Caspase-8 and c-FLIPL associate in lipid rafts with NF-kappaB adaptors during T cell activation.

Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF-kappaB signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIP(L) rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIP(L) at a known caspase-8 cleavage site. The active caspase.c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF-kappaB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF-kappaB regulators PKC theta, CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF-kappaB activation. The current findings define a link among TCR, caspases, and the NF-kappaB pathway that occurs in a sequestered lipid raft environment in T cells.

Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica

The inhalation of airborne pollutants, such as asbestos or silica, is linked to inflammation of the lung, fibrosis, and lung cancer. How the presence of pathogenic dust is recognized and how chronic inflammatory diseases are triggered are poorly understood. Here, we show that asbestos and silica are sensed by the Nalp3 inflammasome, whose subsequent activation leads to interleukin-1beta secretion. Inflammasome activation is triggered by reactive oxygen species, which are generated by a NADPH oxidase upon particle phagocytosis. (NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate.) In a model of asbestos inhalation, Nalp3-/- mice showed diminished recruitment of inflammatory cells to the lungs, paralleled by lower cytokine production. Our findings implicate the Nalp3 inflammasome in particulate matter-related pulmonary diseases and support its role as a major proinflammatory "danger" receptor

Cardiovascular response to beta-adrenergic blockade or activation in 23 inbred mouse strains.

We report the characterisation of 27 cardiovascular-related traits in 23 inbred mouse strains. Mice were phenotyped either in response to chronic administration of a single dose of the beta-adrenergic receptor blocker atenolol or under a low and a high dose of the beta-agonist isoproterenol and compared to baseline condition. The robustness of our data is supported by high trait heritabilities (typically H(2)>0.7) and significant correlations of trait values measured in baseline condition with independent multistrain datasets of the Mouse Phenome Database. We then focused on the drug-, dose-, and strain-specific responses to beta-stimulation and beta-blockade of a selection of traits including heart rate, systolic blood pressure, cardiac weight indices, ECG parameters and body weight. Because of the wealth of data accumulated, we applied integrative analyses such as comprehensive bi-clustering to investigate the structure of the response across the different phenotypes, strains and experimental conditions. Information extracted from these analyses is discussed in terms of novelty and biological implications. For example, we observe that traits related to ventricular weight in most strains respond only to the high dose of isoproterenol, while heart rate and atrial weight are already affected by the low dose. Finally, we observe little concordance between strain similarity based on the phenotypes and genotypic relatedness computed from genomic SNP profiles. This indicates that cardiovascular phenotypes are unlikely to segregate according to global phylogeny, but rather be governed by smaller, local differences in the genetic architecture of the various strains.

Endogenous cannabinoid receptor CB1 activation promotes vascular smooth-muscle cell proliferation and neointima formation.

Percutaneous transluminal angioplasty is frequently used in patients with severe arterial narrowing due to atherosclerosis. However, it induces severe arterial injury and an inflammatory response leading to restenosis. Here, we studied a potential activation of the endocannabinoid system and the effect of FA amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in arterial injury. We performed carotid balloon injury in atherosclerosis-prone apoE knockout (apoE(-/-)) and apoE(-/-)FAAH(-/-) mice. Anandamide levels were systemically elevated in apoE(-/-) mice after balloon injury. ApoE(-/-)FAAH(-/-) mice had significantly higher baseline anandamide levels and enhanced neointima formation compared with apoE(-/-) controls. The latter effect was inhibited by treatment with CB1 antagonist AM281. Similarly, apoE(-/-) mice treated with AM281 had reduced neointimal areas, reduced lesional vascular smooth-muscle cell (SMC) content, and proliferating cell counts. The lesional macrophage content was unchanged. In vitro proliferation rates were significantly reduced in CB1(-/-) SMCs or when treating apoE(-/-) or apoE(-/-)FAAH(-/-) SMCs with AM281. Macrophage in vitro adhesion and migration were marginally affected by CB1 deficiency. Reendothelialization was not inhibited by treatment with AM281. In conclusion, endogenous CB1 activation contributes to vascular SMC proliferation and neointima formation in response to arterial injury.

Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators.

The nuclear peroxisome proliferator-activated receptors (PPARs) alpha, beta, and gamma activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPAR alpha knockout (KO) with PPAR alpha WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPAR alpha in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acid beta-oxidation under conditions of fasting, stress, and exercise.

Fabrication and characterization of silicon position sensitive particle detectors

Position sensitive particle detectors are needed in high energy physics research. This thesis describes the development of fabrication processes and characterization techniques of silicon microstrip detectors used in the work for searching elementary particles in the European center for nuclear research, CERN. The detectors give an electrical signal along the particles trajectory after a collision in the particle accelerator. The trajectories give information about the nature of the particle in the struggle to reveal the structure of the matter and the universe. Detectors made of semiconductors have a better position resolution than conventional wire chamber detectors. Silicon semiconductor is overwhelmingly used as a detector material because of its cheapness and standard usage in integrated circuit industry. After a short spread sheet analysis of the basic building block of radiation detectors, the pn junction, the operation of a silicon radiation detector is discussed in general. The microstrip detector is then introduced and the detailed structure of a double-sided ac-coupled strip detector revealed. The fabrication aspects of strip detectors are discussedstarting from the process development and general principles ending up to the description of the double-sided ac-coupled strip detector process. Recombination and generation lifetime measurements in radiation detectors are discussed shortly. The results of electrical tests, ie. measuring the leakage currents and bias resistors, are displayed. The beam test setups and the results, the signal to noise ratio and the position accuracy, are then described. It was found out in earlier research that a heavy irradiation changes the properties of radiation detectors dramatically. A scanning electron microscope method was developed to measure the electric potential and field inside irradiated detectorsto see how a high radiation fluence changes them. The method and the most important results are discussed shortly.

PPARβ/δ ameliorates fructose-induced insulin resistance in adipocytes by preventing Nrf2 activation

We studied whether PPARβ/δ deficiency modifies the effects of high fructose intake (30% fructose in drinking water) on glucose tolerance and adipose tissue dysfunction, focusing on the CD36-dependent pathway that enhances adipose tissue inflammation and impairs insulin signaling. Fructose intake for 8weeks significantly increased body and liver weight, and hepatic triglyceride accumulation in PPARβ/δ-deficient mice but not in wild-type mice. Feeding PPARβ/δ-deficient mice with fructose exacerbated glucose intolerance and led to macrophage infiltration, inflammation, enhanced mRNA and protein levels of CD36, and activation of the JNK pathway in white adipose tissue compared to those of water-fed PPARβ/δ-deficient mice. Cultured adipocytes exposed to fructose also exhibited increased CD36 protein levels and this increase was prevented by the PPARβ/δ activator GW501516. Interestingly, the levels of the nuclear factor E2-related factor 2 (Nrf2), a transcription factor reported to up-regulate Cd36 expression and to impair insulin signaling, were increased in fructose-exposed adipocytes whereas co-incubation with GW501516 abolished this increase. In agreement with Nrf2 playing a role in the fructose-induced CD36 protein level increases, the Nrf2 inhibitor trigonelline prevented the increase and the reduction in insulin-stimulated AKT phosphorylation caused by fructose in adipocytes. Protein levels of the well-known Nrf2 target gene NAD(P)H: quinone oxidoreductase 1 (Nqo1) were increased in water-fed PPARβ/δ-null mice, suggesting that PPARβ/δ deficiency increases Nrf2 activity; and this increase was exacerbated in fructose-fed PPARβ/δ-deficient mice. These findings indicate that the combination of high fructose intake and PPARβ/δ deficiency increases CD36 protein levels via Nrf2, a process that promotes chronic inflammation and insulin resistance in adipose tissue.

Muscle strength and activation characteristics of power- trained and non-athlete boys and men

During maturation, muscle strength is enhanced through muscle growth, although neuro-muscular factors are also believed to be involved. In adults, training for power sports has been shown to enhance muscle strength and activation. The purpose of this study was to examine muscle strength and activation in power-trained athletes (POW) compared with non-athletes (CON), in boys and in adults. After familiarization subjects performed ten 5-s explosive maximal voluntary contractions for elbow and knee flexion and extension. The adults were stronger then the boys and the adult POW were stronger then the adult CON, even after correction for muscle size. Normalized rate of torque development was higher in the adults then in the boys and higher in the POW then CON boys. The rate of muscle activation was higher in the adults and POW groups. The results suggest that maturation and power-training have an additive effect on muscle activation.

Hot cognition and activation of the medial prefrontal cortex: Self-regulating in contexts involving motivational pressures

The medial prefrontal cortex (mPFC) is involved in performance-monitoring and has been implicated in the generation of several electrocortical responses associated with self-regulation. The error-related negativity (ERN), the inhibitory Nogo N2 (N2), and the feedback-related negativity (FRN) are event-related potential (ERP) components which reflect mPFC activity associated with feedback to behavioural (ERN, N2) and environmental (FRN) consequences. Our main goal was to determine whether or not rnPFC activation varies as a function of motivational context (e.g., those involving performance-related incentives) or the use of internally versus externally generated feedback signals (i.e., errors). Additionally, we assessed medial prefrontal activity in relation to individual differences in personality and temperament. Participants completed a combination of tasks in which performance-related incentives were associated with task performance and feedback generated from internal versus external responses. MPFC activity was indexed using both ERP scalp voltage peaks and intracerebral current source density (CSD) of dorsal and ventral regions. Additionally, participants completed several questionnaires assessing personality and temperament styles. Given previous studies have shown that enhanced mPFC activity to loss (or negative) feedback, we expected that activity in the mPFC would generally be greater during the Loss condition relative to the Win condition for both the ERN and N2. Also, due to the evidence that the (vmPFC) is engaged in arousing contexts, we hypothesized that activity in the ventromedial prefrontal cortex (vmPFC) would be greater than activity in the dorsomedial prefrontal cortex (dmPFC), especially in the Loss condition of the GoNogo task (ERN). Similarly, loss feedback in the BART (FRN) was expected to engage the vmPFC more than the dmPFC. Finally, we predicted that persons rating themselves as more willing to engage in approach-related behaviours or to exhibit rigid cognitive styles would show reduced activity of the mPFC. Overall, our results emphasize the role of affective evaluations of behavioural and environmental consequences when self-regulating. Although there were no effects of context on brain activity, our data indicate that, during the time of the ERN and N2 on the MW Go-Nogo task and the FRN on the BART, the vrnPFC was more active compared to the dmPFC. Moreover, regional recruitment in the mPFC was similar across internally (ERN) and externally (FRN) generated errors signals associated with loss feedback, as reflected by relatively greater activity in the vmPFC than the dmPFC. Our data also suggest that greater activity in the mPFC is associated with better inhibitory control, as reflected by both scalp and CSD measures. Additionally, deactivation of the subgenual anterior cingulate cortex (sgACC) and lower levels of self-reported positive affect were both related to increased voluntary risk-taking on the BART. Finally, persons reporting higher levels of approach-related behaviour or cognitive rigidity showed reduced activity of the mPFC. These results are in line with previous research emphasizing that affect/motivation is central to the processes reflected by mediofrontal negativities (MFNs), that the vmPFC is involved in regulating demands on motivational/affective systems, and that the underlying mechanisms driving these functions vary across both individuals and contexts.

The electromyographic threshold in children: Differences in neuromuscular activation during progressive exercise between boys and men

The electromyographic threshold (EMGTh), defined as an upward inflexion in the rising EMG signal during progressive exercise, is thought to reflect the onset of increased type-II MU recruitment. The study’s objective was to compare the relative exercise intensity at which the EMGTh occurs in boys vs. men. Participants included 21 men (23.4±4.1 yrs) and 23 boys (11.1±1.1 yrs). Ramped cycle-ergometry was conducted to volitional exhaustion with surface EMG recorded from the vastus lateralis muscles. The EMGTh was mathematically determined using a composite of both legs. EMGTh was detected in 95.2% of the men and in 78.3% of the boys (χ2(1, n=44) =2.69, p =.10). The boys’ EMGTh was significantly higher than the men’s (86.4±9.6 vs. 79.7±10.0% of peak power-output at exhaustion; p <.05). These findings suggest that boys activate their type-II MUs to a lesser extent than men during progressive exercise and support the hypothesis of differential child–adult MU activation.

Top-down and Bottom-up influences on ACC activation: Evaluation of a proposed model of the feedback-related negativity

The Feedback-Related Negativity (FRN) is thought to reflect the dopaminergic prediction error signal from the subcortical areas to the ACC (i.e., a bottom-up signal). Two studies were conducted in order to test a new model of FRN generation, which includes direct modulating influences of medial PFC (i.e., top-down signals) on the ACC at the time of the FRN. Study 1 examined the effects of one’s sense of control (top-down) and of informative cues (bottom-up) on the FRN measures. In Study 2, sense of control and instruction-based (top-down) and probability-based expectations (bottom-up) were manipulated to test the proposed model. The results suggest that any influences of medial PFC on the activity of the ACC that occur in the context of incentive tasks are not direct. The FRN was shown to be sensitive to salient stimulus characteristics. The results of this dissertation partially support the reinforcement learning theory, in that the FRN is a marker for prediction error signal from subcortical areas. However, the pattern of results outlined here suggests that prediction errors are based on salient stimulus characteristics and are not reward specific. A second goal of this dissertation was to examine whether ACC activity, measured through the FRN, is altered in individuals at-risk for problem-gambling behaviour (PG). Individuals in this group were more sensitive to the valence of the outcome in a gambling task compared to not at-risk individuals, suggesting that gambling contexts increase the sensitivity of the reward system to valence of the outcome in individuals at risk for PG. Furthermore, at-risk participants showed an increased sensitivity to reward characteristics and a decreased response to loss outcomes. This contrasts with those not at risk whose FRNs were sensitive to losses. As the results did not replicate previous research showing attenuated FRNs in pathological gamblers, it is likely that the size and time of the FRN does not change gradually with increasing risk of maladaptive behaviour. Instead, changes in ACC activity reflected by the FRN in general can be observed only after behaviour becomes clinically maladaptive or through comparison between different types of gain/loss outcomes.

The time-course of lexical activation in Japanese morphographic word recognition: Evidence for a character-driven processing model

This lexical decision study with eye tracking of Japanese two-kanji-character words investigated the order in which a whole two-character word and its morphographic constituents are activated in the course of lexical access, the relative contributions of the left and the right characters in lexical decision, the depth to which semantic radicals are processed, and how nonlinguistic factors affect lexical processes. Mixed-effects regression analyses of response times and subgaze durations (i.e., first-pass fixation time spent on each of the two characters) revealed joint contributions of morphographic units at all levels of the linguistic structure with the magnitude and the direction of the lexical effects modulated by readers’ locus of attention in a left-to-right preferred processing path. During the early time frame, character effects were larger in magnitude and more robust than radical and whole-word effects, regardless of the font size and the type of nonwords. Extending previous radical-based and character-based models, we propose a task/decision-sensitive character-driven processing model with a level-skipping assumption: Connections from the feature level bypass the lower radical level and link up directly to the higher character level.

Genome-wide location analysis and expression studies reveal a role for p110 CUX1 in the activation of DNA replication genes.

Proteolytic processing of the CUX1 transcription factor generates an isoform, p110 that accelerates entry into S phase. To identify targets of p110 CUX1 that are involved in cell cycle progression, we performed genome-wide location analysis using a promoter microarray. Since there are no antibodies that specifically recognize p110, but not the full-length protein, we expressed physiological levels of a p110 isoform with two tags and purified chromatin by tandem affinity purification (ChAP). Conventional ChIP performed on synchronized populations of cells confirmed that p110 CUX1 is recruited to the promoter of cell cycle-related targets preferentially during S phase. Multiple approaches including silencing RNA (siRNA), transient infection with retroviral vectors, constitutive expression and reporter assays demonstrated that most cell cycle targets are activated whereas a few are repressed or not affected by p110 CUX1. Functional classes that were over-represented among targets included DNA replication initiation. Consistent with this finding, constitutive expression of p110 CUX1 led to a premature and more robust induction of replication genes during cell cycle progression, and stimulated the long-term replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV).