Molecular mechanisms involved in cell response to mechanical forces

New devices were designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured. The stimulation setups were able to apply relatively large strains (30%~50%) at high temporal frequencies (140~207 Hz) in a localized subcellular region. One of the advantages of this technique was to be less invasive to the innate cellular functions because there was no direct contact between the stimulating probe and the cell body. A mechanical vibration induced by the device in the substrate gel where cells were seeded could mainly cause global calcium responses of the cells. This global response was initiated by the influx of calcium across the stretch-activated channels in the plasma membrane. The subsequent production of inositol triphosphate (IP3) via phospholipase C (PLC) activation triggered the calcium release from the endoplasmic reticulum (ER) to cause a global intracellular calcium fluctuation over the whole cell body. This global calcium response was also shown to depend on actomyosin contractility and F-actin integrity, probably controlling the membrane stretch-activated channels. The localized nature of the stimulation is one of the most important features of these new designs as it allowed the observation of the calcium signaling propagation by ER calcium release. The next step was to focus on the calcium influx, more specifically the TRPM7 channels. As TRPM7 expression may modulate cell adhesion, an adhesion assay was developed and tested on HUVECs seeded on gel substrates with different treatments: normal treatment on gels showed highest attachment rate, followed by the partially treated gels (only 5% of usual fibronectin amount) and untreated gels, with the lowest attachment rate. The trend of the attachment rates correlated to the magnitude of the calcium signaling observed after mechanical stimulation. TRPM7 expression inhibition by siRNA caused an increased attachment rate when compared to both control and non-targeting siRNA-treated cells, but resulted in an actual weaker response in terms of calcium signaling. It suggests that TRPM7 channels are indeed important for the calcium signaling in response to mechanical stimulation. A complementary study was also conducted consisting in the mechanical stimulation of a dissected Drosophila embryo. Although ionomycin treatment showed calcium influx in the tissue, the mechanical stimulation delivered as a vertical vibration did not elicited calcium signaling in response. One possible reason is the dissection procedure causing desensitization of the tissue due to the scrapings and manipulations to open the embryo.

Development of dendritic cells in the intestine

The intestinal tract is exposed to a large variety of antigens such as food proteins, commensal bacteria and pathogens and contains one of the largest arms of the immune system. The intestinal immune system has to discriminate between harmless and harmful antigens, inducing tolerance to harmless antigens and active immunity towards pathogens and other harmful materials. Dendritic cells (DC) in the mucosal lamina propria (LP) are central to this process, as they sample bacteria from the local environment and constitutively migrate to the draining mesenteric lymph nodes (MLN), where they present antigen to naïve T cells in order to direct an appropriate immune response. Despite their crucial role, understanding the function and phenotype of LP DC has been hampered by the fact that they share phenotypic markers with macrophages (mφ), which are the dominant population of mononuclear phagocyte (MP) in the LP. Recent work in our own and other laboratories has established gating strategies and phenotyping panels that allow precise discrimination between intestinal DC and mφ using the mφ specific markers CD64 and F4/80. In this way four bona fide DC subsets with distinct functions have been identified in adult LP based on their expression of CD11b and CD103 and a major aim of my project was to understand how these subsets might develop in the neonatal intestine. At the beginning of my PhD, the laboratory had used these new methods to show that signal regulatory protein α (SIRPα), an inhibitory receptor expressed by myeloid cells, was expressed by mφ and most DC in the intestine, except for those expressing CD103 alone. In addition, mice carrying a non-signalling mutation in SIRPα (SIRPα mt) had a selective reduction in CD103+CD11b+ DC, a subset which is unique to the intestinal LP. This was the basis for the initial experiments of my project, described in Chapter 3, where I investigated if the phenotype in SIRPα mt mice was intrinsic to haematopoietic cells or not. To explore this, I generated bone marrow (BM) chimeric mice by reconstituting irradiated WT mice with SIRPα mt BM, or SIRPα mt animals with WT BM. These experiments suggested that the defect in CD103+CD11b+ DC was not replicated in DC derived from BM of SIRPα origin. However as this seemed inconsistent with other data, I considered the possibility that 18 the phenotype may have been lost with age, as the BM chimeric mice were considerably older than those used in the original studies of SIRPα function. However a comparison of DC subsets in the intestine of WT and SIRPα mt mice as they aged provided no conclusive evidence to support this idea. As these experiments did show age-dependent effects on DC subsets, in Chapter 4, I went on to investigate how the DC populations appeared in the intestine and other tissues in the neonatal period. These experiments showed there were few CD103+CD11b+ DC present in the LP and migratory DC compartment of the MLN in the neonate and that as this population gradually increased in proportion with age, there was a reciprocal decrease in the relative proportion of CD103-CD11b+ DC. Interestingly, most of the changes in DC numbers in the intestine were found during the second or third week of life when the weaning process began. To validate my findings that there were few CD103+CD11b+ DC in the neonate and that this was not merely an absence of CD103 upregulation, I examined the expression of CD101 and Trem-1, markers that other work in the laboratory had suggested were specific to the CD103+CD11b+ DC lineage. My work showed that CD101 and Trem-1 were co- expressed by most CD103+CD11b+ DC in small intestine (SI) LP, as well as a small subset of CD103-CD11b+ DC in this tissue. Interestingly, Trem-1 was highly specific to the SI LP and migratory DC in the MLN, but absent from the colon and other tissues. CD101 expression was also only found on CD11b+ DC, but showed a less restricted pattern of distribution, being found in several tissues as well as the SI LP. The relative timing of their development suggested there might be a relationship between CD103+CD11b+ and CD103-CD11b+ DC and this was supported by microarray analysis. I hypothesised that the CD103-CD11b+ DC that co-expressed CD101 and Trem-1 may be the cells that developed into CD103+CD11b+ DC. To investigate this I analysed how CD101 and Trem-1 expression changed with age amongst the DC subsets in SI LP, colonic LP (CLP) and MLN. The proportion of CD101+Trem-1+ cells increased amongst CD103+CD11b+ DC in the SI LP and MLN with age, while amongst CD103+CD11b+ DC in the CLP this decreased. This was not the same in CD103-CD11b+ DC, where CD101 and Trem-1 expression was more varied with age in all tissues. CD101 and Trem-1 were not expressed to any great extent on CD103+CD11b- or CD103-CD11b- DC. The phenotypic development of the 19 intestinal DC subsets was paralleled by the gradual upregulation of CD103 expression, while the production of retinoic acid (RA), as assessed by the AldefluorTM assay, was low early in life and did not attain adult levels until after weaning. Thus DC in the neonatal intestine take some time to acquire the adult pattern of phenotypic subsets and are functionally immature compared with their adult counterparts. In Chapter 5, I used CD101 and Trem-1 to explore the ontogeny of intestinal DC subsets in CCR2-/- and SIRPα mt mice, both of which have selective defects in one particular group of DC. The selective defect seen amongst CD103+CD11b+ DC in adult SIRPα mt mice was more profound in mice at D7 and D14 of age, indicating that it may be intrinsic to this population and not highly dependent on environmental factors that change after birth. The expression of CD101 and Trem-1 by both CD103+CD11b+ and CD103-CD11b+ DC was reduced in SIRPα mt mice, again indicating that this entire lineage was affected by the lack of SIRPα signalling. However there was also a generalised defect in the numbers of all DC subsets in many tissues from early in life, suggesting there was compromised development, recruitment or survival of DC in the absence of SIRPα signalling. In contrast to the findings in SIRPα mt mice, more CD103+CD11b+ DC co-expressed CD101 and Trem-1 in CCR2-/- mice, while there were no differences in the expression of these molecules amongst CD103-CD11b+ DC. This may suggest that CCR2+ CD103-CD11b+ DC are not the cells that express CD101 and Trem-1 that are predicted to be the direct precursors of CD103+CD11b+ DC. I also examined the expression of DC growth factor receptors on DC subsets from mice of different ages, but no clear age or subset- related patterns of the expression of mRNA for Csf2ra, Irf4, Tgfbr1 and Rara could be observed. Next, I investigated whether Trem-1 played any role in DC development. Preliminary experiments in Trem-1-/- mice show no differences between any of the DC subsets, nor were there any selective effects on individual subsets when DC development from Trem-1-/- KO and WT BM was compared in competitive chimeras. However these experiments were difficult to interpret due to viability problems and because I found an unexpected defect in the ability of Trem-1-/- BM to generate all DC, irrespective of whether they expressed Trem-1 or not. 20 The final experiments I carried out were to examine the role of the microbiota in driving the differentiation of intestinal DC subsets, based on the hypothesis that this could be one of the environmental factors that might influence events in the developing intestine. To this end I performed experiments in both antibiotic treated and germ free adult mice, both of which showed no significant phenotypic differences amongst any of the DC subsets. However the study of germ free mice was compromised by recent contamination of the colony and may not be the conclusive answer. Together the data in this thesis have shown that the population of CD103+CD11b+ DC, which is unique to the intestine, is not present at birth. These cells gradually increase in frequency over time and as this occurs there is a reciprocal decrease in the frequency of CD103-CD11b+ DC. Along with other results, this leads to the idea that there may be a linear developmental pathway from CD103-CD11b+ DC to CD103+CD11b+ DC that is driven by non-microbial factors that are located preferentially in the small intestine. My project indicates that markers such as CD101 and Trem-1 may assist the dissection of this process and highlights the importance of the neonatal period for these events.

Low spatial frequency filtering modulates early brain processing of affective complex pictures

Recent research on affective processing has suggested that low spatial frequency information of fearful faces provide rapid emotional cues to the amygdala, whereas high spatial frequencies convey fine-grained information to the fusiform gyrus, regardless of emotional expression. In the present experiment, we examined the effects of low (LSF, <15 cycles/image width) and high spatial frequency filtering (HSF, >25 cycles/image width) on brain processing of complex pictures depicting pleasant, unpleasant, and neutral scenes. Event-related potentials (ERP), percentage of recognized stimuli and response times were recorded in 19 healthy volunteers. Behavioral results indicated faster reaction times in response to unpleasant LSF than to unpleasant HSF pictures. Unpleasant LSF pictures and pleasant unfiltered pictures also elicited significant enhancements of P1 amplitudes at occipital electrodes as compared to neutral LSF and unfiltered pictures, respectively; whereas no significant effects of affective modulation were found for HSF pictures. Moreover, mean ERP amplitudes in the time between 200 and 500ms post-stimulus were significantly greater for affective (pleasant and unpleasant) than for neutral unfiltered pictures; whereas no significant affective modulation was found for HSF or LSF pictures at those latencies. The fact that affective LSF pictures elicited an enhancement of brain responses at early, but not at later latencies, suggests the existence of a rapid and preattentive neural mechanism for the processing of motivationally relevant stimuli, which could be driven by LSF cues. Our findings confirm thus previous results showing differences on brain processing of affective LSF and HSF faces, and extend these results to more complex and social affective pictures.

Plasma inflammatory biomarkers response to aerobic versus resisted exercise training for chronic obstructive pulmonary disease patients.

Background: Chronic obstructive pulmonary disease (COPD) is a main risk for morbidity, associated with alterations in systemic inflammation. Recent studies proved that morbidity and mortality of COPD is related to systemic inflammation as it contributes to the pathogenesis of atherosclerosis and cardiovascular disease. However, increase of inflammatory cytokines adversely affects quality of life, alteration in ventilatory and skeletal muscles functions. Moreover, exercise training has many beneficial effects in correction of the adverse effects of COPD. Objective: This study aimed to compare the response of inflammatory cytokines of COPD to aerobic versus resisted exercises. Materials and methods: One hundred COPD diseased patients participated in this study and were randomly included in two groups; the first group received aerobic exercise, whereas the second group received resisted exercise training for 12 weeks. Results: The mean values of TNF-α, Il-2, IL-4, IL-6 and CRP were significantly decreased in both groups. Also; there was a significant difference between both groups at the end of the study with more reduction in patients who received aerobic exercise training. Conclusion: Aerobic exercise is more appropriate than resisted exercise training in modulating inflammatory cytokines level in patients with chronic obstructive pulmonary disease.

Cell surface Glut1 levels distinguish human CD4 and CD8 T lymphocyte subsets with distinct effector functions

International audience

FREQUENCY DOMAIN CHARACTERIZATION OF OPTIC FLOW AND VISION-BASED OCELLAR SENSING FOR ROTATIONAL MOTION

The structure of an animal’s eye is determined by the tasks it must perform. While vertebrates rely on their two eyes for all visual functions, insects have evolved a wide range of specialized visual organs to support behaviors such as prey capture, predator evasion, mate pursuit, flight stabilization, and navigation. Compound eyes and ocelli constitute the vision forming and sensing mechanisms of some flying insects. They provide signals useful for flight stabilization and navigation. In contrast to the well-studied compound eye, the ocelli, seen as the second visual system, sense fast luminance changes and allows for fast visual processing. Using a luminance-based sensor that mimics the insect ocelli and a camera-based motion detection system, a frequency-domain characterization of an ocellar sensor and optic flow (due to rotational motion) are analyzed. Inspired by the insect neurons that make use of signals from both vision sensing mechanisms, advantages, disadvantages and complementary properties of ocellar and optic flow estimates are discussed.

EGCL: an extended G-Code Language with flow control, functions and mnemonic variables

In the context of computer numerical control (CNC) and computer aided manufacturing (CAM), the capabilities of programming languages such as symbolic and intuitive programming, program portability and geometrical portfolio have special importance -- They allow to save time and to avoid errors during part programming and permit code re-usage -- Our updated literature review indicates that the current state of art presents voids in parametric programming, program portability and programming flexibility -- In response to this situation, this article presents a compiler implementation for EGCL (Extended G-code Language), a new, enriched CNC programming language which allows the use of descriptive variable names, geometrical functions and flow-control statements (if-then-else, while) -- Our compiler produces low-level generic, elementary ISO-compliant Gcode, thus allowing for flexibility in the choice of the executing CNC machine and in portability -- Our results show that readable variable names and flow control statements allow a simplified and intuitive part programming and permit re-usage of the programs -- Future work includes allowing the programmer to define own functions in terms of EGCL, in contrast to the current status of having them as library built-in functions

Sparse time-frequency data analysis : a multi-scale approach

In this work, we further extend the recently developed adaptive data analysis method, the Sparse Time-Frequency Representation (STFR) method. This method is based on the assumption that many physical signals inherently contain AM-FM representations. We propose a sparse optimization method to extract the AM-FM representations of such signals. We prove the convergence of the method for periodic signals under certain assumptions and provide practical algorithms specifically for the non-periodic STFR, which extends the method to tackle problems that former STFR methods could not handle, including stability to noise and non-periodic data analysis. This is a significant improvement since many adaptive and non-adaptive signal processing methods are not fully capable of handling non-periodic signals. Moreover, we propose a new STFR algorithm to study intrawave signals with strong frequency modulation and analyze the convergence of this new algorithm for periodic signals. Such signals have previously remained a bottleneck for all signal processing methods. Furthermore, we propose a modified version of STFR that facilitates the extraction of intrawaves that have overlaping frequency content. We show that the STFR methods can be applied to the realm of dynamical systems and cardiovascular signals. In particular, we present a simplified and modified version of the STFR algorithm that is potentially useful for the diagnosis of some cardiovascular diseases. We further explain some preliminary work on the nature of Intrinsic Mode Functions (IMFs) and how they can have different representations in different phase coordinates. This analysis shows that the uncertainty principle is fundamental to all oscillating signals.

Radio Frequency Superconducting Quantum Interference Device Meta-atoms and Metamaterials: Experiment, Theory, and Analysis

Metamamterials are 1D, 2D or 3D arrays of articial atoms. The articial atoms, called "meta-atoms", can be any component with tailorable electromagnetic properties, such as resonators, LC circuits, nano particles, and so on. By designing the properties of individual meta-atoms and the interaction created by putting them in a lattice, one can create a metamaterial with intriguing properties not found in nature. My Ph. D. work examines the meta-atoms based on radio frequency superconducting quantum interference devices (rf-SQUIDs); their tunability with dc magnetic field, rf magnetic field, and temperature are studied. The rf-SQUIDs are superconducting split ring resonators in which the usual capacitance is supplemented with a Josephson junction, which introduces strong nonlinearity in the rf properties. At relatively low rf magnetic field, a magnetic field tunability of the resonant frequency of up to 80 THz/Gauss by dc magnetic field is observed, and a total frequency tunability of 100% is achieved. The macroscopic quantum superconducting metamaterial also shows manipulative self-induced broadband transparency due to a qualitatively novel nonlinear mechanism that is different from conventional electromagnetically induced transparency (EIT) or its classical analogs. A near complete disappearance of resonant absorption under a range of applied rf flux is observed experimentally and explained theoretically. The transparency comes from the intrinsic bi-stability and can be tuned on/ off easily by altering rf and dc magnetic fields, temperature and history. Hysteretic in situ 100% tunability of transparency paves the way for auto-cloaking metamaterials, intensity dependent filters, and fast-tunable power limiters. An rf-SQUID metamaterial is shown to have qualitatively the same behavior as a single rf-SQUID with regards to dc flux, rf flux and temperature tuning. The two-tone response of self-resonant rf-SQUID meta-atoms and metamaterials is then studied here via intermodulation (IM) measurement over a broad range of tone frequencies and tone powers. A sharp onset followed by a surprising strongly suppressed IM region near the resonance is observed. This behavior can be understood employing methods in nonlinear dynamics; the sharp onset, and the gap of IM, are due to sudden state jumps during a beat of the two-tone sum input signal. The theory predicts that the IM can be manipulated with tone power, center frequency, frequency difference between the two tones, and temperature. This quantitative understanding potentially allows for the design of rf-SQUID metamaterials with either very low or very high IM response.

Cross-talk and regulatory interactions between the essential response regulator RpaB and cyanobacterial circadian clock output

The response regulator RpaB (regulator of phycobilisome associated B), part of an essential two-component system conserved in cyanobacteria that responds to multiple environmental signals, has recently been implicated in the control of cell dimensions and of circadian rhythms of gene expression in the model cyanobacterium Synechococcus elongatus PCC 7942. However, little is known of the molecular mechanisms that underlie RpaB functions. In this study we show that the regulation of phenotypes by RpaB is intimately connected with the activity of RpaA (regulator of phycobilisome associated A), the master regulator of circadian transcription patterns. RpaB affects RpaA activity both through control of gene expression, a function requiring an intact effector domain, and via altering RpaA phosphorylation, a function mediated through the N-terminal receiver domain of RpaB. Thus, both phosphorylation cross-talk and coregulation of target genes play a role in the genetic interactions between the RpaA and RpaB pathways. In addition, RpaB∼P levels appear critical for survival under light:dark cycles, conditions in which RpaB phosphorylation is environmentally driven independent of the circadian clock. We propose that the complex regulatory interactions between the essential and environmentally sensitive NblS-RpaB system and the SasA-RpaA clock output system integrate relevant extra- and intracellular signals to the circadian clock.

On the optimaL response of q-vortex

Wingtip vortices represent a hazard for the stability of the following airplane in airport highways. These flows have been usually modeled as swirling jets/wakes, which are known to be highly unstable and susceptible to breakdown at high Reynolds numbers for certain flow conditions, but different to the ones present in real flying airplanes. A very recent study based on Direct Numerical Simulations (DNS) shows that a large variety of helical responses can be excited and amplified when a harmonic inlet forcing is imposed. In this work, the optimal response of q-vortex (both axial vorticity and axial velocity can be modeled by a Gaussian profile) is studied by considering the time-harmonically forced problem with a certain frequency ω. We first reproduce Guo and Sun’s results for the Lamb-Oseen vortex (no axial flow) to validate our numerical code. In the axisymmetric case m = 0, the system response is the largest when the input frequency is null. The axial flow has a weak influence in the response for any axial velocity intensity. We also consider helical perturbations |m| = 1. These perturbations are excited through a resonance mechanism at moderate and large wavelengths as it is shown in Figure 1. In addition, Figure 2 shows that the frequency at which the optimal gain is obtained is not a continuous function of the axial wavenumber k. At smaller wavelengths, large response is excited by steady forcing. Regarding the axial flow, the unstable response is the largest when the axial velocity intensity, 1/q, is near to zero. For perturbations with higher azimuthal wavenumbers |m| > 1, the magnitudes of the response are smaller than those for helical modes. In order to establish an alternative validation, DNS has been carried out by using a pseudospectral Fourier formulation finding a very good agreement.

Effect of Arginine and Oscillatory Ca2+ on Vascular Response Mediated Via Nitric Oxide Signaling in Normal and Salt Sensitive Hypertensive Rat Mesenteric Arterioles

Hypertension, a major risk factor in the cardiovascular system, is characterized by an increase in the arterial blood pressure. High dietary sodium is linked to multiple cardiovascular disorders including hypertension. Salt sensitivity, a measure of how the blood pressure responds to salt intake is observed in more than 50% of the hypertension cases. Nitric Oxide (NO), as an endogenous vasodilator serves many important biological roles in the cardiovascular physiology including blood pressure regulation. The physiological concentrations for NO bioactivity are reported to be in 0-500 nM range. Notably, the vascular response to NO is highly regulated within a small concentration spectrum. Hence, much uncertainty surrounds how NO modulates diverse signaling mechanisms to initiate vascular relaxation and alleviate hypertension. Regulating the availability of NO in the vasculature has demonstrated vasoprotective effects. In addition, modulating the NO release by different means has proved to restore endothelial function. In this study we addressed parameters that regulated NO release in the vasculature, in physiology and pathophysiology such as salt sensitive hypertension. We showed that, in the rat mesenteric arterioles, Ca2+ induced rapid relaxation (time constants 20.8 ± 2.2 sec) followed with a much slower constriction after subsequent removal of the stimulus (time constants 104.8 ± 10.0 sec). An interesting observation was that a fourfold increase in the Ca2+ frequency improved the efficacy of arteriolar relaxation by 61.1%. Our results suggested that, Ca2+ frequency-dependent transient release of NO from the endothelium carried encoded information; which could be translated into different steady state vascular tone. Further, Agmatine, a metabolite of L-arginine, as a ligand, was observed to relax the mesenteric arterioles. These relaxations were NO-dependent and occurred via α-2 receptor activity. The observed potency of agmatine (EC50, 138.7 ± 12.1 µM; n=22), was 40 fold higher than L-arginine itself (EC50, 18.3 ± 1.3 mM; n = 5). This suggested us to propose alternative parallel mechanism for L-arginine mediated vascular relaxation via arginine decarboxylase activity. In addition, the biomechanics of rat mesentery is important in regulation of vascular tone. We developed 2D finite element models that described the vascular mechanics of rat mesentery. With an inverse estimation approach, we identified the elasticity parameters characterizing alterations in normotensive and hypertensive Dahl rats. Our efforts were towards guiding current studies that optimized cardiovascular intervention and assisted in the development of new therapeutic strategies. These observations may have significant implications towards alternatives to present methods for NO delivery as a therapeutic target. Our work shall prove to be beneficial in assisting the delivery of NO in the vasculature thus minimizing the cardiovascular risk in handling abnormalities, such as hypertension.