The role of Malt1 proteolytic activity in T cell activation and lymphoma development

Abstract Long term contact with pathogens induces an adaptive immune response, which is mainly mediated by T and B cells. Antigen-induced activation of T and B cells is an important event, since it facilitates the transition of harmless, low proliferative lymphocytes into powerful and fast expanding cells, which can, if deregulated, be extremely harmful and dangerous for the human body. One of the most important events during lymphocyte activation is the induction of NF-xB activity, a transcription factor that controls not only cytokine secretion, but also lymphocyte proliferation and survival. Recent discoveries identified the CBM complex as the central regulator of NF-xB activity in lymphocytes. The CBM complex consists of the three proteins Carma1, Bcl10 and Malt1, in which Carma1 serves as recruitment platform of the complex and Bcl10 as an adaptor to recruit Malt1 to this platform. But exactly how Malt1 activates NF-x6 is still poorly understood. We discovered that Malt1 is a protease, which cleaves its interaction partner Bcl10 upon T and B cell stimulation. We mapped the Bcl10 cleavage site by single point mutations as well as by a proteomics approach, and used this knowledge to design a fluorogenic Malt1 reporter peptide. With this tool were we able to the first time demonstrate proteolytic activity of Malt1 in vitro, using recombinant Malt1, and in stimulated T cells. Based on similarities to a metacaspase, we designed a Malt1inhibitor, which allowed unto investigate the role of Malt1 activity in T cells. Malt1-inhibited T cells showed a clear defect in NF-xB activity, resulting in impaired IL-2 cytokine secretion levels. We also found a new unexpected role for Bcl10; the blockade of Bcl10 cleavage resulted in a strongly impaired capability of stimulated T cells to adhere to the extracellular matrix protein fibronectin. Because of the central position of the C8M complex, it is not surprising that different lymphomas show abnormal expressions of Carma1, Bcl10 and Malt1. We investigated the role of Malt1 proteolytic activity in the most aggressive subtype of diffuse large B cell lymphomas called ABC, which was described to depend on the expression of Carmal, and frequently carries oncogenic Carmal mutations. We found constitutive high Malt1 activity in all tested ABC cell lines visualized by detection of cleavage products of Malt1 substrates. With the use of the Malt1-inhibitor, we could demonstrate that Malt-inhibition in those cells had two effects. First, the tumor cell proliferation was decreased, most likely because of lower autocrine stimulation by cytokines. Second, we could sensitize the ABC cells towards cell death, which is most likely caused by reduced expression of prosurvival NF-xB target gens. Taken together, we identified Malt1 as a protease in T and B cells, demonstrated its importance for NF-xB signaling and its deregulation in a subtype of diffuse large B cell lymphoma. This could allow the development of a new generation of immunomodulatory and anti-cancer drugs. Résumé Un contact prolongé avec des pathogènes provoque une réponse immunitaire adaptative qui dépend principalement des cellules T et 8. L'activation des lymphocytes T et B, suite à la reconnaissance d'un antigène, est un événement important puisqu'il facilite la transition pour ces cellules d'un état de prolifération limitée et inoffensive à une prolifération soutenue et rapide. Lorsque ce mécanisme est déréglé ìl peut devenir extrêmement nuisible et dangereux pour le corps humain. Un des événement les plus importants lors de l'activation des lymphocytes est l'induction du facteur de transcription NFxB, qui organise la sécrétion de cytokines ainsi que la prolifération et la survie des lymphocytes. Le complexe CBM, composé des trois protéines Carmai, Bc110 et Malt1, a été récemment identifié comme un régulateur central de l'activité de NF-x8 dans les lymphocytes. Carma1 sert de plateforme de recrutement pour ce complexe alors que Bc110 permet d'amener Malt1 dans cette plateforme. Cependant, le rôle exact de Malt1 dans l'activation de NF-tcB reste encore mal compris. Nous avons découvert que Malt1 est une protéase qui clive son partenaire d'interaction BcI10 après stimulation des cellules T et B. Nous avons identifié le site de clivage de BcI10 par une série de mutations ponctuelles ainsi que par une approche protéomique, ce qui nous a permis de fabriquer un peptide reporteur fluorogénique pour mesurer l'activité de Malt1. Grâce à cet outil, nous avons démontré pour la première fois l'activité protéolytique de Malt1 in vitro à l'aide de protéines Malt1 recombinantes ainsi que dans des cellules T stimulées. La ressemblance de Malt1 avec une métacaspase nous a permis de synthétiser un inhibiteur de Malt1 et d'étudier ainsi le rôle de l'activité de Malt1 dans les cellules T. L'inhibition de Malt1 dans les cellules T a révélé un net défaut de l'activité de NF-x8, ayant pour effet une sécrétion réduite de la cytokine IL-2. Nous avons également découvert un rôle inattendu pour Bcl10: en effet, bloquer le clivage de Bcl10 diminue fortement la capacité d'adhésion des cellules T stimulées à la protéine fïbronectine, un composant de la matrice extracellulaire. En raison de la position centrale du complexe CBM, il n'est pas étonnant que le niveau d'expression de Carmai, Bcl10 et Malt1 soit anormal dans plusieurs types de lymphomes. Nous avons examiné le rôle de l'activité protéolytique de Malt1 dans le sous-type le plus agressif des lymphomes B diffus à grandes cellules, appelé sous-type ABC. Ce sous-type de lymphomes dépend de l'expression de Carmai et présente souvent des mutations oncogéniques de Carma1. Nous avons démontré que l'activité de Malt1 était constitutivement élevée dans toutes les lignées cellulaires de type ABC testées, en mettant en évidence la présence de produits de clivage de différents substrats de Malt1. Enfin, l'utilisation de l'inhibiteur de Malt1 nous a permis de démontrer que l'inhibition de Malt1 avait deux effets. Premièrement, une diminution de la prolifération des cellules tumorales, probablement dûe à leur stimulation autocrine par des cytokines fortement réduite. Deuxièmement, une sensibilisation des cellules de type ABC à ia mort cellulaire, vraisemblablement causée par l'expression diminuée de gènes de survie dépendants de NF-tcB. En résumé, nous avons identifié Malt1 comme une protéase dans les cellules T et B, nous avons mis en évidence son importance pour l'activation de NF-xB ainsi que les conséquences du dérèglement de l'activité de Malt1 dans un sous-type de lymphome B diffus à larges cellules. Notre étude ouvre ainsi la voie au développement d'une nouvelle génération de médicaments immunomodulateurs et anti-cancéreux.

The research on radioprotective agents in Chinese materia medica

A series of studies has been carried out in the field of traditional medicine for searching radio-protective agents. According to the theory of traditional Chinese medicine, may prescriptions were tested with experimental animals. Some of them could raise the survival rate of dogs irradiated with lethal dose of Pi-rays by 30-40%. Some symptoms of radiation sickness could be improved. More than one thousand kinds of Chinese herbs were screened. Some of them have pronounced radioprotectice activities. A series of bioactive components wee isolated from these herbs. The mechanism of radiation protection were studied. Having the capability of hemopoietic system and immune system may be the characteristics of these Chinese herbs.

Pessebre virtual: disseny i exportació

Aquest projecte és una part d’un projecte més ampli consistent en estudiar un format gràfic que permeti exportar una escena modelada en Blender i importar aquesta mateixa escena en un entorn interactiu basat en Visual C++ amb OpenGL. D’aquesta forma, disposem de la capacitat de modelat de Blender i de la interacció i visualització de la llibreria OpenGL. Aquest format ha de representar geometria i textures imprescindiblement, i si és possible, d’altres factors importants com il·luminació, visualització i moviment. La part del projecte explicada en aquesta memòria consisteix en estudiar el format gràfic més adient per representar els diferents factors de realisme de l’escena (geometria, textura, etc.) havent triat el format OBJ per la seva capacitat de representació i fàcil edició. Per a provar el format, s’ha dissenyat un diorama de pessebre utilitzant les capacitats de modelatge de Blender. Pel que respecta les figures, aspecte important per a considerar l’escena com a pessebre, s’ha utilitzat un escàner 3D que ha obtingut representacions de malla 3D, a partir de figures reals de pessebre, que posteriorment han estat texturades. S’ha generat un vídeo del diorama de pessebre que permet veure’n tots els detalls navegant amb el punt de vista per l’escena. Aquest vídeo s’ha exposat en la mostra de pessebres de la Associació Pessebrista de Sabadell el Nadal del 2008.

Dengue 2 virus enhancement in asthmatic and non asthmatic individual

During the 1981 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) Cuban epidemic, bronchial asthma (BA) was frequently found as a personal or family antecedent in dengue hemorragic fever patients. Considering that antibody dependent enhancement (ADE) plays an important role in the etiopathogenic mechanism of DHF/DSS, we decide to study the Dengue 2 virus (D2V) capability of replication in peripheral blood leukocytes (PBL) from asthmatic patients and healthy persons. In 90% of asthmatic patients and 53.8% of control group it was obtained PBL with a significant D2V enhancing activity (X² p < 0.01). Power enhancement was higher in asthmatic group. This is the first in vitro study relating BA and the dengue 2 virus immuno enhancement. The results obtained support the role of BA as a risk factor for DHF/DSS as already described on epidemiological data.

Informatització de l'arxiu d'històries clíniques d'un hospital

El sector sanitari és un dels més importants i sensibles dintre de l’economia espanyola, ja que és un element clau del benestar social. Per aquest motiu, el sector de la sanitat s’enfronta a nombrosos reptes, i les Tecnologies de la Informació i les Comunicacions (TIC) obren un ampli ventall de possibilitats de renovació i millora. És per això que Hewlett-Packard està desenvolupant una aplicació informàtica per a hospitals, anomenada HCIS (Health Care Information System). El projecte que es presenta en aquesta memòria és el desenvolupament del mòdul d’HCIS per a gestionar l’Arxiu d’Històries Clíniques d’un hospital, i la seva implantació a la Corporació Sanitària Parc Taulí de Sabadell.

Normal hemopoiesis and lymphopoiesis in the combined absence of numb and numblike.

The mammalian ortholog of the conserved Drosophila adaptor protein Numb (Nb) and its homolog Numblike (Nbl) modulate neuronal cell fate determination at least in part by antagonizing Notch signaling. Because the Notch pathway has been implicated in regulating hemopoietic stem cell self-renewal and T cell fate specification in mammals, we investigated the role of Nb and Nbl in hemopoiesis using conditional gene targeting. Surprisingly simultaneous deletion of both Nb and Nbl in murine bone marrow precursors did not affect the ability of stem cells to self-renew or to give rise to differentiated myeloid or lymphoid progeny, even under competitive conditions in mixed chimeras. Furthermore, T cell fate specification and intrathymic T cell development were unaffected in the combined absence of Nb and Nbl. Collectively our data indicate that the Nb family of adaptor proteins is dispensable for hemopoiesis and lymphopoiesis in mice, despite their proposed role in neuronal stem cell development.

Interactive Epistemology and Reasoning: On the Foundations of Game Theory

Game theory describes and analyzes strategic interaction. It is usually distinguished between static games, which are strategic situations in which the players choose only once as well as simultaneously, and dynamic games, which are strategic situations involving sequential choices. In addition, dynamic games can be further classified according to perfect and imperfect information. Indeed, a dynamic game is said to exhibit perfect information, whenever at any point of the game every player has full informational access to all choices that have been conducted so far. However, in the case of imperfect information some players are not fully informed about some choices. Game-theoretic analysis proceeds in two steps. Firstly, games are modelled by so-called form structures which extract and formalize the significant parts of the underlying strategic interaction. The basic and most commonly used models of games are the normal form, which rather sparsely describes a game merely in terms of the players' strategy sets and utilities, and the extensive form, which models a game in a more detailed way as a tree. In fact, it is standard to formalize static games with the normal form and dynamic games with the extensive form. Secondly, solution concepts are developed to solve models of games in the sense of identifying the choices that should be taken by rational players. Indeed, the ultimate objective of the classical approach to game theory, which is of normative character, is the development of a solution concept that is capable of identifying a unique choice for every player in an arbitrary game. However, given the large variety of games, it is not at all certain whether it is possible to device a solution concept with such universal capability. Alternatively, interactive epistemology provides an epistemic approach to game theory of descriptive character. This rather recent discipline analyzes the relation between knowledge, belief and choice of game-playing agents in an epistemic framework. The description of the players' choices in a given game relative to various epistemic assumptions constitutes the fundamental problem addressed by an epistemic approach to game theory. In a general sense, the objective of interactive epistemology consists in characterizing existing game-theoretic solution concepts in terms of epistemic assumptions as well as in proposing novel solution concepts by studying the game-theoretic implications of refined or new epistemic hypotheses. Intuitively, an epistemic model of a game can be interpreted as representing the reasoning of the players. Indeed, before making a decision in a game, the players reason about the game and their respective opponents, given their knowledge and beliefs. Precisely these epistemic mental states on which players base their decisions are explicitly expressible in an epistemic framework. In this PhD thesis, we consider an epistemic approach to game theory from a foundational point of view. In Chapter 1, basic game-theoretic notions as well as Aumann's epistemic framework for games are expounded and illustrated. Also, Aumann's sufficient conditions for backward induction are presented and his conceptual views discussed. In Chapter 2, Aumann's interactive epistemology is conceptually analyzed. In Chapter 3, which is based on joint work with Conrad Heilmann, a three-stage account for dynamic games is introduced and a type-based epistemic model is extended with a notion of agent connectedness. Then, sufficient conditions for backward induction are derived. In Chapter 4, which is based on joint work with Jérémie Cabessa, a topological approach to interactive epistemology is initiated. In particular, the epistemic-topological operator limit knowledge is defined and some implications for games considered. In Chapter 5, which is based on joint work with Jérémie Cabessa and Andrés Perea, Aumann's impossibility theorem on agreeing to disagree is revisited and weakened in the sense that possible contexts are provided in which agents can indeed agree to disagree.

Manipulating keratinocyte stem cell proliferation and differentiation using RNA interference

ABSTRACT : The epidermis, the outermost compartment of the skin, is a stratified and squamous epithelium that constantly self-renews. Keratinocytes, which represent the main epidermal population, are responsible for its cohesion and barrier function. Epidermal renewal necessitates a fine equilibrium between keratinocyte proliferation and differentiation. The keratinocyte stem cell, located in the basal cell layer, is responsible for epidermal homeostasis and regeneration during the wound healing process. The transcription factor p63 structurally belongs to the p53 superfamily. It is expressed in the basal and supra-basal cell layers of stratified epithelia and is thought to be important for the renewal or the differentiation of keratinocyte stem cells (Yang et al., 1999; Mills et al., 1999). In order to better understand its function, we established an in vitro model of p63 deficient human keratinocyte stem cells using a shp63 mediated RNA interference. Knockdown of endogenous p63 induces downregulation of cell-adhesion genes as previously described (Carroll et al., 2006). Interestingly, the replating of attached p63-knockdown keratinocytes on a feeder layer results in a loss of attachment and proliferation. They are no longer clonogenic. However, if the same population are replated in a fibrin matrix, extended fibrinolysis is reported, a common process in wound healing, suggesting that p63 regulates the fibrinolytic pathway. This result was confirmed by Q-PCR and shows that the urokinase pathway, which mediates fibrinolysis, is upregulated. Altogether, these findings suggest a mechanism in which the fine tuning of p63 expression promotes attachment or release of the keratinocyte stem cell from the basement membrane by inducing genes of adhesion and/or of fibrinolysis. This mechanism may be important for epidermal self-renewal, differentiation as well as wound healing. Its misregulation may be partly responsible for the p63 knockout phenotype. The downregulation of p63 also induces a decrease in LEKTI expression. LEKTI (lymphoepithelial Kazal-type serine protease inhibitor) is a serine protease inhibitor encoded by the Spink5 gene. It is expressed and secreted in the uppermost differentiated layers of stratified epithelia and plays a role in the desquamation process. When this gene is disrupted, humans develop the Netherton syndrome (Chavanas et al., 2000b). It is a dermatosis characterized by hair dysplasias, ichtyosiform erythroderma and impairment in epidermal barrier function promoting inflammation similarly as in psoriasis with inflammatory infiltrate in excess. TNFα (tumor necrosis factor alpha) and EDA1 (ectodysplasin A1) are two transmembraneprecursors that belong to the TNF superfamily, which is involved in immune and inflammation regulation (Smahi et al., 2002). We suggest that the secreted serine protease inhibitor LEKTI plays a role in the regulation of TNFα and EDA1 precursor cleavage and absence of LEKTI induces excess of inflammation. To investigate this hypothesis, we induced downregulation of Spink5 expression in rat keratinocyte stem cells by using a shSpink5 mediated RNA interference approach. Interestingly, expression of TNFα and EDA1 is modified after knockdown of Spink5 by Q-PCR. Moreover, downregulation of Spink5 induces loss of cohesiveness between keratinocytes and colonies adopt a scattered phenotype. Altogether, these preliminary data suggest that downregulation of LEKTI may play a role in the inflammatory response in Netherton syndrome patients, by regulating TNFα expression.

The role of Pten in the Hematopoietic System and the Hematopoietic Stem Cells

Résumé Identification, localisation et activation des cellules souches hématopoiétiques dormantes in vivo Les cellules souches somatiques sont présentes dans la majorité des tissus régénératifs comme la peau, l'épithélium intestinal et le système hématopoiétique. A partir d'une seule cellule, elles ont les capacités de produire d'autres cellules souches du même type (auto-renouvellement) et d'engendrer un ensemble défini de cellules progénitrices différenciées qui vont maintenir ou réparer leur tissu hôte. Les cellules souches adultes les mieux caractérisées sont les cellules souches hématopoiétiques (HSC), localisées dans la moelle osseuse. Un des buts de mon travail de doctorat était de caractériser plus en profondeur la localisation des HSCs endogènes in vivo. Pour ce faire, la technique "label retaining assay", se basant sur la division peu fréquentes et sur la dormance des cellules souches, a été utilisée. Après un marquage des souris avec du BrdU (analogue à l'ADN) suivi d'une longue période sans BrdU, les cellules ayant incorporés le marquage ("label retaining cells" LCRs) ont pu être identifiées dans la moelle osseuse. Ces cellules LCRs étaient enrichies 300 fois en cellules de phenotype HSC et, en utilisant de la cytofluorométrie, il a pu être montré qu'environ 15% de toutes les HSCs d'une souris restent dormantes durant plusieures semaines. Ces HSCs dormantes à long terme ne sont probablement pas impliquées dans la maintenance de 'hématopoièse. Par contre, on assiste à l'activation rapide de ces HSCs dormantes lors d'une blessure, comme une ablation myéloide. Elles re-entrent alors en cycle cellulaire et sont essentielles pour une génération rapide des cellules progénitrices et matures qui vont remplacer les cellules perdues. De plus, la détection des LCRs, combinée avec l'utilisation du marqueur de HSCs c-kit, peut être utilisée pour la localisation des HSCs dormantes présentes dans la paroi endostéale de la cavité osseuse. De manière surprenante, les LCRs c-kit+ ont surtout étés trouvées isolées en cellule unique, suggérant que le micro-environement spécifique entourant et maintenant les HSCs, appelé niche, pourrait être très réduit et abriter une seule HSC par niche. Rôles complexes du gène supresseur de tumeur Pten dans le système hématopoiétique La phosphatase PTEN disparaît dans certains cancers héréditaires ou sporadiques humains, comme les gliomes, les cancers de l'utérus ou du sein. Pten inhibe la voie de signalisation de la PI3-kinase et joue un rôle clé dans l'apoptose, la croissance, la prolifération et la migration cellulaire. Notre but était d'étudier le rôle de Pten dans les HSC normale et durant la formation de leucémies. Pour ce faire, nous avons généré un modèle murin dans lequel le gène Pten peut être supprimé dans les cellules hématopoiétiques, incluant les HSCs. Ceci a été possible en croissant l'allèle conditionnelle ptenflox soit avec le transgène MxCre inductible par l'interféron α soit avec le transgène Scl-CreERt inductible par le tamoxifen. Ceci permet la conversion de l'allèle ptenflox en l'allèle nul PtenΔ dans les HSCs et les autres types cellulaires hématopoiétiques. Les souris mutantes Pten développent une splénomégalie massive causée par une expansion dramatiques de toutes les cellules myéloides. De manière interessante, alors que le nombre de HSCs dans la moelle osseuse diminue progressivement, le nombre des HSCs dans la rate augmente de manière proportionnelle. Etrangement, les analyses de cycle cellulaire ont montrés que Pten n'avait que peu ou pas d'effet sur la dormance des HSCs ou sur leur autorenouvellement. En revanche, une augmentation massive du niveau de la cytokine de mobilisation G-CSF a été détéctée dans le serum sanguin, suggérant que la suppression de Pten stimulerait la mobilisation et la migration des HSC de la moelle osseuse vers la rate. Finallement, la transplantation de moelle osseuse délétée en Pten dans des souris immuno-déficientes montre que Pten fonctionnerait comme un suppresseur de tumeur dans le système hématopoiétique car son absence entraîne la formation rapide de leucémies lymphocytaires. Summary Identification, localization and activation of dormant hematopoietic stun cells in vivo Somatic stem cells are present in most self-renewing tissues including the skin, the intestinal epithelium and the hematopoietic system. On a single cell basis they have the capacity to produce more stem cells of the same phenotype (self-renewal) and to give rise to a defined set of mature differentiated progeny, responsible for the maintenance or repair of the host tissue. The best characterized adult stem cell is the hematopoietic stem cell (HSC) located in the bone marrow. One goal of my thesis work was to further characterize the location of endogenous HSCs in vivo. To do this, a technique called "label retaining assay» was used which takes advantage of the fact that stem cells (including HSCs) divide very infrequently and can be dormant for months. After labeling mice with the DNA analogue BrdU followed by a long BrdU free "chase", BrdU "label retaining cells" (CRCs) could be identified in the bone marrow. These CRCs were 300-fold enriched for phenotypic HSCs and by using flow cytometry analysis it could be shown that about 15% of all HSCs in the mouse are dormant for many weeks. Our results suggest that these long-term dormant HSCs are unlikely to be involved in homeostatic maintenance. However they are rapidly activated and reenter the cell cycle in response to injury signals such as myeloid ablation. In addition, detection of LRCs in combination with the HSC marker c-Kit could be used to locate engrafted dormant HSCs close to the endosteal lining of the bone marrow cavities. Most surprisingly, c-Kit+LRCs were found predominantly as single cells suggesting that the specific stem cell maintaining microenvironment, called niche, has limited space and may house only single HSCs. Complex roles of the tumor suppressor gene Pten in the hematopoietic system. The phosphatase PTEN is lost in hereditary and sporadic forms of human cancers, including gliomas, endometrial and breast cancers. Pten inhibits the PI3-kina.se pathway and plays a key role in apoptosis, cell growth, proliferation and migration. Our aim was to study the role of Pten in normal HSCs and during leukemia formation. To do this, we generated a mouse model in which the Pten gene can be deleted in hematopoietic cells including HSCs. This was achieved by crossing the conditional ptenflox allele with either the interferona inducible MxCre or the tamoxifen inducible Scl-CreERT transgene. This allowed the conversion of the ptenflox allele into a pterr' null allele in HSCs and other hematopoietic cell types. As a result Pten mutant mice developed massive splenomegaly due to a dramatic expansion of all myeloid cells. Interestingly, while the number of bone marrow HSCs progressively decreased, the number of HSCs in the spleen increased to a similar extent. Unexpectedly, extensive cell cycle analysis showed that Pten had little or no effect on HSC dormancy or HSC self-renewal. Instead, dramatically increased levels of the mobilizing cytokine G-CSF were detected in the blood serum suggesting that loss-of Pten stimulates mobilization and migration of HSC from the BM to the spleen. Finally, transplantation of Pten deficient BM cells into immuno-compromised mice showed that Pten can function as a tumor suppressor in the hematopoietic system and that its absence leads to the rapid formation of T cell leukemia.

A general multiserver state-dependent queueing system

The work studies a general multiserver queue in which the service time of an arriving customer and the next interarrival period may depend on both the current waiting time and the server assigned to the arriving customer. Stability of the system is proved under general assumptions on the predetermined distributions describing the model. The proof exploits a combination of the Markov property of the workload process with a regenerative property of the process. The key idea leading to stability is a characterization of the limit behavior of the forward renewal process generated by regenerations. Extensions of the basic model are also studied.

Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein

Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44) identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger) than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

Dinàmica de sistemes mesoscòpics amb correlacions quàntiques mitjançant la formulació de de Broglie-Bohm

Per a determinar la dinàmica espai-temporal completa d’un sistema quàntic tridimensional de N partícules cal integrar l’equació d’Schrödinger en 3N dimensions. La capacitat dels ordinadors actuals permet fer-ho com a molt en 3 dimensions. Amb l’objectiu de disminuir el temps de càlcul necessari per a integrar l’equació d’Schrödinger multidimensional, es realitzen usualment una sèrie d’aproximacions, com l’aproximació de Born–Oppenheimer o la de camp mig. En general, el preu que es paga en realitzar aquestes aproximacions és la pèrdua de les correlacions quàntiques (o entrellaçament). Per tant, és necessari desenvolupar mètodes numèrics que permetin integrar i estudiar la dinàmica de sistemes mesoscòpics (sistemes d’entre tres i unes deu partícules) i en els que es tinguin en compte, encara que sigui de forma aproximada, les correlacions quàntiques entre partícules. Recentment, en el context de la propagació d’electrons per efecte túnel en materials semiconductors, X. Oriols ha desenvolupat un nou mètode [Phys. Rev. Lett. 98, 066803 (2007)] per al tractament de les correlacions quàntiques en sistemes mesoscòpics. Aquesta nova proposta es fonamenta en la formulació de la mecànica quàntica de de Broglie– Bohm. Així, volem fer notar que l’enfoc del problema que realitza X. Oriols i que pretenem aquí seguir no es realitza a fi de comptar amb una eina interpretativa, sinó per a obtenir una eina de càlcul numèric amb la que integrar de manera més eficient l’equació d’Schrödinger corresponent a sistemes quàntics de poques partícules. En el marc del present projecte de tesi doctoral es pretén estendre els algorismes desenvolupats per X. Oriols a sistemes quàntics constituïts tant per fermions com per bosons, i aplicar aquests algorismes a diferents sistemes quàntics mesoscòpics on les correlacions quàntiques juguen un paper important. De forma específica, els problemes a estudiar són els següents: (i) Fotoionització de l’àtom d’heli i de l’àtom de liti mitjançant un làser intens. (ii) Estudi de la relació entre la formulació de X. Oriols amb la aproximació de Born–Oppenheimer. (iii) Estudi de les correlacions quàntiques en sistemes bi- i tripartits en l’espai de configuració de les partícules mitjançant la formulació de de Broglie–Bohm.

Ultrasensitive reporter protein detection in genetically engineered bacteria.

We demonstrate the use of laser-induced fluorescence confocal spectroscopy to measure analyte-stimulated enhanced green fluorescent protein (egfp) synthesis by genetically modified Escherichia coli bioreporter cells. Induction is measured in cell lysates and, since the spectroscopic focal volume is approximately the size of one bioreporter cell, also in individual live bacteria. This is, to our knowledge, the first ever proof-of-concept work utilizing instrumentation with single-molecule detection capability to monitor bioreporter response. Although we use arsenic inducible bioreporters here, the method is extensible to gfp/egfp bioreporters that are responsive to other substances.

Detection of nandrolone metabolites in urine after a football game in professional and amateur players: a Bayesian comparison.

Nandrolone (19-nortestosterone) is a widely used anabolic steroid in sports where strength plays an essential role. Once nandrolone has been metabolised, two major metabolites are excreted in urine, 19-norandrosterone (NA) and 19-noretiocholanolone (NE). In 1997, in France, quite a few sportsmen had concentrations of 19-norandrosterone very close to the IOC cut off limit (2ng/ml). At that time, a debate took place about the capability of the human male body to produce by itself these metabolites without any intake of nandrolone or related compounds. The International Football Federation (FIFA) was very concerned with this problematic, especially because the World Cup was about to start in France. In this respect, a statistical study was held with all football players from the first and second divisions of the Swiss Football National League. All players gave a urine sample after effort and around 6% of them showed traces of 19-norandrosterone. These results were compared with amateur football players (control group) and around 6% of them had very small amounts of 19-norandrosterone and/or 19-noretiocholanolone in urine after effort, whereas none of them had detectable traces of one or the other metabolite before effort. The origin of these compounds in urine after a strenuous physical activity is still unknown, but three hypotheses can be put forward. First, an endogenous production of nandrolone metabolites takes place. Second, nandrolone metabolites are released from the fatty tissues after an intake of nandrolone, some related compounds or some contaminated nutritive supplements. Finally, the sportsmen may have taken something during or just before the football game.

Experimental calcium oxalate crystal production and dissolution by selected wood rot fungi.

Twenty-six species of white-rotting Agaricomycotina fungi (Basidiomycota) were screened for their ability to produce calcium-oxalate (CaOx) crystals in vitro. Most were able to produce CaOx crystals in malt agar medium in the absence of additional calcium. In the same medium enriched with Ca2+, all the species produced CaOx crystals (weddellite or whewellite). Hyphae of four species (Ganoderma lucidum, Polyporus ciliatus, Pycnoporus cinnabarinus, and Trametes versicolor) were found coated with crystals (weddellite/whewellite). The production of CaOx crystals during the growth phase was confirmed by an investigation of the production kinetics for six of the species considered in the initial screening (Pleurotus citrinopileatus, Pleurotus eryngii, Pleurotus ostreatus, P. cinnabarinus, Trametes suaveolens, and T. versicolor). However, the crystals produced during the growth phase disappeared from the medium over time in four of the six species (P. citrinopileatus, P. eryngii, P. cinnabarinus, and T. suaveolens). For P. cinnabarinus, the disappearance of the crystals was correlated with a decrease in the total oxalate concentration measured in the medium from 0.65 μg mm−2 (at the maximum accumulation rate) to 0.30 μg mm−2. The decrease in the CaOx concentration was correlated with a change in mycelia morphology. The oxalate dissolution capability of all the species was also tested in a medium containing calcium oxalate as the sole source of carbon (modified Schlegel medium). Three species (Agaricus blazei, Pleurotus tuberregium, and P. ciliatus) presented a dissolution halo around the growth zone. This study shows that CaOx crystal production is a widespread phenomenon in white-rot fungi, and that an excess of Ca2+ can enhance CaOx crystal production. In addition, it shows that some white-rot fungal species are capable of dissolving CaOx crystals after growth has ceased. These results highlight a diversity of responses around the production or dissolution of calcium oxalate in white-rot fungi and reveal an unexpected potential importance of fungi on the oxalate cycle in the environment.