Mikael Harlén

The Role of Regulators in the Expression of Quorum-Sensing Signals in Pseudomonas aeruginosa

Quorum-sensing systems provide Pseudomonas aeruginosa with a sensitive regulatory mechanism that allows for the induction of several phenotypic genes in a cell density fashion. In this work, a mathematical model of the acylated homoserine lactones regulatory network system in P. aeruginosa has been developed. It is the first integrated model to consider both quorum-sensing systems. The model has allowed us to disentangle the complex behavior exhibited by the system as the concentration of extracellular OdDHL is increased. At either low or high levels of extracellular OdDHL, the bacterium remains in an uninduced or induced state, respectively. At moderate levels, the behavior is characterized by several states. Here, the bacteria can switch suddenly from an uninduced to an induced phenotype in response to small changes in the concentration of extracellular OdDHL. Additionally, we have been able to address the roles of RsaL and Vfr as regulators of the quorum-sensing system. An important result from this analysis suggests that RsaL will increase the concentration of extracellular OdDHL required to induce the system, and it is a key regulator of the inhibition of the quorum-sensing system under low cell densities. Most importantly, our results suggest that Vfr has strong regulatory effects on the system as an increased affinity between the LasR/OdDHL complex, and the lasR promoter leads to significant qualitative changes in induction patterns. We also show experimental data that demonstrate that Vfr is required for signal production in the early phase of growth, but that in the latter stages of growth, the vfr mutant is able to synthesize wild-type levels of signal.

A Theoretical Framework for Quantitative Analysis of the Molecular Basis of Costimulation

We present a theoretical framework for simulating the synaptic accumulation of the costimulatory molecules CD28, CTLA-4, B7-1, and B7-2, based on a system of mean-field, ordinary differential equations, and rigorous biophysical and expression data. The simulations show that binding affinity, stoichiometric properties, expression levels, and, in particular, competition effects all profoundly influence complex formation at cellular interfaces. B7-2 engages 33-fold more CD28 than CTLA-4 at the synapse in contrast to B7-1, which ligates ∼7-fold more CTLA-4 than CD28. Although B7-1 completely dominates interactions with CTLA-4, forming linear arrays of 7-18 receptor-ligand pairs, CTLA-4 is fully engaged by B7-2 when B7-1 is absent. Additional simulations reveal the sensitivity of CD28 interactions to modeled transport processes. The results support the concept that B7-2 and B7-1 are the dominant ligands of CD28 and CTLA-4, respectively, and indicate that the inability of B7-2 to recruit CTLA-4 to the synapse cannot be due to the differential binding properties of B7-1 and B7-2 only. We discuss the apparent redundancy of B7-1 in the context of a potentially dynamic synaptic microenvironment, and in light of functions other than the direct enhancement of T cell inhibition by CTLA-4.

Dynamic modelling of cell death during biofilm development.

Biofilms are currently recognised as the predominant bacterial life-style and it has been suggested that biofilm development is influenced by a number of different processes such as adhesion, detachment, mass transport, quorum sensing, cell death and active dispersal. One of the least understood processes and its effects on biofilm development is cell death. However, experimental studies suggest that bacterial death is an important process during biofilm development and many studies show a relationship between cell death and dispersal in microbial biofilms. We present a model of the process of cell death during biofilm development, with a particular focus on the spatial localisation of cell death or cell damage. Three rules governing cell death or cell damage were evaluated which compared the effects of starvation, damage accumulation, and viability during biofilm development and were also used to design laboratory based experiments to test the model. Results from model simulations show that actively growing biofilms develop steep nutrient gradients within the interior of the biofilm that affect neighbouring microcolonies resulting in cell death and detachment. Two of the rules indicated that high substrate concentrations lead to accelerated cell death, in contrast to the third rule, based on the accumulation of damage, which predicted earlier cell death for biofilms grown with low substrate concentrations. Comparison of the modelling results with experimental results suggests that cell death is favoured under low nutrient conditions and that the accumulation of damage may be the main cause of cell death during biofilm development.

3D computation modelling of the influence of cytokine secretion on Th-cell development suggests that negative selection (inhibition of Th1 cells) is more effective than positive selection by IL-4 for Th2 cell dominance.

Th‐cell development has been suggested to include selective mechanisms in which certain cytokines select either Th1 or Th2 cells to proliferate and grow. The selective theory is based on the observation that Th2 cells secrete IL‐4, a cytokine that promotes Th2 development, whereas Th1 cells secrete interferon‐γ (IFN‐γ) that favours Th1 development, and both positive and negative selective influences have been suggested to operate. In this study, we investigate the role of autocrine secretion and utilization of IL‐4 by Th2 cells and address the question of whether an activated Th2 cell can be positively selected by IL‐4 secreted from other Th2 cells. We present a spatial three dimensional (3D) modelling approach to simulate the interaction between the IL‐4 ligand and its IL‐4 receptors expressed on discrete IL‐4 secreting cells. The simulations, based on existing experimental data on the IL‐4 receptor–ligand system, illustrate how Th‐cell development is highly dependent on the distance between cells that are communicating. The model suggests that a single Th2 cell is likely to communicate with possible target cells within a range of approximately 100 μm and that an activated Th2 cell manages to fill most of its own IL‐4 receptors, even at a low secretion rate. The predictions made by the model suggest that negative selection against Th1 cells is more effective than positive selection by IL‐4 for promoting Th2 dominance.

Mechanical massage and mental training programmes affect employees’ anxiety, stress susceptibility and detachment–a randomised explorative pilot study

BackgroundWorking people’s reduced ability to recover has been proposed as a key factor behind the increase in stress-related health problems. One not yet evidence-based preventive method designed to help employees keep healthy and be less stressed is an armchair with built-in mechanical massage and mental training programmes, This study aimed to evaluate possible effects on employees’ experience of levels of “Anxiety”, “Stress Susceptibility”, “Detachment” and “Social Desirability” when using mechanical massage and mental training programmes, both separately and in combination, during working hours.MethodsEmployees from four different workplaces were randomly assigned to one of the following groups: i) Massage and mental training (sitting in the armchair and receiving mechanical massage while listening to the mental training programmes, n = 19), ii) Massage (sitting in the armchair and receiving mechanical massage only, n = 19), iii) Mental training (sitting in the armchair and listening to the mental training programmes only, n = 19), iv) Pause (sitting in the armchair but not receiving mechanical massage or listening to the mental training programmes, n = 19), v) Control (not sitting in the armchair at all, n = 17). In order to discover how the employees felt about their own health they were asked to respond to statements from the ”Swedish Scale of Personality” (SSP), immediately before the randomisation, after four weeks and after eight weeks (end-of-study).ResultsThere were no significant differences between the five study groups for any of the traits studied (“Somatic Trait Anxiety”, “Psychic Trait Anxiety”, “Stress Susceptibility”, “Detachment” and “Social Desirability”) at any of the occasions. However, the massage group showed a significant decrease in the subscale “Somatic Trait Anxiety” (p = 0.032), during the entire study period. Significant decreases in the same subscale were also observed in the pause group between start and week eight (p = 0.040) as well as between week four and week eight (p = 0.049) and also in the control group between the second and third data collection (p = 0.014). The massage and mental training group showed a significant decrease in “Stress Susceptibility” between week four and week eight (p = 0.022). The pause group showed a significant increase in the subscale “Detachment” (p = 0.044).ConclusionsThere were no significant differences between the five study groups for any of the traits studied. However, when looking at each individual group separately, positive effects in their levels of “Anxiety”, “Stress Susceptibility” and “Detachment” could be seen. Although the results from this pilot study indicate some positive effects, mechanical chair massage and mental training programmes used in order to increase employee’s ability to recover, needs to be evaluated further as tools to increase the employees ability to recover.Australian New Zealand clinical trials registryACTRN12615000020583, Date of registration: 15/01/2015.

Effects of fibre type and diffusion distance on mouse skeletal muscle glycogen content in vitro

In vitro incubation of isolated rodent skeletal muscle is a widely used procedure in metabolic research. One concern with this method is the development of an anoxic state during the incubation period that can cause muscle glycogen depletion. Our aim was to investigate whether in vitro incubation conditions influence glycogen concentration in glycolytic extensor digitorum longus (EDL) and oxidative soleus mouse muscle. Quantitative immunohistochemistry was applied to assess glycogen content in incubated skeletal muscle. Glycogen concentration was depleted, independent of insulin‐stimulation in the incubated skeletal muscle. The extent of glycogen depletion was correlated with the oxidative fibre distribution and with the induction of hypoxia‐induced‐factor‐1‐alpha. Insulin exposure partially prevented glycogen depletion in soleus, but not in EDL muscle, providing evidence that glucose diffusion is not a limiting step to maintain glycogen content. Our results provide evidence to suggest that the anoxic milieu and the intrinsic characteristics of the skeletal muscle fibre type play a major role in inducing glycogen depletion in during in vitro incubations. J. Cell. Biochem. 107: 1189–1197, 2009.

VALIDATION OF THE IN VITRO INCUBATION OF EXTENSOR DIGITORUM LONGUS MUSCLE FROM MICE WITH A MATHEMATICAL MODEL

In vitro incubation of tissues; in particular, skeletal muscles from rodents, is a widely-used experimental method in diabetes research. This experimental method has previously been validated, both experimentally and theoretically. However, much of the methods experimental data remains unclear, including the high-rate of lactate production and the lack of an observable increase in glycogen content, within a given time. The predominant hypothesis explaining the high-rate of lactate production is that this phenomenon is dependent on a mechanism in glycolysis that works as a safety valve, producing lactate when glucose uptake is super-physiological. Another hypothesis is that existing anoxia forces more ATP to be produced from glycolysis, leading to an increased lactate concentration. The lack of an observable increase in glycogen content is assumed to be dependent on limitations in sensitivity of the measuring method used. We derived a mathematical model to investigate which of these hypotheses is most likely to be correct. Using our model, data analysis indicates that the in vitro incubated muscle specimens, most likely are sensing the presence of existing anoxia, rather than an overflow in glycolysis. The anoxic milieu causes the high lactate production. The model also predicts an increased glycogenolysis. After mathematical analyses, an estimation of the glycogen concentration could be made with a reduced model. In conclusion, central anoxia is likely to cause spatial differences in glycogen concentrations throughout the entire muscle. Thus, data regarding total glycogen levels in the incubated muscle do not accurately represent the entire organ. The presented model allows for an estimation of total glycogen, despite spatial differences present in the muscle specimen.