Switchable lanthanide fluorescence probes in homogenous molecular diagnostics

The number of molecular diagnostic assays has increased tremendously in recent years.Nucleic acid diagnostic assays have been developed, especially for the detection of human pathogenic microbes and genetic markers predisposing to certain diseases. Closed-tube methods are preferred because they are usually faster and easier to perform than heterogenous methods and in addition, target nucleic acids are commonly amplified leading to risk of contamination of the following reactions by the amplification product if the reactions are opened. The present study introduces a new closed-tube switchable complementation probes based PCR assay concept where two non-fluorescent probes form a fluorescent lanthanide chelate complex in the presence of the target DNA. In this dual-probe PCR assay method one oligonucleotide probe carries a non-fluorescent lanthanide chelate and another probe a light absorbing antenna ligand. The fluorescent lanthanide chelate complex is formed only when the non-fluorescent probes are hybridized to adjacent positions into the target DNA bringing the reporter moieties in close proximity. The complex is formed by self-assembled lanthanide chelate complementation where the antenna ligand is coordinated to the lanthanide ion captured in the chelate. The complementation probes based assays with time-resolved fluorescence measurement showed low background signal level and hence, relatively high nucleic acid detection sensitivity (low picomolar target concentration). Different lanthanide chelate structures were explored and a new cyclic seven dentate lanthanide chelate was found suitable for complementation probe method. It was also found to resist relatively high PCR reaction temperatures, which was essential for the PCR assay applications. A seven-dentate chelate with two unoccupied coordination sites must be used instead of a more stable eight- or nine-dentate chelate because the antenna ligand needs to be coordinated to the free coordination sites of the lanthanide ion. The previously used linear seven-dentate lanthanide chelate was found to be unstable in PCR conditions and hence, the new cyclic chelate was needed. The complementation probe PCR assay method showed high signal-to-background ratio up to 300 due to a low background fluorescence level and the results (threshold cycles) in real-time PCR were reached approximately 6 amplification cycles earlier compared to the commonly used FRET-based closed-tube PCR method. The suitability of the complementation probe method for different nucleic acid assay applications was studied. 1) A duplex complementation probe C. trachomatis PCR assay with a simple 10-minute urine sample preparation was developed to study suitability of the method for clinical diagnostics. The performance of the C. trachomatis assay was equal to the commercial C. trachomatis nucleic acid amplification assay containing more complex sample preparation based on DNA extraction. 2) A PCR assay for the detection of HLA-DQA1*05 allele, that is used to predict the risk of type 1 diabetes, was developed to study the performance of the method in genotyping. A simple blood sample preparation was used where the nucleic acids were released from dried blood sample punches using high temperature and alkaline reaction conditions. The complementation probe HLA-DQA1*05 PCR assay showed good genotyping performance correlating 100% with the routinely used heterogenous reference assay. 3) To study the suitability of the complementation probe method for direct measurement of the target organism, e.g., in the culture media, the complementation probes were applied to amplificationfree closed-tube bacteriophage quantification by measuring M13 bacteriophage ssDNA. A low picomolar bacteriophage concentration was detected in a rapid 20- minute assay. The assay provides a quick and reliable alternative to the commonly used and relatively unreliable UV-photometry and time-consuming culture based bacteriophage detection methods and indicates that the method could also be used for direct measurement of other micro-organisms. The complementation probe PCR method has a low background signal level leading to a high signal-to-background ratio and relatively sensitive nucleic acid detection. The method is compatible with simple sample preparation and it was shown to tolerate residues of urine, blood, bacteria and bacterial culture media. The common trend in nucleic acid diagnostics is to create easy-to-use assays suitable for rapid near patient analysis. The complementation probe PCR assays with a brief sample preparation should be relatively easy to automate and hence, would allow the development of highperformance nucleic acid amplification assays with a short overall assay time.

Design and validation of stateful composite RESTful web services

A web service is a software system that provides a machine-processable interface to the other machines over the network using different Internet protocols. They are being increasingly used in the industry in order to automate different tasks and offer services to a wider audience. The REST architectural style aims at producing scalable and extensible web services using technologies that play well with the existing tools and infrastructure of the web. It provides a uniform set of operation that can be used to invoke a CRUD interface (create, retrieve, update and delete) of a web service. The stateless behavior of the service interface requires that every request to a resource is independent of the previous ones facilitating scalability. Automated systems, e.g., hotel reservation systems, provide advanced scenarios for stateful services that require a certain sequence of requests that must be followed in order to fulfill the service goals. Designing and developing such services for advanced scenarios with REST constraints require rigorous approaches that are capable of creating web services that can be trusted for their behavior. Systems that can be trusted for their behavior can be termed as dependable systems. This thesis presents an integrated design, analysis and validation approach that facilitates the service developer to create dependable and stateful REST web services. The main contribution of this thesis is that we provide a novel model-driven methodology to design behavioral REST web service interfaces and their compositions. The behavioral interfaces provide information on what methods can be invoked on a service and the pre- and post-conditions of these methods. The methodology uses Unified Modeling Language (UML), as the modeling language, which has a wide user base and has mature tools that are continuously evolving. We have used UML class diagram and UML state machine diagram with additional design constraints to provide resource and behavioral models, respectively, for designing REST web service interfaces. These service design models serve as a specification document and the information presented in them have manifold applications. The service design models also contain information about the time and domain requirements of the service that can help in requirement traceability which is an important part of our approach. Requirement traceability helps in capturing faults in the design models and other elements of software development environment by tracing back and forth the unfulfilled requirements of the service. The information about service actors is also included in the design models which is required for authenticating the service requests by authorized actors since not all types of users have access to all the resources. In addition, following our design approach, the service developer can ensure that the designed web service interfaces will be REST compliant. The second contribution of this thesis is consistency analysis of the behavioral REST interfaces. To overcome the inconsistency problem and design errors in our service models, we have used semantic technologies. The REST interfaces are represented in web ontology language, OWL2, that can be part of the semantic web. These interfaces are used with OWL 2 reasoners to check unsatisfiable concepts which result in implementations that fail. This work is fully automated thanks to the implemented translation tool and the existing OWL 2 reasoners. The third contribution of this thesis is the verification and validation of REST web services. We have used model checking techniques with UPPAAL model checker for this purpose. The timed automata of UML based service design models are generated with our transformation tool that are verified for their basic characteristics like deadlock freedom, liveness, reachability and safety. The implementation of a web service is tested using a black-box testing approach. Test cases are generated from the UPPAAL timed automata and using the online testing tool, UPPAAL TRON, the service implementation is validated at runtime against its specifications. Requirement traceability is also addressed in our validation approach with which we can see what service goals are met and trace back the unfulfilled service goals to detect the faults in the design models. A final contribution of the thesis is an implementation of behavioral REST interfaces and service monitors from the service design models. The partial code generation tool creates code skeletons of REST web services with method pre and post-conditions. The preconditions of methods constrain the user to invoke the stateful REST service under the right conditions and the post condition constraint the service developer to implement the right functionality. The details of the methods can be manually inserted by the developer as required. We do not target complete automation because we focus only on the interface aspects of the web service. The applicability of the approach is demonstrated with a pedagogical example of a hotel room booking service and a relatively complex worked example of holiday booking service taken from the industrial context. The former example presents a simple explanation of the approach and the later worked example shows how stateful and timed web services offering complex scenarios and involving other web services can be constructed using our approach.

Valmistavan tehtaan tuotannon tehostaminen reaaliaikaisen tiedon avulla

Teollinen toimintaympäristö on muuttunut globalisaation seurauksena. Sisäisten ja ulkoisten asiakkaiden vaatimukset ovat entistä haastavampia toimitusajan, laadun ja kustannusten suhteen. Yritysten on automatisoitava toimintoja, jotta voidaan saavuttaa kilpailuetua ja parantaa yrityksen tehokkuutta. Diplomityön tarkoitus on kirjallisuus- ja vertailututkimuksen yhteenvetona koostaa asiakasyrityksen tarpeisiin soveltuva toimintomalli reaaliaikaisen tiedon hyödyntä-miseen valmistavan tehtaan tuotannon tehostamisessa. Kirjallisuustutkimusten avulla pyritään analysoimaan valmistavan teollisuuden erityispiirteet toimintaympäristön osalta, kartoittamaan soveltuvin teknologia haastavaan toimintaympäristöön sekä löytää keinot kuinka reaaliaikaista tietoa hyödynnetään tuotannon tehokkuuden toteutumisessa. Vertailututkimuksessa analysoidaan kahta reaaliaikaista tietoa toiminnoissaan hyödyntävää yritystä, joiden tuloksia verrataan kirjallisuustutkimuksen aineistoon. Työn soveltavassa osuudessa luotiin toimintomalli asiakasyrityksen tarpeisiin, huomioiden tehtaan toiminnot kokonaisuutena. Toimintomallin avulla yritys pystyy toteuttamaan reaaliaikaisen tiedon keräämisen sekä hyödyntämisen yrityksen toi-minnoista. Toimintomallin toimivuus todettiin soveltamalla pilot-projekti asiakasyrityksen tuotantoon ja analysoitiin saavutettuja tuloksia haastattelujen sekä kvalitatiivisten tulosten perusteella. Työn tutkimukset osoittivat haasteet valmistavan teollisuuden toimintaympäristön ja automaatioteknologian soveltamisessa. Yritysten tulee suunnitteella toimintamallin kokonaisuus ja pohtia järjestelmän toimivuutta ongelmatilanteissa. Reaaliaikaisen tiedonkeruun soveltaminen onnistuneesti mahdollistaa hyötyjä monella tapaa. Merkittävimmät ovat tehokkuuden kehittyminen toimintojen nopeutumisena sekä resurssisäästöt toimintojen automatisoinnin johdosta. Reaaliaikaista tietoa voidaan hyödyntää myös suorituskyvyn johtamisessa mittariston avulla.

Sisäisten tuotevirtojen hallinta elintarviketeollisuudessa

Elintarvikealan yritysten tulee kehittää laadukkaita, kustannustehokkaita ja monipuolisen sisällön omaavia tuotteita elintarvikealan standardeja noudattaen nopeammin kuin koskaan. Uusien lopputuotteiden kehittämisen myötä myös puolivalmisteiden kokonaismäärä kasvaa, mikä vaikeuttaa erityisesti sisäisten tuotevirtojen hallittavuutta tehtaalla. Toimintaympäristön muutoksien seurauksena yritysten on automatisoitava toimintoja tehostaakseen tuotantoaan ja saavuttaakseen kilpailuetua. Tutkimuksen tekemiseen käytettiin kvalitatiivista eli laadullista lähestymistapaa, jonka tarkoituksena on tutkia kohdetta mahdollisimman monipuolisesti. Kvalitatiivisen tiedon hankkimiseen käytettiin haastatteluja sekä osallistuvaa ja passiivista tarkkailua. Toimintatutkimuksen tavoin tarkoituksena oli kehittää ratkaisu kohdeyrityksen ongelmaan soveltamalla olemassa olevaa teoreettista tietämystä ja kentällä tehtyjä havaintoja. Tutkimuksen tuloksena löydettiin parhaiten kohdeyrityksen tehdasympäristöön sisäisten tuotevirtojen hallintaan soveltuva seuranta- ja tunnistustekniikka. Lisäksi haastatteluiden pohjalta saatiin määritettyä liikkeellepanevat ja vastustavat voimat, jotka ohjaavat mahdollisen investointisuunnitelman etenemistä ja päätöksentekoa.

Roadmap for Service Charging Development

Corporate support functions are increasingly being concentrated into service centers. Service Management principles guide companies in the transition. Service Financial Management is an integral part in supporting the strategic positioning of the service center. The main goal of this thesis is to create a step-by-step plan to improve and automate the service charging processes for the finance service function of the case company. Automating the service transaction data collection for reporting is expected to improve efficiency, reliability and transparency. Interviews with finance service managers are held to define current processes and areas for improvement. These create the basis for the creation of a development roadmap that takes place in two phases. The first phase is to create an environment where automation is possible, and the second phase is the automation of each finance service. Benchmarking interviews are held with the service centers in three other companies to discover best practices. The service charging processes between the studied companies are found incompatible, and suggestions for process automation cannot be inferred. Some implications of Service Financial Management decisions to the strategy of the service center are identified. The bundling of services and charging them inside or outside of the goal-setting frame of the business unit can be used to support the strategic choice and customer acceptance of the service center.

Online MBPeT dashboard : enabling performance testing as a service in the cloud

In this thesis, tool support is addressed for the combined disciplines of Model-based testing and performance testing. Model-based testing (MBT) utilizes abstract behavioral models to automate test generation, thus decreasing time and cost of test creation. MBT is a functional testing technique, thereby focusing on output, behavior, and functionality. Performance testing, however, is non-functional and is concerned with responsiveness and stability under various load conditions. MBPeT (Model-Based Performance evaluation Tool) is one such tool which utilizes probabilistic models, representing dynamic real-world user behavior patterns, to generate synthetic workload against a System Under Test and in turn carry out performance analysis based on key performance indicators (KPI). Developed at Åbo Akademi University, the MBPeT tool is currently comprised of a downloadable command-line based tool as well as a graphical user interface. The goal of this thesis project is two-fold: 1) to extend the existing MBPeT tool by deploying it as a web-based application, thereby removing the requirement of local installation, and 2) to design a user interface for this web application which will add new user interaction paradigms to the existing feature set of the tool. All phases of the MBPeT process will be realized via this single web deployment location including probabilistic model creation, test configurations, test session execution against a SUT with real-time monitoring of user configurable metric, and final test report generation and display. This web application (MBPeT Dashboard) is implemented with the Java programming language on top of the Vaadin framework for rich internet application development. The Vaadin framework handles the complicated web communications processes and front-end technologies, freeing developers to implement the business logic as well as the user interface in pure Java. A number of experiments are run in a case study environment to validate the functionality of the newly developed Dashboard application as well as the scalability of the solution implemented in handling multiple concurrent users. The results support a successful solution with regards to the functional and performance criteria defined, while improvements and optimizations are suggested to increase both of these factors.