respectively. Afterwards the amplified fragments were cloned into p5RT70ddmycGFPPfMyoAtailTy-HX 15 using Kpn1/SpeI and SpeI/SacII restriction sites. Finally, the DiCre expression construct from p5RT70DiCre-DHFR was placed between the 5′UTR and 3′UTR of ku80 using SpeI. The gene-swap vector p5RT70loxPKillerRedloxPYFP-HX was generated by cloning KillerRed 10 into p5RT70mycGFPPfMyoAtailTy-HX 15 . To introduce a loxP site upstream of KillerRed, the promoter region was amplified using p5RT70 fw and rv primers. The amplified promoter fragment was placed upstream of KillerRed using KpnI and ApaI. To introduce a loxP site and YFP downstream of the KillerRed STOP codon, YFP was amplified from p5RT70ddYFP-CAT 1 using LoxPYFP fw and rv primers. The fragment was then placed downstream of KillerRed using PacI and NotI.

To generate loxPMic2loxP-YFP-HX, the parental vector p5RT70loxPKillerRedloxPYFP-HX was modified such that the mic2 3′UTR was amplified from genomic DNA using the primer pair 3′UTR Mic2 fw/rv, and the PCR fragment was cloned into p5RT70loxPKillerRedloxPYFP-HX via SacI. The mic2 ORF (TGME49_201780) was amplified from cDNA using the primers Mic2 ORF fw/rv, and was cloned into the parental vector p5RT70loxPKillerRedloxPYFP-HX using EcoRI and Pac1. Finally the mic2 5′UTR was amplified from genomic DNA using the primer pair 5′UTR Mic2 fw/rv, and cloned into the final vector using ApaI and EcoRI.

The loxPAct1loxP-YFP-HX construct was generated using the strategy described for loxPMic2loxP-YFP-HX with minor alterations. Firstly, the act1 ORF (TGME49_209030) was amplified from cDNA using the primers Act1 ORF fw/rv and then the resulting PCR product was cloned into the parental vector p5RT70loxPKillerRedloxPYFP-HX using EcoRI and Pac1. To put act1 under the control of the endogenous promoter a 2 kb fragment upstream of the start codon of act1 was amplified from genomic DNA using the oligos 3′UTR Act1 fw/rv and cloned into the parental vector using EcoRI and PacI. Finally the act1 3′UTR was amplified from genomic DNA using the primer pair 3′UTR Act1 fw/rv, and cloned into the final vector using ApaI and EcoRI.

Generation of parasite lines

For the LacZ reporter strain, pLox-P30/11LacZ-Lox-Tub5CAT 17 was inserted randomly into RH hxgprt -genome and positive clones were selected based on chloramphenicol resistance. Subsequently, p5RT70FKBPCre59-DHFR (30 μg DNA) and p5RT70FRBCre60-HX (60 μg DNA) were co-transfected and selected by pyrimethamine, resulting in the strain RH hxgprt -/loxPlacZloxP/FKBPCre59/FRBCre60, (referred to here as BloxP).

To generate a DiCre recipient strain expressing both subunits in the same genomic locus, p5RT70DiCre-HX was transfected into RH hxgprt -(RH hxgprt -/diCre, referred to here as RH DiCre). Expression of Cre subunits was confirmed by Western blot analysis with antibodies against FKBP12 (Thermo Scientific: 1:500 dilution) and FRB (Enzo Life Sciences; 1:1000).

The ku80::diCre recipient strain was generated by replacing hx by diCre in the ku80::hx strain by homologous recombination (ku80::hx::diCre, referred to here as ku80::diCre). The 5′UTR-DiCre-ku80 3′UTR cassette was transfected into ku80::hx strain and subsequently ku80::diCre parasites were selected using 6-thioxanthine (i.e. absence of hx ORF). Integration of diCre into ku80 locus was confirmed by analytical PCR on genomic DNA using ku80::hx fw (1)/ ku80 rv (1′) primer pair to check for the presence of hx in ku80 locus. The conditional myoA KO strain (RH DiCre/myoA::cat/loxPtymyoAloxP, referred to here as loxPMyoA) was generated in three steps. Initially, a floxed transgenic copy of myoA was randomly integrated into RH DiCre recipient strain. To this end, the plasmid p5RT70loxPTyMyoAloxP-DHFR was transfected which confers pyrimethamine resistance. Secondly, the endogenous copy of myoA was replaced with the cat selection cassette by double homologous recombination using pTgMyoA-KO-CAT 3. The resulting loxPMyoA strain carries only one copy of myoA which was confirmed by PCR with the primers MyoA fw and rv that amplify DNA between two exons of myoA. Additionally, Western blotting using α-Ty and α-MyoA antibodies was performed. The floxed myoA can be excised by adding rapamycin (50 nM for 8 hours) resulting in the myoA null mutant (RH DiCre/ myoA::cat/tymyoA -, referred to here as myoA KO. Confirmation of the site-specific recombination leading to the excision of myoA was confirmed by PCR using the following primer sets: MyoA fw (1)/ MyoA rv (1′) and UpstreamLoxP fw (2)/DownstreamLoxP rv (2′). All assays were performed 96 hours post induction of Cre-mediated recombination. Reintroduction of the expression vector p5RT70loxPTyMyoAloxP-DHFR was achieved in absence of drug selection to complement the loss of myoA, resulting in the parasite line RH DiCre/myoA::cat/ tymyoA -/loxPtymyoAloxP (referred to here as loxPMyoA comp).

The conditional mic2 KO strain (ku80::diCre/endogenous mic2::loxPmic2loxP, referred to here as loxPMic2) was generated by transfecting 60 μg of the plasmid loxPMic2loxPYFP-HX into the ku80::diCre parasites to replace the endogenous copy of mic2, and parasites containing stable integration of this construct were selected using XAN and MPA as previously described 19 . The resulting loxPMic2 strain carries only one copy of mic2, which can be excised by adding rapamycin (50 nM in DMSO for 4 hours prior to washout) to generate the mic2 null mutant (ku80::diCre/mic2 -referred to here as mic2 KO). The clonal mic2 KO line was isolated by performing serial dilutions on the clonal induced loxPMic2 strain after 4 hours induction and subsequent removal of rapamycin. Analytical PCR on genomic DNA using the primers intMic2ORF fw (1) and intMic2ORF rv (1′) established the presence / absence of the mic2 ORF. PCR using primers Mic2LoxPF (2) and Mic2 vector 5′ rv (2′) confirmed the 5′ UTR integration in both the loxPMic2 and mic2 KO parasites and Cre-mediated recombination in the mic2 KO parasites. The 3′ UTR integration was checked using the primers HX fw (3) and Mic2 3UTR rv (3′). Additionally, Western blotting using α-MIC2 and α-aldolase antibodies was performed.

The conditional act1 KO strain (ku80::diCre/endogenous act1::loxPact1loxP, referred to here as loxPAct1) was generated by transfecting 50 μg of the plasmid loxPAct1loxPYFP-HX into the ku80::diCre parasites to replace the endogenous copy of act1. After transfection parasites were selected for stable integration using XAN and MPA as described previously 19 . After the selection process the parasite pool was serially diluted to isolate single clones. The resulting LoxPAct1 strain carries only the Cre inducible copy of act1, allowing excision of act1 upon rapamycin addition (50 nM in DMSO for 2 -4 hours prior to washout) to generate the act1 null mutant (ku80::diCre/act1 -referred to here as act1 KO).

To confirm that the endogenous genomic DNA of actin was replaced by the introduced cDNA, analytical PCR was performed using the primers Act1 ORF fw (1) and Act1 ORF rv (1′). Confirmation of 5′ UTR integration and site-specific recombination leading to the excision of act1 was confirmed by PCR using the oligos Act1 5′UTR fw (2) and YFP rv.

The 3′ UTR integration into the correct locus was analysed using the primers HX fw2 (3) and Act1 3UTR rv (3′).

Parasite lines, transfections and selection

T. gondii tachyzoites (RHhxgprt -) were cultured in human foreskin fibroblasts (HFF) cells and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal calf serum, 2 mM glutamine and 25 μg / mL gentamicin. To generate stable transformants, 1 × 10 7 of freshly lysed RH hxgprt -parasites were transfected with 60 μg linearized DNA by electroporation. Selection based on chloramphenicol (1 μM in EtOH; CAT) 20 pyrimethamine (1 μM in EtOH; PYR) 16 , mycophenolic acid (12.5 mg / mL in MeOH; MPA) / xanthine (20 mg / mL in 1M KOH; XAN) 19 and 6-thioxanthine (25 mg / mL in 0.3M NaOH) were performed as described earlier.

PCR, Western blotting and immunofluorescence analysis

In order to extract genomic DNA from T. gondii to use as a PCR template, parasites were pelleted and subsequently washed in PBS. DNA extraction was performed using Qiagen DNeasy blood and tissue kit according to manufacturer's protocol.

Western blot samples were obtained by spinning down extracellular parasites and incubating them with RIPA buffer (50 mM Tris-HCl pH 8; 150 mM NaCl; 1 % Triton X-100; 0.5 % sodium deoxycholate; 0.1 % SDS; 1 mM EDTA) for 20 min on ice to lyse the parasites. Afterwards samples were centrifuged for 30 min at 14,000 rpm at 4 °C and laemmli buffer was added to the supernatant. Unless indicated otherwise 10 6 parasites were loaded onto a SDS acrylamide gel and immunoblot was performed as described previously 1 .

For immunofluorescence analysis, infected HFF monolayers grown on coverslips were fixed in 4 % paraformaldehyde for 20 mins at RT, followed by blocking and permeablisation (2 % BSA & 0.2 % Triton X-100 in PBS). The staining was performed using different combinations of primary antibodies for 1 h and followed by secondary Alexa Fluor 488, Alexa Fluor 350 or Alexa Fluor 594 conjugated antibodies for another 45 min, respectively (1:1000-1:3000, Invitrogen-Molecular Probes).

Equipment and settings

For image acquisition z-stacks of 2 μm increments were collected using a UPLSAPO 100 x oil (1.40NA) objective on a Deltavision Core microscope (Image Solutions -Applied Precision, GE) attached to a CoolSNAP HQ 2 CCD camera. Deconvolution was performed using SoftWoRx Suite 2.0 (Applied Precision, GE) and further processed using ImageJ 1.34r software and Photoshop (Adobe Systems Inc., USA). Image acquisition was also conducted using a 100 x oil objective on a Zeiss Axioskope 2 MOT+ microscope attached to a Hamamatsu Orca-ER digital CCD camera using OpenLab 5.5.2 software (Improvision), and further processed using ImageJ and Adobe Photoshop.

LacZ analysis

To determine the efficiency of DiCre, BloxP parasites were treated with 50 nM rapamycin or 1 μM Shield-1 for 6 hours and grown under normal growth conditions for 48 h. Freshly lysed parasites were then added onto a monolayer of HFF and fixed 20 h later. The presence of LacZ was assessed by adding X-Gal (5-Bromo-4-Chloro-3-Indoyl-β-Dgalactopyranoside) as substrate. Intracellular parasites were then fixed in 4 % paraformaldehyde for 20 min and subsequently washed three times in PBS. Afterwards the samples were incubated with the staining solution for 30 min at 37 °C. The staining solution contains 6 mM potassium ferricyanide, 6 mM potassium ferrocyanide, 2 mM MgCl 2 , 0.2 % Igepal, 0.1 % Na deoxycholate, 1:40 dilution of X-Gal solution (40 mg / mL in N-Ndimethylformamide) in PBS. Afterwards the samples were rinsed 5 times in PBS. Experiment was performed in triplicate and 200 vacuoles were counted using a 100 x objective.

Invasion assay

To investigate the invasion rates of the loxPMyoA parasites (uninduced and induced), parasites were treated with 50 nM rapamycin in DMSO for 8 hours and grown in HFF cell monolayers for 96 hours. Afterwards, uninduced and induced parasites were scratched, filtered and 1 × 10 5 parasites were inoculated onto HFF monolayers per well. Parasites were allowed to invade for one hour at normal growth conditions (37 °C, 5 % CO 2 ), extracellular parasites were washed away with PBS and parasites were incubated for another 24 hours. Cells were fixed in 4 % paraformaldehyde for 20 min followed by immunostaining with α-IMC1 and α-Ty antibodies. The number of invaded parasites was counted in 20 fields using a 100 x objective, and calculated as a percentage value of uninduced loxPMyoA parasites normalised to 100 %.

Egress assay 4 x 10 5 parasites (loxPMyoA uninduced and induced 96 h earlier) were grown in HFF monolayers on coverslips for 36 h. Media was exchanged for pre-warmed, serum-free DMEM supplemented with 2 μM A23187 (in DMSO) in order to artificially induce egress 21 . After 10 min cells were fixed and stained with antibodies as indicated. 200 vacuoles were counted for each condition in three assays.

Plaque assay

1000 freshly lysed parasites were added onto a confluent monolayer of HFF cells and incubated for 5 days. The HFF monolayer was washed in PBS and fixed in ice cold methanol for 20 min. Afterwards, the HFFs were stained with Giemsa (1:10 in water) for 45 min and finally washed in PBS (3 x 5 min). Since methanol destroys the YFP signal, the act1 KO plaque assays were image without fixation to differentiate between YFP positive and negative parasites. To quantify the relative plaque size of the indicated strains after 5 days on HFF monolayers, the average size of 15 plaques was determined and displayed relative to ku80::diCre or RH hxgprt -± s.d, respectively. Plaque area was assessed using Image J 1.34r software.

FACS analysis

Freshly lysed parasites (mixed population expressing p5RT70loxPKillerRedloxPYFP-HX ) were pelleted and washed once in PBS. Parasites were resuspended in 1 ml of 4 % paraformaldehyde and incubated in the dark for 20 min. The suspension was then centrifuged, followed by another PBS wash. Finally, FACS buffer (1 % FCS in PBS supplemented by 1 mM EDTA) was added to achieve a final concentration of 1 x 10 6 parasites per ml. 10,000 events were counted using FACSCalibur Becton Dickinson LSRII.

FSC and SSC were detected as small and large angle scatter of the 488 nm laser. KillerRed was excited by the 561 nm laser and detected by a 585 / 15 band pass filter. The YFP was excited by the 488 nm laser and detected by a 530 / 30 band pass filter and 505 long pass filters. For quantification, total number of fluorescent parasites was considered 100%. μm. (e) Actin is essential for apicoplast replication. loxPAct1 parasites were treated with rapamycin for 4 hours, inoculated on HFF cells, and imaged after the indicated time. Act1 protein levels are significantly reduced in green-fluorescent (act1 KO) parasites after 24 hours and not detectable after 72 hours. The apicoplast, stained with α-HSP60, stops replicating, resulting in huge parasitophorous vacuoles containing few plastids. When parasites are artificially released from the host cell after 72 hours, YFP-expressing parasites can reinvade the host cell in absence of detectable Act1, but fail to replicate the apicoplast (72 h + 24 h). Scale bar: 5 μm.

Figure 1 .

 1 Figure 1. Establishing a conditional Cre-recombinase system in T. gondii (a) Schematic of the DiCre system. Cre recombinase is split: Cre1-59 (green) is fused to FKBP12; Cre60-343 (purple) is fused to FRB. Rapamycin (blue) dimerizes subunits and reconstitute Cre activity. (b) Immunoblot analysis of DiCre expression using α-FKBP12 (19 kDa) and α-FRB antibodies (46 kDa) in loxPlacZloxP reporter parasite line (RH BloxP); α-Catalase (57 kDa) is the loading control. (c) Quantification of Cre-mediated recombination using 50 nM rapamycin or 1 μM Shield-1 for 6 h. Data from three experiments are shown, ±s.d., **: P value of 0.0008 in a two-tailed Student's t-test. (d) Quantification of Cre-mediated recombination over time. RH BloxP parasites expressing FKBP-Cre59 and FRB-Cre60 were treated with 50 nM rapamycin. LacZ expression is determined 72 hours post-induction. Data represents mean values of three independent experiments ±s.d, **: P value < 0.0001 in a two-tailed Student's t-test. (e) Schematic of the gene-swap strategy. The endogenous gene-of-interest (GOI, magenta) is replaced by the indicated cassette; the floxed GOI-cDNA is followed by YFP and the selectable marker (HX). Cre-mediated excision of GOI-cDNA places YFP under control of the promoter (p5RT70), producing green-fluorescent KO parasites. (f,g) Analysis of RH-DiCre transfected with loxPKillerRedloxP-YFP. Expression of KillerRed or YFP was analysed 5 days post-induction (50 nM rapamycin, 8 h in (f) immunofluorescence and (g) FACS analysis. Scale bar: 20 μm; 10,000 events were analysed by FACS.

Figure 2 .

 2 Figure 2. Utilizing the DiCre system to dissect the invasion machinery (a) Current model of apicomplexan gliding and invasion machinery, and summary of KO parasites generated. Immunofluorescence analysis on the indicated KO strains demonstrates the complete absence of MyoA, MIC2 and Act1 respectively using the indicated antibodies. Scale bar: 5 μm. (b) Determination of plaque formation on a HFF monolayer by indicated parasite strains. The respective clonal KO strains (mic2 KO and myoA KO), although viable in vitro, show a significant growth defect. Area of 15 plaques is assessed, **: P value < 0.0001 in a two-tailed Student's t-test. (c) Localisation of other invasion machinery components (MLC1 and GAP45) are not affected in myoA KO parasites, as shown in IFA using the indicated antibodies; scale bar 5 μm. (d) Localisation of M2AP but not AMA1 is disrupted in mic2 KO parasites. IFA was performed using indicated antibodies, scale bar: 5

Nat Methods. Author manuscript; available in PMC 2013 August 01.

Acknowledgments

We thank D. Soldati-Favre (University of Geneva, Switzerland), C.J. Beckers (The University of North Carolina, USA), A. Scherf (Pasteur Institute, France), J.F. Dubremetz (University of Montpellier, France), V. Carruthers (University of Michigan, USA), B. Striepen (University of Georgia, USA) and D.L. Sibley (Washington University, USA) for sharing reagents. We thank the FACS facility of the Institute of Infection, Immunity and Inflammation at Glasgow University for their support. This work was supported by the Wellcome Trust. M.M. is funded by a Wellcome Trust Senior Fellowship [087582/Z/08/Z)]. N.A. is supported by an EviMalaR (European FP7/2007-2013, grant number 242095) PhD fellowship and S.E. was funded via MALSIG. The Wellcome Trust Centre for Molecular Parasitology is supported by core funding from the Wellcome Trust [085349].

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.


Simulation the agglomeration process of solid particles in the presence of a viscous liquid. We are mostly interested in application to iron ore granulation in a horizontal rotating drum. In this work, we use Molecular Dynamics (MD) method to simulate the agglomeration process during the dense granular flows in the rotary drum. In which particles are distributed by an uniform distribution of particle volume fractions.

Granulation (balling) Drum

Agglomeration is the process of particles size enlargement and most commonly refers to the upgrading of material fines into larger particles, such as pellets or granules. Iron ore granulation is an important stage in the steel making. Filling level: Packing fraction:

ROLLING -CASCADING MODEL

-Investigation the agglomeration process of a huge number of particles.

-Comparison between experiment and simulation of agglomeration processes in rotating drum.

Conclusions 1

The effect of size ratio on the granule growth is more crucial than that of rotational speed.

2 Granule growth is an exponential function of size ratio and rotational speed of drum.

3 Kinetic energy normalized by potential energy increases proportional to the rotational speed, but inversely proportional to the size ratio. 4 The wet and contact coordination numbers of agglomerate grains are proportional to size ratio.

m i d 2 s i dt 2 = X (i) ((f n + f c + f vis )n + f t t) + m i g s i g n t m i Liquid bridge R i i m i g f ij c f ij n f ij vis f ik vis f ik c f ij t m k g k R k R j j m j g n ! A B ' ! S L C C R c x z f = S ⇡R 2 c = ' sin ' 2⇡ = V s S ⇤ L = ⌃( 4 3 ⇡R 3 i ) S ⇤ L F r = ! 2 R c g C = AB = 2R c sin ' 2 S = R 2 c 2 (' sin ') LC = AB = 'R c f c = 8 > < > : R, for n < 0 Re n , for 0  n  max n 0, for n max n f vis = 3 2 ⇡R 2 ⌘ 1 n d n dt  = 2⇡ s cos ✓ max n = (1 + 1 2 ✓)V 1/3 b ↵ = R max R min = c h(↵)( V b R 0 ) 1 2 R = p R i R j Diagram of capillary bridge fracture d g
The snapshots of model and force chains of a granule under diametrical compression. Drum Revolutions

  et Génie Civil (LMGC), Université de Montpellier, CNRS, Montpellier, France IATE, UMR1208 INRA -CIRAD -Université de Montpellier -SupAgro, 34060 Montpellier, France <MSE>, UMI 3466 CNRS-MIT, CEE, MIT, 77 Massachusetts Avenue, Cambridge 02139

  kinetic energy normalized by potential energy of granule as function of Fr. Exponential increases of wet & contact coordination numbers (a) and decrease of kinetic energy normalized by potential energy of granule (b), as functions of size ratio 𝞪.

  α=3 V b =7.1.10 -18 (m 3 ), α=3 V b =1.7 10 -17 (m 3 ), α=4 V b =7.1.10 -18 (m 3 ), α=4 V b =1.7 10 -17 (m 3 ), α=5 V b =7.1.10 -18 (m 3 ), α=5

  Normalized peak strength as function of friction coefficient. Packing fraction and coordination number, as functions of size ratio c = s hRi ! Rotational speed (rad/s)


position vector of particle i mass of particle i (kg) vector gravity normal unit vector tangential unit vector

We investigate the agglomeration process of solid particles in the presence of a viscous liquid. We are mostly interested in application to iron ore granulation in a horizontal rotating drum. In this work, we use Molecular Dynamics (MD) method to simulate the agglomeration process during the dense granular flows in the rotary drum. In which particles are distributed by an uniform distribution of particle volume fractions.

Granulation (balling) Drum

Agglomeration is the process of particles size enlargement and most commonly refers to the upgrading of material fines into larger particles, such as pellets or granules. Iron ore granulation is an important stage in the steel making. Filling level: Packing fraction:

ROLLING -CASCADING MODEL

-Investigation the agglomeration process of a huge number of particles.

-Comparison between experiment and simulation of agglomeration processes in rotating drum.

Conclusions 1

The effect of size ratio on the granule growth is more crucial than that of rotational speed. 2 Granule growth is an exponential function of size ratio and rotational speed of drum. 3 Kinetic energy normalized by p o t e n t i a l e n e r g y i n c r e a s e s proportional to the rotational speed, but inversely proportional to the size ratio. 4 The wet and contact coordination numbers of agglomerate grains are proportional to size ratio.

m i d 2 s i dt 2 = X (i) ((f n + f c + f vis )n + f t t) + m i g s i g n t mi ! A B ' ! S L C C R c x z f = S ⇡R 2 c = ' sin ' 2⇡ = Vs S ⇤ L = ⌃( 4 3 ⇡R 3 i ) S ⇤ L F r = ! 2 Rc g C = AB = 2Rc sin ' 2 S = R 2 c 2 (' sin ') LC = AB = 'Rc fc = 8 > < > : R, for n < 0 Re n , for 0  n  max n 0, for n max n fvis = 3 2 ⇡R 2 ⌘ 1 n d n dt  = 2⇡ s cos ✓ max n = (1 + 1 2 ✓)V 1/3 b ↵ = Rmax Rmin = c h(↵)( Vb R 0 ) 1 2 R = p

  Granular material flow & granule growth in the cascading regime 28 th ALERT Workshop Exponential increase of granule for different Froude numbers. Exponential increase of granule for different size ratios Exponential increase of kinetic energy normalized by potential energy of granule as function of Fr. Exponential increases of wet & contact coordination numbers (a) and decrease of kinetic energy normalized by potential energy of granule (b), as functions of size ratio .

  et Génie Civil (LMGC), Université de Montpellier, CNRS, Montpellier, France. Bridge and Road Department, Danang Architecture University, 566 Nui Thanh St, Hai Chau Dist, Danang, Vietnam. IATE, UMR1208 INRA -CIRAD -Université de Montpellier -SupAgro, 34060 Montpellier, France. <MSE>, UMI 3466 CNRS-MIT, CEE, MIT, 77 Massachusetts Avenue, Cambridge 02139, USA.