The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease. Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells. S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients. Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity. Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.

Cell cultivation in bioreactors would benefit from developed monitoring systems with online real-time imaging to evaluate cell culture conditions and processes. This opportunity can be provided with the newly developed in situ Microscope also called ISM. The ISM probe is mounted into the wall of a bioreactor and consists of a measurement zone with an illuminating light source to obtain real-time images of moving cells in suspension. The instrument is linked to advanced imaging analysis software which can be specifically adapted for the objects in study. The aim of this project is to analyze the T lymphocyte cell line Jurkat T cells using the ISM equipment and identify specific features of the cells that can be obtained. The results show that the equipment and linked software are suitable for monitoring cell density, cell size distribution and cell surface analysis of the Jurkat cells during cultivation. The ISM could also detect induced changes in cell size caused by osmotic shifts and the course of an infection occurring in the cell suspension using a developed software for online real-time monitoring.

The freshwater crayfish, Pacifastacus leniusculus, has been used as a model for studying hematopoiesis or blood cell production or hematopoiesis and immunity. The work of this thesis aims to investigate the impact of factors such as ROS signaling, Ast1, and the PVF/PVR signaling pathway in controlling stem cell behavior during hematopoiesis and specifically the role of the crosslinking enzyme transglutaminase (TGase) in regulation of hematopoiesis. The role of ROS in crayfish hematopoiesis was characterized by using the antioxidant named NAC to inhibit ROS production. Low ROS level resulted in a prolonged decrease in hemocyte numbers and a combined injection of LPS and NAC caused a slower rate of new hemocyte production. A low ROS level in cell cultures supplemented with crude Ast1 was found to inhibit cell spreading and a high extracellular TGase activity was detected on the surfaces of APC and HPT cells. We suggest that ROS serves as a prime signal to control proliferation and differentiation of progenitor cells by affecting extracellular TGase activity. We reported an inhibitory effect of Ast1 on TGase enzyme activity and on its crosslinking activity and consequently Ast1 affects the clot formation and thus coagulation by inhibiting the crosslinking activity of the TGase enzyme. Secretion of the clot protein (CP) and the production of CP filament network between spreading cells were observed in HPT cell cultures in vitro. In the presence of CP together with Ast1 in 3D-collagen-I cultures, HPT cells were found to be more elongated and they formed chains of cells throughout the surrounding matrix. In the HPT tissue, CP was located around the HPT cells or around the lobules of HPT, and thus, CP was demonstrated to be a part of ECM and to possibly function together with collagen in generating a suitable environment for HPT progenitor cells. The inhibition of PVF/PVR downstream signaling pathway by Sunitinib malate resulted in a dramatic change of cell morphology and induction of an increase cell surface area during cell culture. The addition of crude Ast1 into the cell cultures in vitro enhanced this effect. Consequently, cell migration was stimulated and a high extracellular TGase activity on HPT cell surface was found after this inhibition. In conclusion, the work in this thesis provides new insight in understanding the role of the extracellular matrix (ECM) and extracellular TGase activity in controlling stem cell activity.

Olika typer av blodcancer är några av de vanligast förekommande cancerformerna i världen. Ju tidigare sjukdomen upptäcks och beroende på vilken typ av blodcancer någon drabbats av ser prognosen i många fall god ut då dagens behandlingsmetoder under flera år effektiviserats. Dock finns det fall där patienter inte svarar på traditionella terapier som cytostatika och stålning eller gånger där sjukdomen kommer tillbaka i aggressivare former. I dessa fall krävs andra metoder för att försöka behandla och bota cancern.   En metod som på senare tid fått mycket uppmärksamhet i forskarvärlden är användandet av kroppens egna immunceller som behandlingsform mot blodcancer. Genom att rena fram en patients egna T-celler från ett blodprov, kan dessa sedan genetiskt modifieras till att känna igen specifika tumörassocierade antigener (TAA) som bara vissa typer av cancerceller uttrycker. Metoden går ut på att T-cellerna får chimära antigen-receptorer (CAR), som uttrycks på cellytan, där CAR-T cellerna sedan injiceras tillbaka till patienten. CAR-T cellerna känner igen cancercellerna och attackerar sedan, med målet att patienten efter behandlingen inte ska ha några cancerceller kvar i kroppen.   Kliniska försök gjorda på patienter med olika typer av blodcancer har visat lovande resultat, särskilt gällande patienter som fått återfall av blodcancertypen Akut Lymfatisk Leukemi (ALL). De som fått delta i studierna har haft mycket dåliga prognoser och har innan blivit behandlade med de konventionella behandlingsterapierna, men utan eller med mycket dåligt resultat. Förhoppningar som väckts från dessa forskningsresultat har lett till diskussion att CAR-T celler kan komma att förändra cancervården och i framtiden kanske vara en lika vanlig behandlingsmetod som strålning eller kemoterapi. Dock kvarstår många problem som forskarna måste lyckas lösa innan CAR-T celler kan räknas som konventionell. Bland annat finns stora risker att patienter vid behandling kan drabbas av cytokinfrisläppningssyndrom (CRS, eng. cytokine release syndrome), där immunförsvaret kan attackera kroppens egna organ som i värsta fall kan leda till döden. Cancercellerna kan även komma tillbaka efterbehandling, då de har utvecklat en resistens mot CAR-T cellerna. Metoder för att undvika dessa toxiska responser och göra T-cellerna mer effektiva är bara några av de problem som kvarstår.

This study deals with the use of 3D-dynamic visualizations for teaching complex molecular cell biology concepts. The focus is on signal transduction, which is a concept that constitutes an important part of biological systems. 3D-dynamic visualizations (animations) were produced and shown for a total of 24 students attending a course in molecular cell biology at Karlstad University, Sweden. Data were collected by questionnaires and interviews which were structured around the understandability and usefulness of the animations. The results indicate that animations are useful for teaching life science concepts and can serve as a complement to lectures. They are useful for visualizing continuous time-dependent processes like signal transduction chains. Several connections between students' issues of understanding and layout-issues of the animations were established. A number of implications follow from the study. Basic understanding of animations is fundamental for understanding of advanced concepts, which should be kept in mind in the design phase of production. The level of realism of different factors in animations, like molecule speed and distances, has to be set to strike a balance between conceptual understanding and scientific correctness. Visualization of 3D-structure of molecules provides an understanding of molecule and systemic function. The study reinforces the need to use visualizations in life science teaching. / Denna studie behandlar användningen av 3D-dynamiska visualiseringar för att lära ut komplexa koncept i molekylär cellbiologi. Fokus är på signalöverföring, vilket är ett koncept som är en viktig del av biologiska system. 3D-dynamiska visualiseringar producerades och visades för totalt 24 studenter närvarande på en kurs i molekylär cellbiologu vid Karlstad Universitet, Sverige. Data samlades genom frågeformulär och intervjuer strukturerade runt förståelse och användning av animeringarna. Resultaten indikerar att animeringar är användbara för att lära ut koncept inom livsvetenskap och kan vara ett komplement till lektioner. De är användbara för att visualisera kontinuerliga tidsberoende processer som signalöverföringskedjor. Flera kopplingar mellan frågeställningar för studenternas förståelse och layout-frågeställningar för animeringarna fastställdes. Studien medför ett antal följder. Grundläggande förståelse av animeringar är fundamentalt för förståelse av avancerade koncept, vilket ska betänkas vid designfasen av produktion. Nivån av realism av olika faktorer i animeringarna, som molekylhastighet och avstånd, måste sättas för få balans mellan konceptuell förståelse och vetenskaplig riktighet. Visualisering av molekylers 3D-struktur ger förståelse av molekyl och systemisk funktion. Studien stärker behovet av att använda visualisering i undervisning av livsvetenskap. / <p>Chemistry education, kemididaktik</p>

This thesis describes the use of antibodies and immunofluorescence for subcellular localization of proteins. The key objective is the creation of an open-source atlas with information on the subcellular location of every human protein. Knowledge of the spatial distribution and the precise location of a protein within a cell is important for its functional characterization, and describing the human proteome in terms of compartment proteomes is important to decipher cellular organization and function.   Immunofluorescence and confocal microscopy of cultured cells were used for high-resolution detection of proteins on a high-throughput scale. Critical to immunofluorescence results are sample preparation and specific antibodies. Antibody staining of cells requires fixation and permeabilization, both of which can result in loss or redistribution of proteins and masking of epitopes. A high-throughput approach demands a standardized protocol suitable for the majority of proteins across cellular compartments. Paper I presents an evaluation of sample preparation techniques from which such a single fixation and permeabilization protocol was optimized. Paper II describes the results from applying this protocol to 4000 human proteins in three cell lines of different origin.   Paper III presents a strategy for application-specific antibody validation. Antibodies are the key reagents in immunofluorescence, but all antibodies have potential for off-target binding and should be validated thoroughly. Antibody performance varies across sample types and applications due to the competition present and the effect of the sample preparation on antigen accessibility. In this paper application-specific validation for immunofluorescence was conducted using colocalization with fluorescently tagged protein in transgenic cell lines. / <p>QC 20160509</p>

Cell assay that evaluates a biologics’ potency and efficacy is an important part in the discovery and development of drug candidates. However, it requires regular maintenance of cell cultures, and the cell assays can only be performed when the cells have reached 70-80% confluency. By instead using assay-ready cells, the drugs can be screened at any time by simply thawing the cells. This creates a more flexible assay, while saving time, labor and materials in addition to removing day-to-day variability. In this report, the freezing conditions for HEK-Blue™ CD40L cells are evaluated using the assay-ready cell method compared to a continuous culture. Via colorimetric detection, the CD40 receptors’ activation can be determined and a dose-response curve of a CD40 agonist can be produced. The optimal freezing condition for the assay-ready cells were determined to be 10% DMSO and a cell concentration between 1-30 million cells/mL. After reproducibility, robustness and screening tests, it could be concluded that the method generally produced results that had no significant difference to a continuous culture. Some of the assay-ready cells display a higher background which can affect the value of the efficacy. The source of the background will have to be evaluated in future studies. The potency, on the other hand, is stable regardless of cell method or high background.

No description available.

A crucial step during apoptosis is the accumulation of the pro-apoptotic protein Bax on the mitochondria where it triggers permeabilization of the outer membrane. This causes the release of cytochrome c into the cytosol and is considered a point of no return in programmed cell death. Endophilin B1, also known as Bax-interacting factor 1 (Bif-1) stimulates mitochondrial recruitment of Bax during apoptosis and loss of endophilin B1 is noted in many cancer types. Despite the importance of their interaction its role and function during cell death remains unclear. To examine the molecular mechanism behind their interaction this project aimed at solving the structure of endophilin B1-Bax complexes when bound to membrane mimicking platforms known as nanodiscs (NDs). NDs are composed of a lipid bilayer held together by a membrane scaffolding protein (MSP) that encircles the bilayer creating a disc-shaped structure. By designing NDs that resembles the mitochondrial outer membrane (MOM), this study intended to stimulate complex formation and stable binding to nanodiscs with the ambition of visualising their interaction using Cryo-EM. Due to difficulties of expressing and purifying Bax as well as time consuming optimization of ND assembly the final goal could not be reached. By establishing an optimized protocol for NDs using the MSP variant MSP2N2 and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) lipids as well as identifying challenges of expressing and purifying Bax this study lays ground for future structural studies that aims at elucidating the molecular mechanism behind the interaction.

No description available.