Profiling of signal transduction in human memory T cells

Résumé pour un large public: La vaccination a eu un impact énorme sur la santé mondiale. Mais, quel est le principe d'un vaccin? Il est basé sur la 'mémoire immunologique', qui est une particularité exclusive des systèmes immunitaires des organismes évolués. Suite à une infection par un pathogène, des cellules spécialisées de notre système immunitaire (les lymphocytes) le reconnaissent et initient une réaction immunitaire qui a pour but son élimination. Pendant cette réaction se développent aussi des cellules, appelées cellules lymphocytaires mémoire, qui persistent pour longue durée et qui ont la capacité de stimuler une réaction immunitaire très efficace immédiatement après une seconde exposition à ce même pathogène. Ce sont ces cellules mémoires (lymphocytes B et T) qui sont à la base de la 'mémoire immunologique' et qui sont stimulées lors de la vaccination. Chez l'homme, deux populations distinctes des lymphocytes T mémoires ont été identifiées: les cellules centrales (CM) et effectrices (EM) mémoires. Ces populations sont fonctionnellement hétérogènes et exercent des rôles distincts et essentiels dans l'immunité protectrice. Typiquement, les cellules effectrices mémoires sont capables de tuer immédiatement le pathogène tandis que les cellules centrales mémoires sont responsables d'initier une réponse immunitaire complète. Pourtant, les mécanismes biochimiques qui contrôlent les fonctions de ces cellules ont été jusqu'à présent peu étudiés à cause de la faible fréquence de ces cellules et de la quantité limitée de tissus humains disponibles pour les analyses. La compréhension de ces mécanismes est cruciale pour la réalisation de vaccins efficaces et pour le développement de nouveaux médicaments capables de moduler la réponse immunitaire lymphocytaire. Dans cette thèse, nous avons d'abord développé et amélioré une technologie appelée 'protéine array en phase inverse' qui possède un niveau de sensibilité beaucoup plus élevé par rapport aux technologies classiquement utilisées dans l'étude des protéines. Grâce à cette technique, nous avons pu comparer la composition protéique du système de transmission des signaux d'activation des cellules CM et EM humaines. L'analyse de 8 à 13 sujets sains a montré que ces populations des cellules mémoires possèdent un système de signalisation protéique différent. En effet, les cellules EM possèdent, par rapport aux cellules CM, des niveaux réduits d'une protéine régulatrice (appelée c-Cbl) que nous avons démontré comme étant responsable des fonctions spécifiques de ces cellules. En effet, en augmentant artificiellement l'expression de cette protéine régulatrice dans les cellules EM jusqu'au niveau de celui des cellules CM, nous avons induit dans les cellules EM des capacités fonctionnelles caractéristiques des cellules CM. En conclusion, notre étude a identifié, pour la première fois chez l'homme, un mécanisme biochimique qui contrôle les fonctions des populations des cellules mémoires. Résumé en Français: Les cellules mémoires persistent inertes dans l'organisme et produisent des réactions immunitaires rapides et robustes contre les pathogènes précédemment rencontrés. Deux populations distinctes des cellules mémoires ont été identifiées chez l'homme: les cellules centrales (CM) et effectrices (EM) mémoires. Ces populations sont fonctionnellement hétérogènes et exercent des rôles distincts et critiques dans l'immunité protectrice. Les mécanismes biochimiques qui contrôlent leurs fonctions ont été jusqu'à présent peu étudiés, bien que leur compréhension soit cruciale pour le développement des vaccins et des nouveaux traitements/médicaments. Les limites majeures à ces études sont la faible fréquence de ces populations et la quantité limitée de tissus humains disponibles. Dans cette thèse nous avons d'abord développé et amélioré la technologie de 'protéine array en phase inverse' afin d'analyser les molécules de signalisation des cellules mémoires CD4 et CD8 humaines isolées ex vivo. L'excellente sensibilité, la reproductibilité et la linéarité de la détection, ont permis de quantifier des variations d'expression protéiques supérieures à 20% dans un lysat équivalent à 20 cellules. Ensuite, grâce à l'analyse de 8 à 13 sujets sains, nous avons prouvé que les cellules mémoires CD8 ont une composition homogène de leur système de signalisation tandis que les cellules CD4 EM expriment significativement de plus grandes quantités de SLP-76 et des niveaux réduits de c-Cbl, Syk, Fyn et LAT par rapport aux cellules CM. En outre, l'expression réduite du régulateur négatif c-Cbl est corrélée avec l'expression des SLP-76, PI3K et LAT uniquement dans les cellules EM. L'évaluation des propriétés fonctionnelles des cellules mémoires a permis de démontrer que l'expression réduite du c-Cbl dans les cellules EM est associé à une diminution de leur seuil d'activation. En effet, grâce a la technique de transduction cytosolique, nous avons augmenté la quantité de c-Cbl des cellules EM à un niveau comparable à celui des cellules CM et constaté une réduction de la capacité des cellules EM à proliférer et sécréter des cytokines. Ce mécanisme de régulation dépend principalement de l'activité d'ubiquitine ligase de c-Cbl comme démontré par l'impact réduit du mutant enzymatiquement déficient de c-Cbl sur les fonctions de cellules EM. En conclusion, cette thèse identifie c-Cbl comme un régulateur critique des réponses fonctionnelles des populations de cellules T mémoires et fournit, pour la première fois chez l'homme, un mécanisme contrôlant l'hétérogénéité fonctionnelle des ces cellules. De plus, elle valide l'utilisation combinée des 'RPP arrays' et de la transduction cytosolique comme outil puissant d'analyse quantitative et fonctionnel des protéines de signalisation. Summary : Memory cells persist in a quiescent state in the body and mediate rapid and vigorous immune responses toward pathogens previously encountered. Two subsets of memory cells, namely central (CM) and effector (EM) memory cells, have been identified in humans. These subsets display high functional heterogeneity and assert critical and distinct roles in the control of protective immunity. The biochemical mechanisms controlling their functional properties remain so far poorly investigated, although their clarification is crucial for design of effective T-cell vaccine and drug development. Major limitations to these studies lie in the low frequency of memory T cell subsets and the limited amount of human specimen available. In this thesis we first implemented the innovative reverse phase protein array approach to profile 15 signalling components in human CD8 and CD4 memory T cells isolated ex vivo. The high degree of sensitivity, reproducibility and linearity achieved, allowed an excellent quantification of variations in protein expression higher than 20% in as few as 20-cell equivalent per spot. Based on the analysis of 8 to 13 healthy subjects, we showed that CD8 memory cells have a homogeneous composition of their signaling machinery while CD4 EM cells express statistically significant increased amounts of SLP-76 and reduced levels of c- Cbl, Syk, Fyn and LAT as compared to CM cells. Moreover, in EM but not CM cells, reduced expression of negative regulator c-Cbl correlated with the expression of SLP-76, PI3K and LAT. Subsequently, we demonstrated that the higher functional properties and the lower functional threshold of EM cells is associated with reduced expression of c-Cbl. Indeed, by increasing c-Cbl content of EM cells to the same level of CM cells using cytosolic transduction, we impaired their proliferation and cytokine production. This regulatory mechanism was primarily dependent on c-Cbl E3 ubiquitin ligase activity as evidenced by the weaker impact of enzymatically deficient c-Cbl C381A mutant on EM cell functions. Together, these results identify c-Cbl as a critical regulator of the functional responses of memory T cell subsets and provides, for the first time in humans, a mechanism controlling the functional heterogeneity of memory CD4 cells. Moreover it validates the combined use of RPP arrays and cytosolic transduction approaches as a powerful tool to quantitatively analyze signalling proteins and functionally assess their roles.

Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests.

Given the multiplicity of nanoparticles (NPs), there is a requirement to develop screening strategies to evaluate their toxicity. Within the EU-funded FP7 NanoTEST project, a panel of medically relevant NPs has been used to develop alternative testing strategies of NPs used in medical diagnostics. As conventional toxicity tests cannot necessarily be directly applied to NPs in the same manner as for soluble chemicals and drugs, we determined the extent of interference of NPs with each assay process and components. In this study, we fully characterized the panel of NP suspensions used in this project (poly(lactic-co-glycolic acid)-polyethylene oxide [PLGA-PEO], TiO2, SiO2, and uncoated and oleic-acid coated Fe3O4) and showed that many NP characteristics (composition, size, coatings, and agglomeration) interfere with a range of in vitro cytotoxicity assays (WST-1, MTT, lactate dehydrogenase, neutral red, propidium iodide, (3)H-thymidine incorporation, and cell counting), pro-inflammatory response evaluation (ELISA for GM-CSF, IL-6, and IL-8), and oxidative stress detection (monoBromoBimane, dichlorofluorescein, and NO assays). Interferences were assay specific as well as NP specific. We propose how to integrate and avoid interference with testing systems as a first step of a screening strategy for biomedical NPs.

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing.

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

The relative salience of facial features when differentiating faces based on an interference paradigm

Research on face recognition and social judgment usually addresses the manipulation of facial features (eyes, nose, mouth, etc.). Using a procedure based on a Stroop-like task, Montepare and Opeyo (J Nonverbal Behav 26(1):43-59, 2002) established a hierarchy of the relative salience of cues based on facial attributes when differentiating faces. Using the same perceptual interference task, we established a hierarchy of facial features. Twenty-three participants (13 men and 10 women) volunteered for the experiment to compare pairs of frontal faces. The participants had to judge if the eyes, nose, mouth and chin in the pair of images were the same or different. The factors manipulated were the target-distractive factor (4 face components 9 3 distractive factors), interference (absent vs. present) and correct answer (the same vs. different). The analysis of reaction times and errors showed that the eyes and mouth were processed before the chin and nose, thus highlighting the critical importance of the eyes and mouth, as shown by previous research.

Using music as a signal for biofeedback

Studies on the potential benefits of conveying biofeedback stimulus using a musical signal have appeared in recent years with the intent of harnessing the strong effects that music listening may have on subjects. While results are encouraging, the fundamental question has yet to be addressed, of how combined music and biofeedback compares to the already established use of either of these elements separately. This experiment, involving young adults (N = 24), compared the effectiveness at modulating participants' states of physiological arousal of each of the following conditions: A) listening to pre-recorded music, B) sonification biofeedback of the heart rate, and C) an algorithmically modulated musical feedback signal conveying the subject's heart rate. Our hypothesis was that each of the conditions (A), (B) and (C) would differ from the other two in the extent to which it enables participants to increase and decrease their state of physiological arousal, with (C) being more effective than (B), and both more than (A). Several physiological measures and qualitative responses were recorded and analyzed. Results show that using musical biofeedback allowed participants to modulate their state of physiological arousal at least equally well as sonification biofeedback, and much better than just listening to music, as reflected in their heart rate measurements, controlling for respiration-rate. Our findings indicate that the known effects of music in modulating arousal can therefore be beneficially harnessed when designing a biofeedback protocol.

Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes.

Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses.

Sähkökäyttöisen konejärjestelmän vuorovaikutteinen simulointi

Työn tavoitteena oli toteuttaa simulointimalli, jolla pystytään tutkimaan kestomagnetoidun tahtikoneen aiheuttaman vääntömomenttivärähtelyn vaikutuksia sähkömoottoriin liitetyssä mekaniikassa. Tarkoitus oli lisäksi selvittää kuinka kyseinen simulointimalli voidaan toteuttaa nykyaikaisia simulointiohjelmia käyttäen. Saatujen simulointitulosten oikeellisuus varmistettiin tätä työtä varten rakennetulla verifiointilaitteistolla. Tutkittava rakenne koostui akselista, johon kiinnitettiin epäkeskotanko. Epäkeskotankoon kiinnitettiin massa, jonka sijaintia voitiin muunnella. Massan asemaa muuttamalla saatiin rakenteelle erilaisia ominaistaajuuksia. Epäkeskotanko mallinnettiin joustavana elementtimenetelmää apuna käyttäen. Mekaniikka mallinnettiin dynamiikan simulointiin tarkoitetussa ADAMS –ohjelmistossa, johon joustavana mallinnettu epäkeskotanko tuotiin ANSYS –elementtimenetelmäohjelmasta. Mekaniikan malli siirrettiin SIMULINK –ohjelmistoon, jossa mallinnettiin myös sähkökäyttö. SIMULINK –ohjelmassa mallinnettiin sähkökäyttö, joka kuvaa kestomagnetoitua tahtikonetta. Kestomagnetoidun tahtikoneen yhtälöt perustuvat lineaarisiin differentiaaliyhtälöihin, joihin hammasvääntömomentin vaikutus on lisätty häiriösignaalina. Sähkökäytön malli tuottaa vääntömomenttia, joka syötetään ADAMS –ohjelmistolla mallinnettuun mekaniikkaan. Mekaniikan mallista otetaan roottorin kulmakiihtyvyyden arvo takaisinkytkentänä sähkömoottorin malliin. Näin saadaan aikaiseksi yhdistetty simulointi, joka koostuu sähkötoimilaitekäytöstä ja mekaniikasta. Tulosten perusteella voidaan todeta, että sähkökäyttöjen ja mekaniikan yhdistetty simulointi on mahdollista toteuttaa valituilla menetelmillä. Simuloimalla saadut tulokset vastaavat hyvin mitattuja tuloksia.

GPS-signal Source Based on Direct Digital Synthesis

This thesis presents the design and implementation of a GPS-signal source suitable for receiver measurements. The developed signal source is based on direct digital synthesis which generates the intermediate frequency. The intermediate frequency is transfered to the final frequency with the aid of an Inphase/Quadrature modulator. The modulating GPS-data was generated with MATLAB. The signal source was duplicated to form a multi channel source. It was shown that, GPS-signals ment for civil navigation are easy to generate in the laboratory. The hardware does not need to be technically advanced if navigation with high level of accuracy is not needed. It was also shown that, the Inphase/Quadrature modulator can function as a single side band upconverter even with a high intermediate frequency. This concept reduces the demands required for output filtering.

Microstructure of the superior longitudinal fasciculus predicts stimulation-induced interference with on-line motor control.

A cortical visuomotor network, comprising the medial intraparietal sulcus (mIPS) and the dorsal premotor area (PMd), encodes the sensorimotor transformations required for the on-line control of reaching movements. How information is transmitted between these two regions and which pathways are involved, are less clear. Here, we use a multimodal approach combining repetitive transcranial magnetic stimulation (rTMS) and diffusion tensor imaging (DTI) to investigate whether structural connectivity in the 'reaching' circuit is associated to variations in the ability to control and update a movement. We induced a transient disruption of the neural processes underlying on-line motor adjustments by applying 1Hz rTMS over the mIPS. After the stimulation protocol, participants globally showed a reduction of the number of corrective trajectories during a reaching task that included unexpected visual perturbations. A voxel-based analysis revealed that participants exhibiting higher fractional anisotropy (FA) in the second branch of the superior longitudinal fasciculus (SLF II) suffered less rTMS-induced behavioral impact. These results indicate that the microstructural features of the white matter bundles within the parieto-frontal 'reaching' circuit play a prominent role when action reprogramming is interfered. Moreover, our study suggests that the structural alignment and cohesion of the white matter tracts might be used as a predictor to characterize the extent of motor impairments.

A cellular system for spatial signal decoding in chemical gradients.

Directional cell growth requires that cells read and interpret shallow chemical gradients, but how the gradient directional information is identified remains elusive. We use single-cell analysis and mathematical modeling to define the cellular gradient decoding network in yeast. Our results demonstrate that the spatial information of the gradient signal is read locally within the polarity site complex using double-positive feedback between the GTPase Cdc42 and trafficking of the receptor Ste2. Spatial decoding critically depends on low Cdc42 activity, which is maintained by the MAPK Fus3 through sequestration of the Cdc42 activator Cdc24. Deregulated Cdc42 or Ste2 trafficking prevents gradient decoding and leads to mis-oriented growth. Our work discovers how a conserved set of components assembles a network integrating signal intensity and directionality to decode the spatial information contained in chemical gradients.