Antismooth Muscle and Antiactin Antibodies Are Indirect Markers of Histological and Biochemical Activity of Autoimmune Hepatitis

Reactivity and titers of autoantibodies vary during the course of autoimmune hepatitis (AIH), and some autoantibodies have been associated with disease activity and adverse outcomes after treatment. The aim of this study was to assess the autoantibody behavior in AIH and its significance as predictors of biochemical and histological remission. A total of 117 patients with AIH (mean age 18.6 [4-69] years) were evaluated and tested for auto- antibodies at disease onset and successively (mean 3.2 [2-6] times) after a mean follow-up evaluation of 70 [20-185] months. Antismooth muscle (ASMA), antiliver kidney micro- some type 1 (anti-LKM1), antiliver cytosol type 1 (anti-LC1), antimitochondrial, antinu- clear (ANA), and antiactin antibodies (AAA) were determined at disease onset and 379 other times during the follow-up evaluation through indirect immunofluorescence in rodent tissues, HEp-2 cells, and human fibroblasts. Anti-SLA/LP were assessed 45 times in the follow-up evaluation of 19 patients using enzyme-linked immunosorbent assay (ELISA). Upon admission, AIH types 1 and 2 were observed in 95 and 17 patients, respectively. Five subjects had AIH with anti-SLA/LP as the sole markers. Patients initially negative for AAA did not develop these antibodies thereafter. ANA were detected de novo in six and three subjects with AIH types 1 and 2, respectively. After treatment, only ASMA ( > 1:80) and AAA ( > 1:40) were significantly associated with biochemical (76.9% and 79.8%) and histological features (100% and 100%) of disease activity ( P < 0.001). Conclusion: With the exception of ANA, the autoantibody profile does not markedly vary in the course of AIH. The persistence of high titers of ASMA and/or AAA in patients with AIH is associated with disease activity.

The juvenile three-spined stickleback : model organism for the study of estrogenic and androgenic endocrine disruption in laboratory and field

Industrial and domestic sewage effluents have been found to cause reproductive disorders in wild fish, often as a result of the interference of compounds in the effluents with the endocrine system. This thesis describes laboratory-based exposure experiments and a field survey that were conducted with juveniles of the three-spined stickleback, Gasterosteus aculeatus. This small teleost is a common fish in Swedish coastal waters and was chosen as an alternative to non-native test species commonly used in endocrine disruption studies, which allows the comparison of field data with results from laboratory experiments. The aim of this thesis was to elucidate 1) if genetic sex determination and differentiation can be disturbed by natural and synthetic steroid hormones and 2) whether this provides an endpoint for the detection of endocrine disruption, 3) to evaluate the applicability of specific estrogen- and androgen-inducible marker proteins in juvenile three-spined sticklebacks, 4) to investigate whether estrogenic and/or androgenic endocrine disrupting activity can be detected in effluents from Swedish pulp mills and domestic sewage treatment plants and 5) whether such activity can be detected in coastal waters receiving these effluents. Laboratory exposure experiments found juvenile three-spined sticklebacks to be sensitive to water-borne estrogenic and androgenic steroid substances. Intersex – the co-occurrence of ovarian and testicular tissue in gonads – was induced by 17β-estradiol (E2), 17α-ethinylestradiol (EE2), 17α-methyltestosterone (MT) and 5α-dihydrotestosterone (DHT). The first two weeks after hatching was the phase of highest sensitivity. MT was ambivalent by simultaneously eliciting masculinizing and feminizing effects. When applying a DNA-based method for genetic sex identification, it was found that application of MT only during the first two weeks after hatching caused total and apparently irreversible development of testis in genetic females. E2 caused gonad type reversal from male to female. E2 and EE2 induced vitellogenin - the estrogen-responsive yolk precursor protein, while DHT and MT induced spiggin – the androgen-responsive glue protein of the stickleback. None of the effluents from two pulp mills and two domestic sewage treatment plants had any estrogenic or androgenic activity. Juvenile three-spined sticklebacks were collected during four subsequent summers at the Swedish Baltic Sea coast in recipients of effluents from pulp mills and a domestic sewage treatment plant as well as remote reference sites. No sings of endocrine disruption were observed at any site, when studying gonad development or marker proteins, except for a deviation of sex ratios at a reference site. The three-spined stickleback – with focus on the juvenile stage – was found to be a sensitive species suitable for the study of estrogenic and androgenic endocrine disruption.

Effect of temperature and food concentration on the relationship between growth and AARS activity in Paracartia grani nauplii

[EN]The in situ activity of the enzymes aminoacyl-tRNA synthetases (AARS) and the growth rates of naupliar stages of the planktonic marine copepod Paracartia grani were measured in the laboratory under different temperature and food concentrations. We assessed the effect of these parameters on growth and protein synthesis rates of P. grani nauplii. Growth and protein synthesis rates of P. grani nauplii depended on temperature and food concentration. AARS activity is valid as index of somatic growth for P. grani nauplii when growth is not limited by food availability. However, the relationship between protein-specific AARS activity and nauplii growth varied according to food availability levels. The degradation of proteins during starvation and/or the ß-oxidation of fatty acids affected the relationship between specific AARS activity and growth rates. The results presented here add to previous studies showing that the AARS activity is a useful tool for estimating somatic growth of this and other key copepod species. Nevertheless, further research is required to elucidate the validity of AARS activity as a universal proxy for growth.

Diazotrophic activity of Trichodesmium and unicellular cyanobacteria in the subtropical NE Atlantic

Programa de Doctorado en Oceanografía

Mathematical models of cognitive processes

The research activity carried out during the PhD course was focused on the development of mathematical models of some cognitive processes and their validation by means of data present in literature, with a double aim: i) to achieve a better interpretation and explanation of the great amount of data obtained on these processes from different methodologies (electrophysiological recordings on animals, neuropsychological, psychophysical and neuroimaging studies in humans), ii) to exploit model predictions and results to guide future research and experiments. In particular, the research activity has been focused on two different projects: 1) the first one concerns the development of neural oscillators networks, in order to investigate the mechanisms of synchronization of the neural oscillatory activity during cognitive processes, such as object recognition, memory, language, attention; 2) the second one concerns the mathematical modelling of multisensory integration processes (e.g. visual-acoustic), which occur in several cortical and subcortical regions (in particular in a subcortical structure named Superior Colliculus (SC)), and which are fundamental for orienting motor and attentive responses to external world stimuli. This activity has been realized in collaboration with the Center for Studies and Researches in Cognitive Neuroscience of the University of Bologna (in Cesena) and the Department of Neurobiology and Anatomy of the Wake Forest University School of Medicine (NC, USA). PART 1. Objects representation in a number of cognitive functions, like perception and recognition, foresees distribute processes in different cortical areas. One of the main neurophysiological question concerns how the correlation between these disparate areas is realized, in order to succeed in grouping together the characteristics of the same object (binding problem) and in maintaining segregated the properties belonging to different objects simultaneously present (segmentation problem). Different theories have been proposed to address these questions (Barlow, 1972). One of the most influential theory is the so called “assembly coding”, postulated by Singer (2003), according to which 1) an object is well described by a few fundamental properties, processing in different and distributed cortical areas; 2) the recognition of the object would be realized by means of the simultaneously activation of the cortical areas representing its different features; 3) groups of properties belonging to different objects would be kept separated in the time domain. In Chapter 1.1 and in Chapter 1.2 we present two neural network models for object recognition, based on the “assembly coding” hypothesis. These models are networks of Wilson-Cowan oscillators which exploit: i) two high-level “Gestalt Rules” (the similarity and previous knowledge rules), to realize the functional link between elements of different cortical areas representing properties of the same object (binding problem); 2) the synchronization of the neural oscillatory activity in the γ-band (30-100Hz), to segregate in time the representations of different objects simultaneously present (segmentation problem). These models are able to recognize and reconstruct multiple simultaneous external objects, even in difficult case (some wrong or lacking features, shared features, superimposed noise). In Chapter 1.3 the previous models are extended to realize a semantic memory, in which sensory-motor representations of objects are linked with words. To this aim, the network, previously developed, devoted to the representation of objects as a collection of sensory-motor features, is reciprocally linked with a second network devoted to the representation of words (lexical network) Synapses linking the two networks are trained via a time-dependent Hebbian rule, during a training period in which individual objects are presented together with the corresponding words. Simulation results demonstrate that, during the retrieval phase, the network can deal with the simultaneous presence of objects (from sensory-motor inputs) and words (from linguistic inputs), can correctly associate objects with words and segment objects even in the presence of incomplete information. Moreover, the network can realize some semantic links among words representing objects with some shared features. These results support the idea that semantic memory can be described as an integrated process, whose content is retrieved by the co-activation of different multimodal regions. In perspective, extended versions of this model may be used to test conceptual theories, and to provide a quantitative assessment of existing data (for instance concerning patients with neural deficits). PART 2. The ability of the brain to integrate information from different sensory channels is fundamental to perception of the external world (Stein et al, 1993). It is well documented that a number of extraprimary areas have neurons capable of such a task; one of the best known of these is the superior colliculus (SC). This midbrain structure receives auditory, visual and somatosensory inputs from different subcortical and cortical areas, and is involved in the control of orientation to external events (Wallace et al, 1993). SC neurons respond to each of these sensory inputs separately, but is also capable of integrating them (Stein et al, 1993) so that the response to the combined multisensory stimuli is greater than that to the individual component stimuli (enhancement). This enhancement is proportionately greater if the modality-specific paired stimuli are weaker (the principle of inverse effectiveness). Several studies have shown that the capability of SC neurons to engage in multisensory integration requires inputs from cortex; primarily the anterior ectosylvian sulcus (AES), but also the rostral lateral suprasylvian sulcus (rLS). If these cortical inputs are deactivated the response of SC neurons to cross-modal stimulation is no different from that evoked by the most effective of its individual component stimuli (Jiang et al 2001). This phenomenon can be better understood through mathematical models. The use of mathematical models and neural networks can place the mass of data that has been accumulated about this phenomenon and its underlying circuitry into a coherent theoretical structure. In Chapter 2.1 a simple neural network model of this structure is presented; this model is able to reproduce a large number of SC behaviours like multisensory enhancement, multisensory and unisensory depression, inverse effectiveness. In Chapter 2.2 this model was improved by incorporating more neurophysiological knowledge about the neural circuitry underlying SC multisensory integration, in order to suggest possible physiological mechanisms through which it is effected. This endeavour was realized in collaboration with Professor B.E. Stein and Doctor B. Rowland during the 6 months-period spent at the Department of Neurobiology and Anatomy of the Wake Forest University School of Medicine (NC, USA), within the Marco Polo Project. The model includes four distinct unisensory areas that are devoted to a topological representation of external stimuli. Two of them represent subregions of the AES (i.e., FAES, an auditory area, and AEV, a visual area) and send descending inputs to the ipsilateral SC; the other two represent subcortical areas (one auditory and one visual) projecting ascending inputs to the same SC. Different competitive mechanisms, realized by means of population of interneurons, are used in the model to reproduce the different behaviour of SC neurons in conditions of cortical activation and deactivation. The model, with a single set of parameters, is able to mimic the behaviour of SC multisensory neurons in response to very different stimulus conditions (multisensory enhancement, inverse effectiveness, within- and cross-modal suppression of spatially disparate stimuli), with cortex functional and cortex deactivated, and with a particular type of membrane receptors (NMDA receptors) active or inhibited. All these results agree with the data reported in Jiang et al. (2001) and in Binns and Salt (1996). The model suggests that non-linearities in neural responses and synaptic (excitatory and inhibitory) connections can explain the fundamental aspects of multisensory integration, and provides a biologically plausible hypothesis about the underlying circuitry.

Modulation Techniques for Multi-Phase Converters and Control Strategies for Multi-Phase Electric Drives

The ever-increasing spread of automation in industry puts the electrical engineer in a central role as a promoter of technological development in a sector such as the use of electricity, which is the basis of all the machinery and productive processes. Moreover the spread of drives for motor control and static converters with structures ever more complex, places the electrical engineer to face new challenges whose solution has as critical elements in the implementation of digital control techniques with the requirements of inexpensiveness and efficiency of the final product. The successfully application of solutions using non-conventional static converters awake an increasing interest in science and industry due to the promising opportunities. However, in the same time, new problems emerge whose solution is still under study and debate in the scientific community During the Ph.D. course several themes have been developed that, while obtaining the recent and growing interest of scientific community, have much space for the development of research activity and for industrial applications. The first area of research is related to the control of three phase induction motors with high dynamic performance and the sensorless control in the high speed range. The management of the operation of induction machine without position or speed sensors awakes interest in the industrial world due to the increased reliability and robustness of this solution combined with a lower cost of production and purchase of this technology compared to the others available in the market. During this dissertation control techniques will be proposed which are able to exploit the total dc link voltage and at the same time capable to exploit the maximum torque capability in whole speed range with good dynamic performance. The proposed solution preserves the simplicity of tuning of the regulators. Furthermore, in order to validate the effectiveness of presented solution, it is assessed in terms of performance and complexity and compared to two other algorithm presented in literature. The feasibility of the proposed algorithm is also tested on induction motor drive fed by a matrix converter. Another important research area is connected to the development of technology for vehicular applications. In this field the dynamic performances and the low power consumption is one of most important goals for an effective algorithm. Towards this direction, a control scheme for induction motor that integrates within a coherent solution some of the features that are commonly required to an electric vehicle drive is presented. The main features of the proposed control scheme are the capability to exploit the maximum torque in the whole speed range, a weak dependence on the motor parameters, a good robustness against the variations of the dc-link voltage and, whenever possible, the maximum efficiency. The second part of this dissertation is dedicated to the multi-phase systems. This technology, in fact, is characterized by a number of issues worthy of investigation that make it competitive with other technologies already on the market. Multiphase systems, allow to redistribute power at a higher number of phases, thus making possible the construction of electronic converters which otherwise would be very difficult to achieve due to the limits of present power electronics. Multiphase drives have an intrinsic reliability given by the possibility that a fault of a phase, caused by the possible failure of a component of the converter, can be solved without inefficiency of the machine or application of a pulsating torque. The control of the magnetic field spatial harmonics in the air-gap with order higher than one allows to reduce torque noise and to obtain high torque density motor and multi-motor applications. In one of the next chapters a control scheme able to increase the motor torque by adding a third harmonic component to the air-gap magnetic field will be presented. Above the base speed the control system reduces the motor flux in such a way to ensure the maximum torque capability. The presented analysis considers the drive constrains and shows how these limits modify the motor performance. The multi-motor applications are described by a well-defined number of multiphase machines, having series connected stator windings, with an opportune permutation of the phases these machines can be independently controlled with a single multi-phase inverter. In this dissertation this solution will be presented and an electric drive consisting of two five-phase PM tubular actuators fed by a single five-phase inverter will be presented. Finally the modulation strategies for a multi-phase inverter will be illustrated. The problem of the space vector modulation of multiphase inverters with an odd number of phases is solved in different way. An algorithmic approach and a look-up table solution will be proposed. The inverter output voltage capability will be investigated, showing that the proposed modulation strategy is able to fully exploit the dc input voltage either in sinusoidal or non-sinusoidal operating conditions. All this aspects are considered in the next chapters. In particular, Chapter 1 summarizes the mathematical model of induction motor. The Chapter 2 is a brief state of art on three-phase inverter. Chapter 3 proposes a stator flux vector control for a three- phase induction machine and compares this solution with two other algorithms presented in literature. Furthermore, in the same chapter, a complete electric drive based on matrix converter is presented. In Chapter 4 a control strategy suitable for electric vehicles is illustrated. Chapter 5 describes the mathematical model of multi-phase induction machines whereas chapter 6 analyzes the multi-phase inverter and its modulation strategies. Chapter 7 discusses the minimization of the power losses in IGBT multi-phase inverters with carrier-based pulse width modulation. In Chapter 8 an extended stator flux vector control for a seven-phase induction motor is presented. Chapter 9 concerns the high torque density applications and in Chapter 10 different fault tolerant control strategies are analyzed. Finally, the last chapter presents a positioning multi-motor drive consisting of two PM tubular five-phase actuators fed by a single five-phase inverter.

Neural basis of visual deficits recovery: visual residual functions and multisensory integration.

The ability of integrating into a unified percept sensory inputs deriving from different sensory modalities, but related to the same external event, is called multisensory integration and might represent an efficient mechanism of sensory compensation when a sensory modality is damaged by a cortical lesion. This hypothesis has been discussed in the present dissertation. Experiment 1 explored the role of superior colliculus (SC) in multisensory integration, testing patients with collicular lesions, patients with subcortical lesions not involving the SC and healthy control subjects in a multisensory task. The results revealed that patients with collicular lesions, paralleling the evidence of animal studies, demonstrated a loss of multisensory enhancement, in contrast with control subjects, providing the first lesional evidence in humans of the essential role of SC in mediating audio-visual integration. Experiment 2 investigated the role of cortex in mediating multisensory integrative effects, inducing virtual lesions by inhibitory theta-burst stimulation on temporo-parietal cortex, occipital cortex and posterior parietal cortex, demonstrating that only temporo-parietal cortex was causally involved in modulating the integration of audio-visual stimuli at the same spatial location. Given the involvement of the retino-colliculo-extrastriate pathway in mediating audio-visual integration, the functional sparing of this circuit in hemianopic patients is extremely relevant in the perspective of a multisensory-based approach to the recovery of unisensory defects. Experiment 3 demonstrated the spared functional activity of this circuit in a group of hemianopic patients, revealing the presence of implicit recognition of the fearful content of unseen visual stimuli (i.e. affective blindsight), an ability mediated by the retino-colliculo-extrastriate pathway and its connections with amygdala. Finally, Experiment 4 provided evidence that a systematic audio-visual stimulation is effective in inducing long-lasting clinical improvements in patients with visual field defect and revealed that the activity of the spared retino-colliculo-extrastriate pathway is responsible of the observed clinical amelioration, as suggested by the greater improvement observed in patients with cortical lesions limited to the occipital cortex, compared to patients with lesions extending to other cortical areas, found in tasks high demanding in terms of spatial orienting. Overall, the present results indicated that multisensory integration is mediated by the retino-colliculo-extrastriate pathway and that a systematic audio-visual stimulation, activating this spared neural circuit, is able to affect orientation towards the blind field in hemianopic patients and, therefore, might constitute an effective and innovative approach for the rehabilitation of unisensory visual impairments.

Stimulus-induced gamma activity in the electrocorticogram of freely moving telemetric implanted rats: the neuronal signature of novelty detection

During this thesis a new telemetric recording system has been developed allowing ECoG/EEG recordings in freely behaving rodents (Lapray et al., 2008; Lapray et al., in press). This unit has been shown to not generate any discomfort in the implanted animals and to allow recordings in a wide range of environments. In the second part of this work the developed technique has been used to investigate what cortical activity was related to the process of novelty detection in rats’ barrel cortex. We showed that the detection of a novel object is accompanied in the barrel cortex by a transient burst of activity in the γ frequency range (40-47 Hz) around 200 ms after the whiskers contact with the object (Lapray et al., accepted). This activity was associated to a decrease in the lower range of γ frequencies (30-37 Hz). This network activity may represent the optimal oscillatory pattern for the propagation and storage of new information in memory related structures. The frequency as well as the timing of appearance correspond well with other studies concerning novelty detection related burst of activity in other sensory systems (Barcelo et al., 2006; Haenschel et al., 2000; Ranganath & Rainer, 2003). Here, the burst of activity is well suited to induce plastic and long-lasting modifications in neuronal circuits (Harris et al., 2003). The debate is still open whether synchronised activity in the brain is a part of information processing or an epiphenomenon (Shadlen & Movshon, 1999; Singer, 1999). The present work provides further evidence that neuronal network activity in the γ frequency range plays an important role in the neocortical processing of sensory stimuli and in higher cognitive functions.

New synthetic bile acid analogue agonists of FXR and TGR5 receptors: Analytical methodologies for the study of their physico-chemical properties, pharmacokinetic activity and metabolism.

This thesis reports an integrated analytical approach for the study of physicochemical and biological properties of new synthetic bile acid (BA) analogues agonists of FXR and TGR5 receptors. Structure-activity data were compared with those previous obtained using the same experimental protocols on synthetic and natural occurring BA. The new synthetic BA analogues are classified in different groups according also to their potency as a FXR and TGR5 agonists: unconjugated and steroid modified BA and side chain modified BA including taurine or glycine conjugates and pseudo-conjugates (sulphonate and sulphate analogues). In order to investigate the relationship between structure and activity the synthetic analogues where admitted to a physicochemical characterization and to a preliminary screening for their pharmacokinetic and metabolism using a bile fistula rat model. Sensitive and accurate analytical methods have been developed for the quali-quantitative analysis of BA in biological fluids and sample used for physicochemical studies. Combined High Performance Liquid Chromatography Electrospray tandem mass spectrometry with efficient chromatographic separation of all studied BA and their metabolites have been optimized and validated. Analytical strategies for the identification of the BA and their minor metabolites have been developed. Taurine and glycine conjugates were identified in MS/MS by monitoring the specific ion transitions in multiple reaction monitoring (MRM) mode while all other metabolites (sulphate, glucuronic acid, dehydroxylated, decarboxylated or oxo) were monitored in a selected-ion reaction (SIR) mode with a negative ESI interface by the following ions. Accurate and precise data where achieved regarding the main physicochemical properties including solubility, detergency, lipophilicity and albumin binding . These studies have shown that minor structural modification greatly affect the pharmacokinetics and metabolism of the new analogues in respect to the natural BA and on turn their site of action, particularly where their receptor are located in the enterohepatic circulation.

Neural network activity in the neonatal acute slice, slice culture and cell culture

Information processing and storage in the brain may be presented by the oscillations and cell assemblies. Here we address the question of how individual neurons associate together to assemble neural networks and present spontaneous electrical activity. Therefore, we dissected the neonatal brain at three different levels: acute 1-mm thick brain slice, cultured organotypic 350-µm thick brain slice and dissociated neuronal cultures. The spatio-temporal properties of neural activity were investigated by using a 60-channel Micro-electrode arrays (MEA), and the cell assemblies were studied by using a template-matching method. We find local on-propagating as well as large- scale propagating spontaneous oscillatory activity in acute slices, spontaneous network activity characterized by synchronized burst discharges in organotypic cultured slices, and autonomous bursting behaviour in dissociated neuronal cultures. Furthermore, repetitive spike patterns emerge after one week of dissociated neuronal culture and dramatically increase their numbers as well as their complexity and occurrence in the second week. Our data indicate that neurons can self-organize themselves, assembly to a neural network, present spontaneous oscillations, and emerge spatio-temporal activation patterns. The spontaneous oscillations and repetitive spike patterns may serve fundamental functions for information processing and storage in the brain.

LPS-induced modifications in spontaneous network activity causes neuronal apoptosis in neonatal cerebral cortex

During the perinatal period the developing brain is most vulnerable to inflammation. Prenatal infection or exposure to inflammatory factors can have a profound impact on fetal neurodevelopment with long-term neurological deficits, such as cognitive impairment, learning deficits, perinatal brain damage and cerebral palsy. Inflammation in the brain is characterized by activation of resident immune cells, especially microglia and astrocytes whose activation is associated with a variety of neurodegenerative disorders like Alzheimer´s disease and Multiple sclerosis. These cell types express, release and respond to pro-inflammatory mediators such as cytokines, which are critically involved in the immune response to infection. It has been demonstrated recently that cytokines also directly influence neuronal function. Glial cells are capable of releaseing the pro-inflammatory cytokines MIP-2, which is involved in cell death, and tumor necrosis factor alpha (TNFalpha), which enhances excitatory synaptic function by increasing the surface expression of AMPA receptors. Thus constitutively released TNFalpha homeostatically regulates the balance between neuronal excitation and inhibition in an activity-dependent manner. Since TNFalpha is also involved in neuronal cell death, the interplay between neuronal activity MIP-2 and TNFalpha may control the process of cell death and cell survival in developing neuronal networks. An increasing body of evidence suggests that neuronal activity is important in the regulation of neuronal survival during early development, e.g. programmed cell death (apoptosis) is augmented when neuronal activity is blocked. In our study we were interested on the impact of inflammation on neuronal activity and cell survival during early cortical development. To address this question, we investigated the impact of inflammation on neuronal activity and cell survival during early cortical development in vivo and in vitro. Inflammation was experimentally induced by application of the endotoxin lipopolysaccharide (LPS), which initiates a rapid and well-characterized immune response. I studied the consequences of inflammation on spontaneous neuronal network activity and cell death by combining electrophysiological recordings with multi-electrode arrays and quantitative analyses of apoptosis. In addition, I used a cytokine array and antibodies directed against specific cytokines allowing the identification of the pro-inflammatory factors, which are critically involved in these processes. In this study I demonstrated a direct link between inflammation-induced modifications in neuronal network activity and the control of cell survival in a developing neuronal network for the first time. Our in vivo and in vitro recordings showed a fast LPS-induced reduction in occurrence of spontaneous oscillatory activity. It is indicated that LPS-induced inflammation causes fast release of proinflammatory factors which modify neuronal network activity. My experiments with specific antibodies demonstrate that TNFalpha and to a lesser extent MIP-2 seem to be the key mediators causing activity-dependent neuronal cell death in developing brain. These data may be of important clinical relevance, since spontaneous synchronized activity is also a hallmark of the developing human brain and inflammation-induced alterations in this early network activity may have a critical impact on the survival of immature neurons.

In vivo and in vitro investigation of the tissue-specific activity of the human CYP3A4 and CYP3A5 promoters

The human cytochrome P450 3A4 (CYP3A4), the predominant but variably expressed cytochrome P450 in adult liver and small intestine is involved in the metabolism of over 50% of currently used drugs. Its paralog CYP3A5 plays a crucial role in the disposition of several drugs with low therapeutic index, including tacrolimus. Limited information is available for the CYP3A5 transcriptional regulation and its induction by xenobiotics remains controversial. In the first part of this study, we analysed the CYP3A5 transcriptional regulation and its induction by xenobiotics in vivo using transgenic mice. To this end, two transgenic strains were established by pronuclear injection of a plasmid, expressing firefly luciferase driven by a 6.2 kb of the human CYP3A5 promoter. A detailed analysis of both strains shows a tissue distribution largely reflecting that of CYP3A5 transcripts in humans. Thus, the highest luciferase activity was detected in the small intestine, followed by oesophagus, testis, lung, adrenal gland, ovary, prostate and kidney. However, no activity was observed in the liver. CYP3A5-luc transgenic mice were similarly induced in both sexes with either PCN or TCPOBOP in small intestine in a dose-dependent manner. Thus, the 6.2 kb upstream promoter of CYP3A5 mediates the broad tissue activity in transgenic mice. CYP3A5 promoter is inducible in the small intestine in vivo, which may contribute to the variable expression of CYP3A in this organ. rnThe hepato-intestinal level of the detoxifying oxidases CYP3A4 and CYP3A5 is adjusted to the xenobiotic exposure mainly via the xenosensor and transcriptional factor PXR. CYP3A5 is additionally expressed in several other organs lacking PXR, including kidney. In the second part of this study, we investigated the mechanism of the differential expression of CYP3A5 and CYP3A4 and its evolutionary origin using renal and intestinal cells, and comparative genomics. For this examination, we established a two-cell line models reflecting the expression relationships of CYP3A4 and CYP3A5 in the kidney and small intestine in vivo. Our data demonstrate that the CYP3A5 expression in renal cells was enabled by the loss of a suppressing Yin Yang 1 (YY1)-binding site from the CYP3A5 promoter. This allowed for a renal CYP3A5 expression in a PXR-independent manner. The YY1 element is retained in the CYP3A4 gene, leading to its suppression, perhaps via interference with the NF1 activity in renal cells. In intestinal cells, the inhibition of CYP3A4 expression by YY1 is abrogated by a combined activating effect of PXR and NF1 acting on their respective response elements located adjacent to the YY1-binding site on CYP3A4 proximal promoter. CYP3A4 expression is further facilitated by a point mutation attenuating the suppressing effect of YY1 binding site. The differential expression of CYP3A4 and CYP3A5 in these organs results from the loss of the YY1 binding element from the CYP3A5 promoter, acting in concert with the differential organ expression of PXR, and with the higher accumulation of PXR response elements in CYP3A4. rn

Secondary prevention through cardiac rehabilitation: physical activity counselling and exercise training: key components of the position paper from the Cardiac Rehabilitation Section of the European Association of Cardiovascular Prevention and Rehabilitation

Cardiac patients after an acute event and/or with chronic heart disease deserve special attention to restore their quality of life and to maintain or improve functional capacity. They require counselling to avoid recurrence through a combination of adherence to a medication plan and adoption of a healthy lifestyle. These secondary prevention targets are included in the overall goal of cardiac rehabilitation (CR). Cardiac rehabilitation can be viewed as the clinical application of preventive care by means of a professional multi-disciplinary integrated approach for comprehensive risk reduction and global long-term care of cardiac patients. The CR approach is delivered in tandem with a flexible follow-up strategy and easy access to a specialized team. To promote implementation of cardiac prevention and rehabilitation, the CR Section of the EACPR (European Association of Cardiovascular Prevention and Rehabilitation) has recently completed a Position Paper, entitled 'Secondary prevention through cardiac rehabilitation: A condition-oriented approach'. Components of multidisciplinary CR for seven clinical presentations have been addressed. Components include patient assessment, physical activity counselling, exercise training, diet/nutritional counselling, weight control management, lipid management, blood pressure monitoring, smoking cessation, and psychosocial management. Cardiac rehabilitation services are by definition multi-factorial and comprehensive, with physical activity counselling and exercise training as central components in all rehabilitation and preventive interventions. Many of the risk factor improvements occurring in CR can be mediated through exercise training programmes. This call-for-action paper presents the key components of a CR programme: physical activity counselling and exercise training. It summarizes current evidence-based best practice for the wide range of patient presentations of interest to the general cardiology community.

The link between visual exploration and neuronal activity: A multi-modal study combining eye tracking, functional magnetic resonance imaging and transcranial magnetic stimulation

In the present multi-modal study we aimed to investigate the role of visual exploration in relation to the neuronal activity and performance during visuospatial processing. To this end, event related functional magnetic resonance imaging er-fMRI was combined with simultaneous eye tracking recording and transcranial magnetic stimulation (TMS). Two groups of twenty healthy subjects each performed an angle discrimination task with different levels of difficulty during er-fMRI. The number of fixations as a measure of visual exploration effort was chosen to predict blood oxygen level-dependent (BOLD) signal changes using the general linear model (GLM). Without TMS, a positive linear relationship between the visual exploration effort and the BOLD signal was found in a bilateral fronto-parietal cortical network, indicating that these regions reflect the increased number of fixations and the higher brain activity due to higher task demands. Furthermore, the relationship found between the number of fixations and the performance demonstrates the relevance of visual exploration for visuospatial task solving. In the TMS group, offline theta bursts TMS (TBS) was applied over the right posterior parietal cortex (PPC) before the fMRI experiment started. Compared to controls, TBS led to a reduced correlation between visual exploration and BOLD signal change in regions of the fronto-parietal network of the right hemisphere, indicating a disruption of the network. In contrast, an increased correlation was found in regions of the left hemisphere, suggesting an intent to compensate functionality of the disturbed areas. TBS led to fewer fixations and faster response time while keeping accuracy at the same level, indicating that subjects explored more than actually needed.

Inhibition of SIRT1 Impairs the Accumulation and Transcriptional Activity of HIF-1α Protein under Hypoxic Conditions

Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models in vitro and in in vivo models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions.