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In biology, it is common to study cultured cells (in vitro) with fluorescence time-lapse microscopy. The cells are recorded for longer period of time and can later be viewed at an accelerated speed. During the acquisition some live cells tend to migrate. This can be a problem if the cell’s migration speed is high enough to move outside the field of view (FOV) during the acquisition time. The cells that moves outside the FOV can no longer be recorded and the information about them will be lost. This thesis presents scripts that have been developed for ZEN (blue) to be able to track a specific migrating cell of interest in real-time with automated control of imaging parameters. The microscope stage position is modified on-the-fly to have the tracked cell in the center of the FOV for the whole experiment. Three different types of experiments to track migrating NK cells were performed with the scripts. The results show that the scripts were able to track one NK cell for more than 1 hour in both conventional wide-field and lattice light-sheet microscopy. The segmentation was inaccurate when one or more objects were in close proximity to the tracked cell. By applying a watershed algorithm the segmentation result can be improved in some cases. / Inom cellulär biologi är det vanligt att studera odlade celler (in vitro) med time- lapse-mikroskopi. Flertals bilder tas på cellerna under en längre tidsperiod och när experimentet är klart så kan man titta på bilderna som en video. Under förvärvet av bilderna så tenderar vissa levande celler att migrera. Ett problem som kan uppstå är om cellens migrationshastighet är tillräckligt hög för att röra sig utanför synfältet under anskaffningstiden. De celler som rör sig utanför synfältet kan inte längre avbildas och informationen om dem kommer att gå förlorad. I denna avhandling presenteras programmeringskoder som har utvecklats för ZEN (blue) som kan spåra en specifik migrerande cell i realtid med automatiserad kontroll av bildbehandlings parametrar. Mikroskopets scenposition modifieras under experimentets gång för att få den spårade cellen kontinuerligt i mitten av synfältet. Tre olika sorters experiment i kombination med programmeringskoderna utfördes för att spåra NK-celler. Resultaten visar att programmeringskoderna lyckades spåra en NK-cell i mer än 1 timme i både ett bredfältsfluorescensmikroskop och ett lattice light-sheet mikroskop. Segmenteringen var felaktig när ett eller flera objekt var i närheten av den spårade cellen. Genom att tillämpa en watershed algoritm kan segmenteringsresultatet förbättras i vissa fall.

Oocyte activation deficiency (OAD) is an infertile condition observed in patients who have experienced recurrent total fertilisation failure (TFF) following intracytoplasmic sperm injection treatment. This condition was considered to be an idiopathic factor for a long time but strong clinical evidence now suggests that dysfunctional forms of phospholipase C zeta (PLCζ) may be predominant causative factors for OAD. Genetic contribution has played a role in patients suspected of having OAD, as four PLCζ exonic mutations have been discovered and characterised as being the cause of infertility. In this study, a novel nonsense mutation, PLCζK322Stop, was identified in the PLCζ XY-linker region of Patient LR. This variant results in the truncation of approximately half of PLCζ, therefore was non-functional when activity was tested. Patient LR, which also exhibited a previously reported mutation, PLCζH233L, may suggest that the patient is sub-fertile, as opposed to being infertile, as initially expected. Although research has purely focused upon the coding regions of PLCζ, it was obvious that our knowledge of PLCζ regulatory elements remain very limited. Next generation sequencing (NGS) was therefore employed to detect variants in the non-coding regions of PLCζ, promoter and introns, which may have resulted in the observed phenotypic diversity of PLCζ expression in fertile and infertile patients. As a result of mapping failure, an alternative approach was considered to identify variants within human PLCζ, and this involved using the single nucleotide polymorphism (SNP) database. Over 2500 SNPs were localised in the intronic regions of PLCζ and thus, it could be speculated that these variants may help elucidate the wide variation of PLCζ expression reported. Additionally, two particular patients with TFF (79 and 107) were investigated in this study to identify an association with PLCζ and their infertile state. For Patient 79, multiple PLCζ immunofluorescence analysis was performed and a significant improvement in PLCζ expression was observed one year after his first investigation. This may have been the result of an external factor, which influenced protein expression. As for Patient 107, a novel substitution mutation, PLCζV193E, was identified and was predicted to affect PLCζ stability and folding. There is global interest to create a safer and alternative OAD therapy, namely a human recombinant PLCζ protein (hrPLCζ). The first method, using a bacterial cell line resulted in successful purification and identification but the product proved to be inactive following mouse oocyte microinjection. The second method involved production of a mammalian-expressed hrPLCζ, which was successfully purified and identified but due to time restrictions, could not be tested for functionality. Concurrently, the findings in this thesis have reinforced the association between PLCζ and OAD, and provided improved options for the diagnosis and treatment of OAD.

A l’avenir, l'agriculture et l'environnement vont pouvoir bénéficier de plus en plus de données hétérogènes collectées par des réseaux de capteurs sans fil (RCSF). Ces données alimentent généralement des outils d’aide à la décision (OAD). Dans cette thèse, nous nous intéressons spécifiquement aux systèmes sensibles et adaptatifs au contexte basés sur un RCSF et un OAD, dédiés au suivi de phénomènes naturels. Nous proposons ainsi une formalisation pour la conception et la mise en œuvre de ces systèmes. Le contexte considéré se compose de données issues du phénomène étudié mais également des capteurs sans fil (leur niveau d’énergie par exemple). Par l’utilisation des ontologies et de techniques de raisonnement, nous visons à maintenir le niveau de qualité de service (QdS) des données collectées (en accord avec le phénomène étudié) tant en préservant le fonctionnement du RCSF. Pour illustrer notre proposition, un cas d'utilisation complexe, l'étude des inondations dans un bassin hydrographique, est considéré. Cette thèse a produit un logiciel de simulation de ces systèmes qui intègre un système de simulation multi-agents (JADE) avec un moteur d’inférence à base de règles (Jess). / In the future, agriculture and environment will rely on more and more heterogeneous data collected by wireless sensor networks (WSN). These data are generally used in decision support systems (DSS). In this dissertation, we focus on adaptive context-aware systems based on WSN and DSS, dedicated to the monitoring of natural phenomena. Thus, a formalization for the design and the deployment of these kinds of systems is proposed. The considered context is established using the data from the studied phenomenon but also from the wireless sensors (e.g., their energy level). By the use of ontologies and reasoning techniques, we aim to maintain the required quality of service (QoS) level of the collected data (according to the studied phenomenon) while preserving the resources of the WSN. To illustrate our proposal, a complex use case, the study of floods in a watershed, is described. During this PhD thesis, a simulator for context-aware systems which integrates a multi-agent system (JADE) and a rule engine (Jess) has been developed.Keywords: ontologies, rule-based inferences, formalization, heterogeneous data, sensors data streams integration, WSN, limited resources, DSS, adaptive context-aware systems, QoS, agriculture, environment.