| 1. |
- Andersson, Martin, et al.
(author)
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Thin film metal sensors in fusion bonded glass chips for high-pressure microfluidics
- 2017
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In: Journal of Micromechanics and Microengineering. - : IOP Publishing. - 0960-1317 .- 1361-6439. ; 27:1
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Journal article (peer-reviewed)abstract
- High-pressure microfluidics offers fast analyses of thermodynamic parameters for compressed process solvents. However, microfluidic platforms handling highly compressible supercritical CO2 are difficult to control, and on-chip sensing would offer added control of the devices. Therefore, there is a need to integrate sensors into highly pressure tolerant glass chips. In this paper, thin film Pt sensors were embedded in shallow etched trenches in a glass wafer that was bonded with another glass wafer having microfluidic channels. The devices having sensors integrated into the flow channels sustained pressures up to 220 bar, typical for the operation of supercritical CO2. No leakage from the devices could be found. Integrated temperature sensors were capable of measuring local decompression cooling effects and integrated calorimetric sensors measured flow velocities over the range 0.5-13.8 mm/s. By this, a better control of high-pressure microfluidic platforms has been achieved.
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| 2. |
- Di Martino, Maria Letizia, et al.
(author)
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Barcoded Consortium Infections Resolve Cell Type-Dependent Salmonella enterica Serovar Typhimurium Entry Mechanisms
- 2019
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In: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 10:3
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Journal article (peer-reviewed)abstract
- Bacterial host cell invasion mechanisms depend on the bacterium's virulence factors and the properties of the target cell. The enteropathogen Salmonella enterica serovar Typhimurium (S. Tm) invades epithelial cell types in the gut mucosa and a variety of immune cell types at later infection stages. The molecular mechanism(s) of host cell entry has, however, been studied predominantly in epithelial cell lines. S. Tm uses a type three secretion system (TTSS-1) to translocate effectors into the host cell cytosol, thereby sparking actin ruffle-dependent entry. The ruffles also fuel cooperative invasion by bystander bacteria. In addition, several TTSS-1-independent entry mechanisms exist, involving alternative S. Tm virulence factors, or the passive uptake of bacteria by phagocytosis. However, it remains ill-defined how S. Tm invasion mechanisms vary between host cells. Here, we developed an internally controlled and scalable method to map S. Tm invasion mechanisms across host cell types and conditions. The method relies on host cell infections with consortia of chromosomally tagged wild-type and mutant S. Tm strains, where the abundance of each strain can be quantified by qPCR or amplicon sequencing. Using this methodology, we quantified cooccurring TTSS-1-dependent, cooperative, and TTSS-1-independent invasion events in epithelial, monocyte, and macrophage cells. We found S. Tm invasion of epithelial cells and monocytes to proceed by a similar MOI-dependent mix of TTSS-1-dependent and cooperative mechanisms. TTSS-1-independent entry was more frequent in macrophages. Still, TTSS-1-dependent invasion dominated during the first minutes of interaction also with this cell type. Finally, the combined action of the SopB/SopE/SopE2 effectors was sufficient to explain TTSS-1-dependent invasion across both epithelial and phagocytic cells. IMPORTANCE Salmonella enterica serovar Typhimurium (S. Tm) is a widespread and broad-host-spectrum enteropathogen with the capacity to invade diverse cell types. Still, the molecular basis for the host cell invasion process has largely been inferred from studies of a few selected cell lines. Our work resolves the mechanisms that Salmonellae employ to invade prototypical host cell types, i.e., human epithelial, monocyte, and macrophage cells, at a previously unattainable level of temporal and quantitative precision. This highlights efficient bacterium-driven entry into innate immune cells and uncovers a type III secretion system effector module that dominates active bacterial invasion of not only epithelial cells but also monocytes and macrophages. The results are derived from a generalizable method, where we combine barcoding of the bacterial chromosome with mixed consortium infections of cultured host cells. The application of this methodology across bacterial species and infection models will provide a scalable means to address host-pathogen interactions in diverse contexts.
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| 3. |
- Ek, Robert, 1974-
(musician, creator_code:cre_t)
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Ek
- 2023
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Artistic work (peer-reviewed)abstract
- Uruppförande vid festivalen NYTT - Festival för nutida kammarmusik och konst, den 18 Augusti 2023. Festivalen äger rum i Lessebo och Växjö kommun.Verket för basklarinett är komponerat av Viktor Varela och tillägnat Robert Ek.
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| 4. |
- Ek, Viktor, et al.
(author)
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A motile doublet form of Salmonella Typhimurium diversifies target search behavior at the epithelial surface
- 2022
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In: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 117:5, s. 1156-1172
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Journal article (peer-reviewed)abstract
- The behaviors of infectious bacteria are commonly studied in bulk. This is effective to define the general properties of a given isolate, but insufficient to resolve subpopulations and unique single-microbe behaviors within the bacterial pool. We here employ microscopy to study single-bacterium characteristics among Salmonella enterica serovar Typhimurium (S.Tm), as they prepare for and launch invasion of epithelial host cells. We find that during the bacterial growth cycle, S.Tm populations switch gradually from fast planktonic growth to a host cell-invasive phenotype, characterized by flagellar motility and expression of the Type-three-secretion-system-1. The indistinct nature of this shift leads to the establishment of a transient subpopulation of S.Tm "doublets"-waist-bearing bacteria anticipating cell division-which simultaneously express host cell invasion machinery. In epithelial cell culture infections, these S.Tm doublets outperform their "singlet" brethren and represent a hyperinvasive subpopulation. Atop both glass and enteroid-derived monolayers, doublets swim along markedly straighter trajectories than singlets, thereby diversifying search patterns and improving the surface exploration capacity of the total bacterial population. The straighter swimming, combined with an enhanced cell-adhesion propensity, suffices to account for the hyperinvasive doublet phenotype. This work highlights bacterial cell length heterogeneity as a key determinant of target search patterns atop epithelia.
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| 5. |
- Ek, Viktor
(author)
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It Takes Two to Tango : Bacterial heterogeneity and host cell features govern Salmonella infection
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Salmonella enterica serovar Typhimurium (S.Tm) causes enterocolitis with significant worldwide morbidity and mortality. The general aim of this thesis is to investigate variation in host cell invasion mechanisms used by S.Tm across different host cell contexts, as well as the influence of bacterial cell-cell heterogeneity on invasion-relevant S.Tm behaviours. The thesis is divided into four sub-projects, each a section in the presented work.First, a genetic barcoding technique for tracking bacteria in mixed consortium infections was developed and applied to evaluate the dependency on the type three secretion system 1 (T3SS-1) and its effectors for host cell entry. It was found that S.Tm invasion of cultured epithelial cells and monocytes is mainly mediated by T3SS-1, or by cooperative uptake of bystander bacteria. T3SS-1-independent entry was possible in cultured macrophages, although T3SS-1-dependent entry was predominant also there. In fact, active invasion was promoted by the same T3SS-1 effectors in all three cell types. Second, an in-depth comparison of S.Tm infections in cell line cultures and in the mouse gut mucosa in vivo highlighted a “discreet-invasion” modality in vivo, in sharp contrast to the prevailing “ruffle” model for host cell invasion. While ruffle-mediated entry into epithelial cell lines was driven by the T3SS-1 effectors SopBEE2, discreet-invasion into the murine gut absorptive epithelium is driven predominantly by the SipA effector, as well as the SiiE adhesin. Furthermore, discreet-invasion targeted apicolateral “hot spots” near cell-cell junctions, dependent on the local cell neighbourhood, which was further charted in the final two sub-projects. Third, single-bacterium characteristics among S.Tm populations were studied using time-lapse microscopy. The indistinct nature of the shift from growth to virulence induction spawned a transient subpopulation of S.Tm “doublets”, cell division intermediates also exhibiting pronounced swimming and host cell invasion aptitude. The longer doublets also displayed a different search pattern during near-surface swimming, highlighting bacterial cell length heterogeneity as a key determinant of target search atop epithelia. Fourth, the morphogenic impact of clinically relevant antibiotics were explored, in context of the previous data. Even S.Tm bacteria with the most extreme morphological abnormalities (e.g. highly filamentous or coccoid individuals), induced by chloramphenicol, ciprofloxacin, nitrofurantoin, and meropenem, could robustly swim and invade epithelial host cells. While high concentrations of these antibiotics were effective at suppressing growth and virulence, a range of low, sub-inhibitory concentrations even enhanced host cell invasion capacity and affected the near-surface swimming behaviour among surviving bacteria. In summary, the present investigation highlights the pivotal importance of taking both host cell features and bacterial heterogeneity into account when studying infection processes.
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| 6. |
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| 7. |
- Fattinger, Stefan A., et al.
(author)
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Salmonella Typhimurium discreet-invasion of the murine gut absorptive epithelium
- 2020
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In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 16:5
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Journal article (peer-reviewed)abstract
- Bacterial pathogens can use secreted effector molecules to drive entry into host cells. Studies of the intestinal pathogen S.Tm have been central to uncover the mechanistic basis for the entry process. More than two decades of research have resulted in a detailed model for how S.Tm invades gut epithelial cells through effector triggering of large Rho-GTPase-dependent actin ruffles. However, the evidence for this model comes predominantly from studies in cultured cell lines. These experimental systems lack many of the architectural and signaling features of the intact gut epithelium. Our study surprisingly reveals that in the intact mouse gut, S.Tm invades absorptive epithelial cells through a process that does not require the Rho-GTPase-activating effectors and can proceed in the absence of the prototypical ruffling response. Instead, S.Tm exploits another effector, SipA, to sneak in through discreet entry structures close to cell-cell junctions. Our results challenge the current model for S.Tm epithelial cell entry and emphasizes the need of taking a physiological host cell context into account when studying bacterium-host cell interactions. Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host's gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through "discreet-invasion". This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context.
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| 8. |
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| 9. |
- Solyom, Peter, et al.
(author)
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Further investigatiions on the influence of sediment-associated phthalate esters (DEHP and DINP) on hatching and survival of the moorfrog Rana arvalis
- 2001
-
Reports (other academic/artistic)abstract
- The hatchability and survival of eggs from Moorfrog (Rana arvalis) and condition and growth of hatched larvae were examined in presence of various concentrations of di-2-ethylhexyl phtalate (DEHP) in the sediment. Two types of sediment have been used in the biological assay. Fine sediment comprising mostly degraded organic material and a coarse type consisting of higher degree at two temperatures 10 and S°C. Each test series consisted of three different concentrations of spiked sediments, 1000, 300 and 100 mg DEHP/k:g dw together with solvent (acetone) control and control. Five replicants were examined for each of the different treatments. Exposure to DEHP conducted at 1ooc showed no significant effect on the hatching frequency and median hatching time for all tested concentrations, compared to the control groups. soc, except that the lower temperature gave a significant decrease in hatching frequency for all tested concentrations compared to the 10°C series. The lower hatchability at the lower temperature indicated that an unnaturally high stress factor was added to the experiments when exposure was conducted at the lower temperature. Larval mortality was less than 2 percent, in all tested concentrations and was not correlated to the temperature. There was no significant difference in mortality among the various treatments. The visual morphological deformities were also low in all tested concentrations and no significant difference could be established. Growth measurements showed no significant difference between the sediment types or a dose related difference within the 10°C and S°C experiments. Parallel to DEHP, an identical experiment with diisononyl phthalate (DINP) was performed in the same manner at 10°C. Exposure to DINP showed no significant effect on the hatching frequency and median hatching time for all tested concentrations, compared to the control groups. There was no significant difference in mortality and growth among the various treatments. Visual morphological deformities were also low in the controls as well as in all tested concentrations and no significant difference could be established.
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