The activity of protest movements in 1956-1977 in Denmark and Sweden

Bachelor thesis "The Activity of Protest Movements in 1956-1977 in Denmark and Sweden" deals with the activities of social movements that originated in the territory of Denmark and Sweden in connection with the Western wave of protest activism and the overall radicalization of youth in the 1960s and the 1970s. It was especially a movement against nuclear weapons and the war in Vietnam and the student revolt of 1968. The paper analyzes the circumstances, the protest actions and subsequent consequences of activity of these movements in both countries and its objective is a complex comparison of the Danish and the Swedish cases. This comparison subsequently highlights the differences between the protest movements of Denmark and Sweden in various aspects, which are described in the text. One of them is the development of left-wing movement after 1956 and the emergence of new intellectual currents that influenced the direction of the protest movements. Other parts are devoted to the course and the extent of demonstrations of particular movements and their content, goals and expressions, for example through alternative culture. The thesis also provides the characteristics of the socio-political situation in Denmark and Sweden in the given period, which imply the specifics of the development of protest..

MALDI-TOF mass spectrum of the AMB-PEG fraction from RPC.

<p>The major mass peaks observed had masses corresponding to that of the AMB-PEG conjugate, thereby verifying the identity of that peak fraction. AMB-PEG mass peaks were absent from the collected unconjugated AMB fraction.</p

Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity.

<p>20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A₃₄₈/A₄₀₉ ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.</p

MIC₅₀ (μM) of various fungal species.

<p>MIC₅₀ (μM) of various fungal species.</p

LIVE/DEAD staining of HEK293 and IMR-90 cells after exposure to AMB-PEG 1, 2 and unconjugated AMB for 24 hours.

<p>Live cells are stained green and dead cells stained red. AMB-PEG did not cause cell death at concentrations of 139 μM in HEK293 cells and 277 μM in IMR-90 cells. The molar ratio of AMB to PEG did not have any visible effect on cell toxicity. Conversely, unconjugated AMB caused extensive cell death at concentrations above 4.33 μM in both cell lines. Experiment was performed twice, each time with three independently prepared AMB-PEG formulations.</p

Identification of Autocrine Growth Factors Secreted by CHO Cells for Applications in Single-Cell Cloning Media

Chinese hamster ovary (CHO) cell lines are widely used for the expression of therapeutic recombinant proteins, including monoclonal antibodies and other biologics. For manufacturing, cells derived from a single-cell clone are typically used to ensure product consistency. Presently, fetal bovine serum (FBS) is commonly used to support low cell density cultures to obtain clonal cell populations because cells grow slowly, or even do not survive at low cell densities in protein-free media. However, regulatory authorities have discouraged the use of FBS to reduce the risk of contamination by adventitious agents from animal-derived components. In this study, we demonstrated how a complementary mass spectrometry-based shotgun proteomics strategy enabled the identification of autocrine growth factors in CHO cell-conditioned media, which has led to the development of a fully defined single-cell cloning media that is serum and animal component-free. Out of 290 secreted proteins that were identified, eight secreted growth factors were reported for the first time from CHO cell cultures. By supplementing a combination of these growth factors to protein-free basal media, single cell growth of CHO cells was improved with cloning efficiencies of up to 30%, a 2-fold improvement compared to unsupplemented basal media. Complementary effects of these autocrine growth factors with other paracrine growth factors were also demonstrated when the mixture improved cloning efficiency to 42%, similar to that for the conditioned medium

Production of Functional Soluble Dectin-1 Glycoprotein Using an IRES-Linked Destabilized-Dihydrofolate Reductase Expression Vector

<div><p>Dectin-1 (CLEC7A) is a C-type lectin receptor that binds to β-glucans found in fungal cell walls to act as a major pattern recognition receptor (PRR). Since β-glucans epitope is not present in human cells, we are of the opinion that Dectin-1 can have therapeutic functions against fungal infections. We thus set out to produce a soluble extracellular domain of murine Dectin-1 (called sDectin-1) in sufficient titers to facilitate such studies in mouse models. Since sDectin-1 has previously been shown to be glycosylated, we chose to produce this protein using Chinese Hamster Ovary (CHO) cells, a mammalian host cell line suitable for the high-titer production of recombinant glycoproteins. To ensure a high titer production of sDectin-1 and minimize the effects of gene fragmentation, we constructed a mammalian expression vector with a PEST-destabilized dhfr amplifiable marker downstream of an attenuated IRES element, which was in turn downstream of the sDectin-1 gene and a CMV IE promoter. Stably transfected and MTX-amplified cell pools were generated using this vector, and maximum sDectin-1 titers of 246 mg/l and 598 mg/l were obtained in shake flask batch culture and bioreactor fed-batch culture respectively. The purified recombinant sDectin-1 was shown to be glycosylated. Protein functionality was also demonstrated by its ability to bind to zymosan particles and to the cell wall of <em>Saccharomyces cerevisiae</em>. We describe for the first time the use of an attenuated IRES-linked PEST-destabilized dhfr amplifiable marker for the production of recombinant proteins with stably amplified cell pools. With our process, we reached the highest reported titer for producing recombinant proteins smaller than 50 kDa in cell pools. sDectin-1 protein produced is glycosylated and functional. This vector design can thus be used efficiently for the high-titer production of functional recombinant proteins.</p> </div

Binding of cHis-sDectin-1 to <i>Saccharomyces cerevisiae</i> cells.

<p>Purified cHis-sDectin-1 was diluted to a concentration of 25 µg/ml in blocking buffer with 500 µg/ml, 100 µg/ml or no laminarin. This was added to <i>Saccharomyces cerevisiae</i> yeast cells from overnight culture in blocking buffer (PBS with 3% FBS). The cells were then probed with an mDectin-1 goat polyclonal antibody (1∶200; AF1756; R&D Systems) and AlexaFluor546-conjugated anti-goat antibody (1∶100; Catalog Number A-11056; Molecular Probes). After which, the cells were washed with PBS and fixed using 4% paraformaldehyde. The cells were then resuspended in PBS and visualized by phase contrast and fluorescent microscopy at (A) 200× magnification and (B) 600× magnification. Images are cropped or scaled to fit the illustration. cHis-sDectin-1 stained yeast cells was also analyzed by flow cytometry (C).</p

Design of sDectin-1 expression vector.

<p>(A) Vector map of pDec1-nHis and pDec1-cHis, and illustration of its design to enhance sDectin-1 production. (B) Amino acid sequence of mDectin-1 from bone-marrow derived macrophage cells of C57BL/6 mice. The peptide fragment expressed in sDectin-1, the transmembrane region, the C-type lectin domain and the two potential N-linked glycosylation sites are indicated on the sequence based on alignment to protein sequence from Uniprot Accession Q6QLQ4.</p

MTX amplification and characterization of sDectin-1 cell pools.

<p>(A) Western blotting of supernatant samples from cultures adapted to different MTX concentrations using an mDectin-1 goat polyclonal antibody (1∶1000; AF1756; R&D Systems) with a HRP conjugated anti-goat antibody (1∶2000; Catalog number V8051; Promega). cHis pool and nHis pool are cell pools producing sDectin-1 with histidine tagged at the C- and N-terminals respectively. (B) Shake flask batch culture cell growth and sDectin-1 production profiles of sDectin-1 producing cell pools. Values shown represent mean values obtained from three replicate flasks. Error bars indicate the standard deviation of the experiment.</p