Les mastocytoses systémiques (SM) constituent un groupe hétérogène de maladies rares, caractérisées par l’accumulation anormale de mastocytes malins dans la moelle osseuse et dans d’autres organes extra-cutanés. La majorité des patients avec SM ont une mutation activatrice du gène KIT, le plus souvent la mutation KIT D816V, retrouvée chez plus de 90% de tous les patients. Cette mutation induit l’activation constitutive du récepteur KIT en déclenchant de façon aberrante une cascade de voies de signalisation, dont la voie PI3K/AKT et JAK/STAT5, aboutissant à l’inhibition de l’apoptose et à l’augmentation de la prolifération et de la survie des mastocytes malins. Cependant, l’efficacité des inhibiteurs de tyrosines kinases (ITKs) sur cette mutation est limitée à cause de la résistance et/ou de toxicité liée à un manque de spécificité. Il est donc nécessaire de trouver de nouvelles approches thérapeutiques afin de contourner cette résistance au cours des SM KIT D816V+ avancées. Nous avons utilisé une approche consistant à cibler de façon combinée des molécules activées en aval de KIT D816V, comme AKT et STAT5, par des inhibiteurs pharmacologiques. Ceci nous a permis d’identifier une combinaison synergique entre un inhibiteur d’AKT (GSK690693) et un inhibiteur de STAT5 (BP-1-102). Ces composés sont capables d’inhiber la prolifération des cellules KIT D816V+ seuls ou en combinaison, mais à de très fortes concentrations, malheureusement non utilisables en thérapeutique. Néanmoins, ces premiers résultats ont permis de valider AKT et STAT5 comme cibles potentielles dans le traitement des SM avancées. La seconde approche employée a été de cibler directement le récepteur KIT D816V par des inhibiteurs pharmacologiques. A l’issu d’un criblage, nous avons identifié trois composés - BLU2317, BLU2718 et DCC-2618 - capables d’inhiber sélectivement la phosphorylation de KIT D816V. Ces composés inhibent la prolifération des cellules ROSAKIT D816V et HMC-1.2, et induisent l’apoptose des cellules de façon dose-dépendante. Bien que les effets de ces trois composés soient similaires, DCC-2618 agit à des concentrations plus faibles par rapport aux composés BLU2317 et BLU2718. Afin d’apprécier l’efficacité in vivo de DCC-2618, nous avons d’abord établi un nouveau modèle de SM basé sur l’injection intraveineuse des cellules ROSAKIT D816V-Gluc exprimant la Gaussia luciferase (Gluc) dans des souris NSG. La présence de la Gluc sécrétée par les cellules ROSAKIT D816V-Gluc facilite la mise en évidence de prise de greffe et permet un contrôle précis de la progression de la maladie. Ce modèle reproduit, au bout de 4 semaines, chez toutes les souris greffées, une SM avancée similaire à celle retrouvée chez l’homme, avec atteinte de la moelle osseuse, du sang, de la rate et du foie, tandis que la dégradation de l’état général des souris n’est observée qu’à partir de 12 semaines. Ce nouveau modèle offre suffisamment de temps pour explorer la cinétique de la progression de la maladie et surtout pour effectuer des études pharmacologiques précliniques. L’évaluation de l’effet de DCC-2618 in vivo a été réalisée sur ce modèle. Etonnamment, DCC-2618 n’a pas été capable d’inhiber la progression de la maladie chez les souris traitées, bien qu’atteignant des concentrations élevées dans la moelle osseuse et le plasma des souris traitées. Néanmoins, DCC-2618 s’est montré capable d’inhiber la phosphorylation de KIT dans les cellules issues de la moelle osseuse des souris traitées. En revanche, contrairement aux effets observés in vitro, DCC-2618 a induit une surexpression de phospho-ERK1/2 dans les cellules malignes des souris greffées. Ceci suggère qu’ERK1/2 joue un rôle important dans la résistance au composé DCC-2618 et éventuellement à d’autres ITKs, indépendamment du récepteur KIT. ERK1/2 pourrait donc être une nouvelle cible thérapeutique d’intérêt dans le traitement des SM résistantes aux ITKs / Systemic mastocytosis (SM) is a heterogeneous group of rare diseases characterized by abnormal accumulation of malignant mast cells (MCs) in the bone marrow (BM) and other extra-cutaneous organs. The majority of SM patients have an activating mutation in the KIT gene, usually the D816V point mutation, which is found in more than 90% of all patients. This mutation induces constitutive activation of the KIT receptor by triggering a cascade of signaling pathways, including the PI3K/AKT and the JAK/STAT5 pathways, resulting in the inhibition of apoptosis and increased survival and proliferation of malignant mast cells. However, the efficacy of the tyrosine kinase inhibitors (TKIs) on this mutation is limited due to resistance and/or toxicity associated with a lack of specificity. It is therefore critical to find new therapeutic approaches to overcome this resistance to TKIs, particularly for advanced KIT D816V+ SM. In the present thesis, we have used an approach consisting in targeting molecules activated downstream of KIT D816V, such as AKT and STAT5, using pharmacological inhibitors in combination. This allowed us to identify a synergistic combination of an AKT inhibitor (GSK690693) and an inhibitor of STAT5 (BP-1-102). These compounds are able to inhibit proliferation of KIT D816V+ cells, alone or in combination, but at very high concentrations, unfortunately not useful therapeutically. Nevertheless, these initial results have validated STAT5 and AKT as potential targets for the treatment of advanced SM. The second approach used was to target directly the KIT D816V receptor by pharmacological inhibitors. After a large screening, we identified three compounds - BLU2317, BLU2718 and DCC-2618 - which selectively inhibit the phosphorylation of KIT D816V. These compounds inhibit the proliferation of ROSAKIT D816V and HMC-1.2 cells, and induce apoptosis of these cells in a dose-dependent manner. Although the effects of these three compounds are similar, the DCC-2618 compound acts at lower concentrations relative to BLU2317 and BLU2718 compounds. In order to assess the in vivo efficacy of DCC-2618, we first established a new model of SM based on intravenous injection of cells expressing Gaussia luciferase (Gluc), ROSAKIT D816V-Gluc cells, in NSG mice. The presence of the secreted Gluc in ROSAKIT D816V-Gluc cells facilitates the detection of engraftment and allows precise monitoring of disease progression. This model reproduced within four weeks, in all grafted mice, an advanced SM similar to the one found in humans, with neoplastic MCs infiltration in BM, blood, spleen and liver, while the terminal deterioration of the clinical condition of the mice was observed after 12 weeks. Thus, this new in vivo model allows modulating the aggressiveness of the disease by varying the number of injected cells. It provides sufficient time to explore the kinetics of disease progression and especially to conduct preclinical pharmacological studies. We then evaluated the effect of DCC-2618 compound in vivo on this model. Surprisingly, DCC-2618 was not able to inhibit disease progression in treated mice, although it reached high concentrations in the BM and the plasma of treated mice. Nevertheless, we showed that the compound was able to inhibit the phosphorylation of the KIT receptor in cells derived from the BM of treated mice. In addition, contrasting to the effects observed in vitro, DCC-2618 induced an over-expression of phospho-ERK1/2 in the malignant cells of transplanted mice. This suggests that ERK1/2 may play a critical role in the resistance to DCC-2618, and possibly to other TKIs, independently of the KIT receptor. ERK1/2 could thus be a new interesting therapeutic target in the treatment of advanced SM resistant to TKIs

TCR signal strength is critical for CD8+ T cell clonal expansion after antigen stimulation. Levels of the transcription factor IRF4 control the magnitude of this process through induction of genes involved in proliferation and glycolytic metabolism. The signaling mechanism connecting graded TCR signaling to the generation of varying amounts of IRF4 is not well understood. Here, using multiple methods to vary TCR signal strength and measure changes in transcriptional activation in single CD8+ T cells, we connect antigen potency to the kinetics of NFAT activation and Irf4 mRNA expression. T cells that transduce weaker TCR signals exhibit a marked delay in Irf4 mRNA induction resulting in decreased overall IRF4 expression in individual cells and increased heterogeneity within the clonal population. The activity of the tyrosine kinase ITK acts as a signaling catalyst that accelerates the rate of the cellular response to TCR stimulation, controlling the time to onset of Irf4 gene transcription. These findings provide insight into the signal transduction pathway accounting for the reduced clonal expansion of low affinity CD8+ T cells following infection. We also describe another context for ITK activity, autoreactive T cell migration. Here, we connect TCR signaling strength to modulation of selectin binding and autoreactive T cell-mediated pathology in an adoptive transfer model system of autoimmune disease. Understanding the signaling mechanisms linking changes in TCR signaling to CD8 T cell function is important in furthering the understanding of vaccine development and T cell adoptive immunotherapy.

Mitt syfte med examensarbetet har varit att undersöka hållningen till IT pedagogiken samt genusperspektivet utifrån några lärares och elevers erfarenheter med perspektiv på historieundervisningen på högstadiet. Arbetet har ställts i relation till tidigare forskning kring IT-pedagogik och genus samt kursplanerna för historia i högstadiet. I resultatdelen presenteras genomförda intervjuer med tre lärare samt en elevgrupp bestående av fyra elever. I genomförandet av intervjuerna har det används två olika metoder. Främst har jag utgått ifrån en gruppintervju med utgångspunkt i den kvalitativa forskingen samt tre enskilda intervjuer för att uppnå mitt resultat. Resultatet visar på olika erfarenheter och tankar kring IT och genus i undervisningen där olika perspektiv kring möjligheterna av IT-pedagogiken samt genusstrukturer presenteras. Nya möjligheter som presenteras är referenspunkter till nytt material samt potentialen att väcka elevernas motivation och utveckla deras erfarenheter. Även en problematisk kommunikation mellan pedagoger och elever uppvisas som följd detta av mitt resultat. Resultatet kring genusperspektivet i klassrummet samt IT-pedagogiken uppvisar även en situation där sociala och kulturella konstruktioner skapar en komplex bild mellan flickor och pojkar. Slutsatsen, är att det krävs en förändring kring de sociala konstruktionerna som finns mellan pojkar och flickor och en bättre kommunikation samt initiativtagande bland pedagoger som kan skapa en befogad plats för IT-pedagogik i undervisningen.

The High Luminosity upgrade of the Large Hadron Collider (HL-LHC), necessitates that the ATLAS experiment replace their current Inner Detector (ID) system. The new Inner Tracker (ITk) will be an all silicon detector, utilising both pixel and strip sensors, with the aim of performing as well, or better than the current system - but in a much more challenging environment. The ITk Strip detector will consist of 17888 modules, ∼ 700 of which will be produced in the Scandinavian ITk Cluster - a collaboration of Copenhagen, Lund, Oslo and Uppsala university and our industrial partner NOTE. This work encompasses the journey from individual components through industrial scale module assembly and on to performance evaluation studies at the DESY II testbeam facility. Optimisation studies were performed of the correlated multi-variable calibration necessary for a glue robot to precisely and reliably dispense the two component epoxy used in the bonding of front-end electronics to the silicon sensor. Procedures and tools were developed for integrating this process into an industrial workflow, and to account for future fundamental changes, such as a switch in the epoxy utilised. To demonstrate sufficient tracking performance of ITk strip modules, even at end-of-life, testbeam campaigns of pre-irradiated modules are conducted. These campaign serve as vi-tal feasibility studies for the ITk as a whole. Reconstruction of end-cap type modules have been historically tricky, due to their complex geometry. This work presents the full integration of semi-automated end-cap type module reconstruction in the Corryvreckan testbeam analysis framework. This represent a major improvement in turnover time from raw data to final result, making the previously impossible concept of live reconstruction during testbeam campaigns within reach.

After ten years of massive success, the Large Hadron Collider (LHC) at CERN is going for an upgrade to the next phase, The High Luminosity Large Hadron Collider (HL-LHC) which is planned to start its operation in 2029. This is expected to have a fine boost to its performance, with an instantaneous luminosity of 5.0×1034 cm-2s -1 (ultimate value 7.5×1034 cm-2s -1 ) with 200 average interactions per bunch crossing which will increase the fluences up to more than 1016 neq/ cm2 , resulting in high radiation damage in ATLAS detector. To withstand this situation, it was proposed to make the innermost layer with 3D silicon sensors, which will have radiation tolerance up to 2×1016 neq/cm2 with a Total Ionization Dose of 9.9 MGy. Two-pixel geometries have been selected for 3D sensors, 50 × 50 μm2 for Endcap (ring), which will be produced by FBK (Italy) and SINTEF (Norway), and 25 × 100 μm2 for Barrel (stave), will be produced by CNM (Spain). A discussion is made in this thesis about the production of FBK on both geometries, as they have made a breakthrough with their Stepper lithography process. The yield improved, specifically for the geometry 25 × 100 μm2 with two electrode readouts, which was problematic in the mask aligner approach. Their sensors were characterized electrically at waferlevel as well as after integration with RD53a readout chip (RoC) on single-chip cards (SCC) and were verified against Innermost Tracker criteria. The SCCs were sent for irradiation up to 1×1016 neq/cm2 and were tested under electron test beam, and a hit efficiency of 97% was presented. Some more SCCs have been sent to Los Alamos for irradiating them up to 1.5×1016 neq/cm2 fluence. As the 3D sensors will be mounted as Triplets, a discussion is also made on their assembly and QA/QC process. A reception testing and electrical testing setup both at room temperature and the cold temperature was made and discussed, with results from some early RD53a RoC-based triplets. The pre-production sensors are already evaluated, and soon they will be available bump-bonded with ITkPixV1 RoC for further testing.

The ATLAS detector at CERN's Large Hadron Collider has been instrumental in scientific discoveries,including the Higgs particle. As part of the High Luminosity (HL) upgrade, the current Inner Detector (ID) isbeing replaced by the new IInner Tracker (ITk), and Uppsala University is collaborating with industrypartner NOTE to produce around 700 modules for the ITk. The production process involves gluing hybridsto sensors using a glue robot, which currently lacks a complete metrology system needed to verify thegeometry of produced items. This project aims to integrate a Micro Epsilon ILD1900 laser sensor with theglue robot to enable accurate measurements in the z-axis. In order to reach this goal, a few differenttasks had to be completed. These included creating a measurement API to enable the utilisation of thelaser sensor, recommending modifications to the glue robot syringe and camera holder in order to holdthe laser sensor, performing validation tests of the laser sensor's measuring capability, developing a lasersensor mount for a mock-up glue robot used during the validation process and developing a holder for aUSB/RS422 converter necessary for communication with the laser sensor. As a result of the project, themeasurement API was successfully created and utilised, appropriate modifications to the syringe holderwere made, validation tests were conducted though deemed insufficient due to limitations of the setup,and, finally, the laser sensor mount for the mock-up glue robot and the USB/RS422 holder weresuccessfully designed and produced.

Image segmentation is a process to identify regions of interest from digital images. Image segmentation plays an important role in medical image processing which enables a variety of clinical applications. It is also a tool to facilitate the detection of abnormalities such as cancerous lesions in the brain. Although numerous efforts in recent years have advanced this technique, no single approach solves the problem of segmentation for the large variety of image modalities existing today. Consequently, brain MRI segmentation remains a challenging task. The purpose of this thesis is to demonstrate brain MRI segmentation for delineation of tumors, ventricles and other anatomical structures using Insight Segmentation and Registration Toolkit (ITK) routines as the foundation. ITK is an open-source software system to support the Visible Human Project. Visible Human Project is the creation of complete, anatomically detailed, three-dimensional representations of the normal male and female human bodies. Currently under active development, ITK employs leading-edge segmentation and registration algorithms in two, three, and more dimensions. A goal of this thesis is to implement those algorithms to facilitate brain segmentation for a brain cancer research scientist.

Syftet med denna rapport är att få mer kunskap om hur digitala läromedel kan främja elevers kunskapsbildning i matematikundervisningen. Denna kvalitativa c-uppsats omfattar två delar. Den första delen består i att utveckla ett verktyg för att kunna analysera datorprogram gjorda för matematikundervisning. Detta verktyg baseras på tidigare analysverktyg, litteratur samt egna erfarenheter. Den andra delen består i att analysera tre olika programvaror med hjälp av verktyget för att se hur det fungerar samt se på vilka sätt programvarorna främjar matematikundervisningen. Resultatet visar att verktyget fungerar bra och att de testade programmen är mycket olika och att alla har sina egna styrkor för att underlätta matematikundervisningen. Alla program har självklart även svagheter vilka främst består i att de inte tillräckligt tagit tillvara på de möjligheter det digitala mediet ger programmen.

T lymphocytes express either a preTCR, or a clonotyoic γδ TCR or αβ TCR together with the CD3-complex and the associated ζ-chain. TCR:CD3:ζ-signalling is crucial for T cell development and antigen-specific activation including proliferation, differentiation, effector functions and apoptosis of mature T cells. Protein tyrosine kinase (PTK) cascades lie at the heart of proximal TCR:CD3:ζ-signalling. The CSK-, SRC-, SYK- and TEC-family members C-terminal SRC kinase (CSK), lymphocyte-specific protein tyrosine kinase (LCK), ζ-chain associated protein tyrosine kinase of 70 kDa (ZAP-70) and interleukin-2-inducible T cell kinase (ITK), respectively, are the major T cell players. After TCR:CD3:ζ-complex triggering, activation of PTKs result in tyrosine phosphorylation signals. These include phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ-chains, adaptor proteins that nucleate the proximal LAT:SLP-76-signalosome controlling almost all TCR:CD3:ζ-induced signalling events. These events initiate Ca2+-flux, activation of mitogen-activated protein kinases (MAPKs), activation of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB), activation of nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) as well as actin reorganization, cell-adhesion and motility.Througout the last five decades, the immune system has been extensively investigated in vitro and in animal models such as the murine system. Additionally, studying and taking care of human primary immunodeficiency diseases (PIDs) has been seminal for our understanding of the human immune system as animal models not always recapitulates the subtleties found in men.In my doctoral thesis I report the first case of autosomal recessive human LCK-deficiency, a novel autosomal recessive mutation leading to human ZAP-70-deficiency and a novel autosomal recessive mutation leading to human ITK-deficiency. I provide detailed clinical, immunological and biochemical analyses especially of TCR:CD3:ζ-signalling and compare my findings to the well-established Lck-/-, Zap-70-/- and Itk-/- murine models.

T lymphocytes express either a preTCR, or a clonotypic γδ TCR or αβ TCR together with the CD3-complex and the associated ζ-chain. TCR:CD3:ζ-signalling is crucial for T cell development and antigen-specific activation including proliferation, differentiation, effector functions and apoptosis of mature T cells. Protein tyrosine kinase (PTK) cascades lie at the heart of proximal TCR:CD3:ζ-signalling. The CSK-, SRC-, SYK- and TEC-family members C-terminal SRC kinase (CSK), lymphocyte-specific protein tyrosine kinase (LCK), ζ-chain associated protein tyrosine kinase of 70 kDa (ZAP-70) and interleukin-2-inducible T cell kinase (ITK), respectively, are the major T cell players. After TCR:CD3:ζ-complex triggering, activation of PTKs results in tyrosine phosphorylation signals. These include phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ-chains, and adaptor proteins that nucleate the proximal LAT:SLP-76-signalosome controlling almost all TCR:CD3:ζ-induced signalling events. These events initiate Ca2+-flux, activation of mitogen-activated protein kinases (MAPKs), activation of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB), activation of nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) as well as actin reorganization, cell-adhesion and motility. Throughout the last five decades, the immune system has been extensively investigated in vitro and in animal models such as the murine system. Additionally, studying and taking care of human primary immunodeficiency diseases (PIDs) has been seminal for our understanding of the human immune system as animal models not always recapitulate the subtleties found in men. In my doctoral thesis I report the first case of autosomal recessive human LCK-deficiency, a novel autosomal recessive mutation leading to human ZAP-70-deficiency and a novel autosomal recessive mutation leading to human ITK-deficiency. I provide detailed clinical, immunological and biochemical analyses especially of TCR:CD3:ζ-signalling and compare my findings to the well-established Lck-/-, Zap-70-/- and Itk-/- murine models.