| 1. |
- Nilsson, Sara, 1990-
(författare)
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How requirements development could support design of effective and resource-efficient offerings
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- What a company offers its customers has to fulfil several different needs, desires, constraints, which can originate from multiple different sources that affect the offering throughout its life cycle. All these criteria have to come together and be translated into statements that can support the designer’s understanding of the offering’s purpose. This translation is done through a requirements development process to provide a controlled process to define statements that describe what the offering is supposed to fulfil.This research provides insights on key challenges and success factors in requirements development to support the design of effective and resource-efficient offerings. Namely, it identifies crucial sources and aspects to be considered, and a requirements development process demonstrating how to overcome identified challenges. By getting the requirements right from the beginning, sub-optimisation and unnecessary time and risks can be avoided. The consideration of accurate sources and aspects is considered to be one of the most important factors for the successful design of offerings. It is also in the earliest phases of design, that is to say requirements development, where one has the greatest possibility to affect the environmental impact of the offering. What is missing, however, is sufficient and appropriate support in industry on how to do so.The gap between the three areas of effectiveness and resource efficiency, design of integrated offerings, and requirements development has been investigated. Results are based on findings in the literature and in industry, identified primarily by qualitative studies. In the research, 15 different companies have been included through a number of interviews and discussions.Key sources and aspects to consider in the requirements development process are identified along with challenges, and success factors that can be utilised to overcome the identified challenges. This research’s final results include an adapted requirements development process that considers the earlier-mentioned sources and aspect, challenges, and success factors. Such a requirements development process should support the design of effective and resource-efficient offerings.
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| 2. |
- Starrsjö, Sara, et al.
(författare)
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Reduction of adsorbable organically bound halogens (AOX) formation at near-neutral pH chlorine dioxide bleaching of softwood kraft pulp
- 2020
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Ingår i: Holzforschung. - : Walter de Gruyter GmbH. - 0018-3830 .- 1437-434X. ; 74:6, s. 597-604
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Tidskriftsartikel (refereegranskat)abstract
- Recently, a new type of bleaching sequence, Elemental Chlorine Free (ECF) light with one D stage, has been developed. It combines the efficiency and high selectivity of chlorine dioxide (ClO2) bleaching with more environmental friendly oxygen based bleaching chemicals. This work examines the effect of pH on the formation of adsorbable organically bound halogens (AOX) in an intermediate D stage - a single ClO2 stage at the middle of an ECF light bleaching sequence. Carbon dioxide (CO2) is used to generate a bicarbonate buffer in situ, stabilizing the pH during the bleaching. Near-neutral pH is hypothesized to decrease the formation of strongly chlorinating species, so that the AOX formation is reduced. The results indicate that a near-neutral pH D stage can reduce the AOX content in the effluents with up to 30%. The ISO brightness was unchanged to a lower ClO2 consumption. The pulp viscosity was slightly higher after near-neutral pH D stage, but to its disadvantage a lesser delignification and removal of HexA was obtained. The degradation of HexA correlated well with the AOX, affirming earlier theories that HexA has a major impact on the AOX formation. The higher amounts of residual HexA and lignin resulted in more thermal yellowing of the pulps bleached with a near-neutral pH D stage.
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| 3. |
- Friedman, Mikaela, et al.
(författare)
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Engineering and characterization of a bispecific HER2 x EGFR-binding affibody molecule
- 2009
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 54, s. 121-131
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Tidskriftsartikel (refereegranskat)abstract
- HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G(4)S)(3) [(Gly(4)-Ser)(3)]-encoding gene fragment. The encoded 30 kDa affibody construct (Z(HER2))(2)-(G(4)S)(3)-(Z(EGFR))(2), with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.
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| 4. |
- Grönwall, Caroline, et al.
(författare)
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Selection and characterization of Affibody ligands binding to Alzheimer amyloid beta peptides
- 2007
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 128:1, s. 162-183
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Tidskriftsartikel (refereegranskat)abstract
- Affibody (Affibody) ligands specific for human amyloid beta (Abeta) peptides (40 or 42 amino acid residues in size), involved in the progress of Alzheimer's disease, were selected by phage display technology from a combinatorial protein library based on the 58-amino acid residue staphylococcal protein A-derived Z domain. Post-selection screening of 384 randomly picked clones, out of which 192 clones were subjected to DNA sequencing and clustering, resulted in the identification of 16 Affibody variants that were produced and affinity purified for ranking of their binding properties. The two most promising Affibody variants were shown to selectively and efficiently bind to Abeta peptides, but not to the control proteins. These two Affibody ligands were in dimeric form (to gain avidity effects) coupled to affinity resins for evaluation as affinity devices for capture of Abeta peptides from human plasma and serum. It was found that both ligands could efficiently capture Abeta that were spiked (100 microgml(-1)) to plasma and serum samples. A ligand multimerization problem that would yield suboptimal affinity resins, caused by a cysteine residue present at the binding surface of the Affibody ligands, could be circumvented by the generation of second-generation Affibody ligands (having cysteine to serine substitutions). In an epitope mapping effort, the preferred binding site of selected Affibody ligands was mapped to amino acids 30-36 of Abeta, which fortunately would indicate that the Affibody molecules should not bind the amyloid precursor protein (APP). In addition, a significant effort was made to analyze which form of Abeta (monomer, dimer or higher aggregates) that was most efficiently captured by the selected Affibody ligand. By using Western blotting and a dot blot assay in combination with size exclusion chromatography, it could be concluded that selected Affibody ligands predominantly bound a non-aggregated form of analyzed Abeta peptide, which we speculate to be dimeric Abeta. In conclusion, we have successfully selected Affibody ligands that efficiently capture Abeta peptides from human plasma and serum. The potential therapeutic use of these optimized ligands for extracorporeal capture of Abeta peptides in order to slow down or reduce amyloid plaque formation, is discussed.
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| 5. |
- Guldevall, Karolin, et al.
(författare)
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Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays
- 2010
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:11, s. e15453-
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Tidskriftsartikel (refereegranskat)abstract
- New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.
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| 6. |
- Lindström, Sara, et al.
(författare)
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A microwell array device with integrated microfluidic components for enhanced single-cell analysis
- 2009
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 30:24, s. 4166-4171
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Tidskriftsartikel (refereegranskat)abstract
- Increasing awareness of the importance of cell heterogeneity in many biological and medical contexts is prompting increasing interest in systems that allow single-cell analysis rather than conventional bulk analysis (which provides average values for variables of interest from large numbers of cells). Recently, we presented a microwell chip for long-term, high-throughput single-cell analysis. The chip has proved to be useful for purposes such as screening individual cancer and stem cells for protein/gene markers. However, liquids in the wells can only be added or changed by manually rinsing the chip, or parts of it. This procedure has several well-known drawbacks - including risks of cross-contamination, large dead volumes and laboriousness - but there have been few previous attempts to integrate liquid rinsing/switching channels in "ready-to-use" systems for single-cell analysis. Here we present a microwell system designed (using flow simulations) for single-cell analysis with integrated microfluidic components (microchannels, magnetically driven micropumps and reservoirs) for supplying the cell culture wells with reagents, or rinsing, thus facilitating controlled, directed liquid handling. It can be used totally independently, since tubing is not essential. The practical utility of the integrated system has been demonstrated by culturing endothelial cells in the microwells, and successfully applying live-cell Calcein AM staining. Systems such as this combining high-density microwell chips with microfluidic components have great potential in numerous screening applications, such as exploring the important, but frequently undetected, heterogeneity in drug responses among individual cells.
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| 7. |
- Lindström, Sara, et al.
(författare)
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High-Density Microwell Chip for Culture and Analysis of Stem Cells
- 2009
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Ingår i: PLos ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 4:9, s. e6997-
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Tidskriftsartikel (refereegranskat)abstract
- With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.
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| 8. |
- Lindström, Sara, et al.
(författare)
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Overview of single-cell analyses : microdevices and applications
- 2010
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 10:24, s. 3363-3372
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Forskningsöversikt (refereegranskat)abstract
- Numerous microdevices developed for single-cell analyses have been presented in the last decades. Practical usefulness in biological and clinical settings has become an important focus during the development and implementation of new structures and assays. Single-cell analysis has been applied in intracellular research, gene-and protein content and expression, PCR, cell culture and division, clone formation, differentiation, morphology, lysis, separation, sorting, cytotoxicity and fluorescence screens, antibody secretion, etc. as discussed here along with brief descriptions of the technical devices used for the studies, e. g. well-, trap-, pattern-, and droplet-based structures. This review aims to serve as an overview of available techniques for single-cell analysis by describing the different biological single-cell assays that have been performed to date and how each individual application requires a particular device design.
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| 9. |
- Lindström, Sara, et al.
(författare)
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PCR amplification and genetic analysis in a microwell cell culturing chip
- 2009
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; , s. 3465-3471
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Tidskriftsartikel (refereegranskat)abstract
- We have previously described a microwell chip designed for high throughput, long-term single-cell culturing and clonal analysis in individual wells providing a controlled way of studying high numbers of individual adherent or non-adherent cells. Here we present a method for the genetic analysis of cells cultured on-chip by PCR and minisequencing, demonstrated using two human adherent cell lines: one wild type and one with a single-base mutation in the p53 gene. Five wild type or mutated cells were seeded per well (in a defined set of wells, each holding 500 nL of culture medium) in a 672-microwell chip. The cell chip was incubated overnight, or cultured for up to five days, depending on the desired colony size, after which the cells were lysed and subjected to PCR directly in the wells. PCR products were detected, in the wells, using a biotinylated primer and a fluorescently labelled primer, allowing the products to be captured on streptavidin-coated magnetic beads and detected by a fluorescence microscope. In addition, to enable genetic analysis by minisequencing, the double-stranded PCR products were denatured and the immobilized strands were kept in the wells by applying a magnetic field from the bottom of the wells while the wells were washed, a minisequencing reaction mixture was added, and after incubation in appropriate conditions the expected genotypes were detected in the investigated microwells, simultaneously, by an array scanner. We anticipate that the technique could be used in mutation frequency screening, providing the ability to correlate cells' proliferative heterogeneity to their genetic heterogeneity, in hundreds of samples simultaneously. The presented method of single-cell culture and DNA amplification thus offers a potentially powerful alternative to single-cell PCR, with advantageous robustness and sensitivity
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| 10. |
- Lindström, Sara, et al.
(författare)
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Towards high-throughput single cell/clone cultivation and analysis
- 2008
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 29, s. 1219-1227
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Tidskriftsartikel (refereegranskat)abstract
- In order to better understand cellular processes and behavior, a controlled way of studying high numbers of single cells and their clone formation is greatly needed. Numerous ways of ordering single cells into arrays have previously been described, but platforms in which each cell/clone can be addressed to an exact position in the microplate, cultivated for weeks and treated separately in a high-throughput manner have until now been missing. Here, a novel microplate developed for high-throughput single cell/clone cultivation and analysis is presented. Rapid single cell seeding into microwells, using conventional flow cytometry, allows several thousands of single cells to be cultivated, short-term (72 h) or long-term (10-14 days), and analyzed individually. By controlled sorting of individual cells to predefined locations in the microplate, analysis of single cell heterogeneity and clonogenic properties related to drug sensitivity can be accomplished. Additionally, the platform requires remarkably low number of cells, a major advantage when screening limited amounts of patient cell samples. By seeding single cells into the microplate it is possible to analyze the cells for over 14 generations, ending up with more than 10 000 cells in each well. Described here is a proof-of-concept on compartmentalization and cultivation of thousands of individual cells enabling heterogeneity analysis of various cells/clones and their response to different drugs.
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