Survival responses in stressed organs

Apoptosis or programmed cell death is a regulated form of cell suicide executed by cysteine proteases, or "caspases", to maintain proper tissue homeostasis in multicellular organisms. Dysregulation of apoptosis leads to pathological complications including cancer, autoimmunity, neurodegenerative, and heart diseases. Beside their known function as the key executioners of apoptotic cell death, caspases were reported to mediate non-apoptotic functions. In this report we study the survival signals conveyed through caspase-3-mediated cleavage of Ras GTPase-activating proteins (RasGAP). Ubiquitously expressed, RasGAP senses caspase activity and controls the cell death/survival switch. RasGAP is cleaved once at low caspase activity and the generated N-terminal fragment (fragment N) induces a survival response by activating Ras/PI3K/Akt pathway. However, high caspase activity associated with increased stress leads to fragment Ν cleavage into fragments that do not mediate any detectable survival signals. In this thesis project we studied the role of fragment Ν in protecting stressed organs as well as in maintenance of their functionality. In response to stress in different organs, we found that mice lacking caspase-3 or unable to cleave RasGAP (Knock-In mice), and therefore unable to generate fragment N, were deficient in Akt activation and experienced increased apoptosis compared to wild-type mice. Augmented tissue damage and organ dysfunction in those mice highlight the importance of fragment Ν in activating Akt-mediated prosurvival pathway and in protection of organs during episodes of stress. In parallel we investigated the role of fragment Ν in regulating the activation of transcription factor NF-kB, a master regulator of inflammation. Sustained NF-kB activation may be detrimental by directly causing apoptosis or leading to a persistent damaging inflammation response. We found that fragment Ν is a potent inhibitor of NF-kB by favoring its nuclear export. Therefore, fragment Ν regulates NF-kB activity and contributes to a controlled response as well as maintenance of homeostasis in stressed cells. Importantly, these findings introduce new insights of how activated caspase-3 acts as a stress intensity sensor that controls cell fate by either initiating a fragment N- dependent cell resistance program or a cell suicide response. This identifies the pivotal role of fragment Ν in protection against patho-physiological damage, and encourages the development of therapies which aim to increase cell resistance to vigorous treatment. - L'apoptose, ou mort cellulaire programmée, est une forme contrôlée de suicide cellulaire exécuté par des protéines appelées caspases, dans le but de maintenir l'homéostasie des tissus sains dans les organismes multicellulaires. Un mauvais contrôle de l'apoptose peut mener à des pathologies comme le cancer, la neurodégénération et les maladies cardiaques et auto-immunes. En dehors de leur rôle connu d'exécutrices de l'apoptose, les caspases ont aussi été identifiées dans d'autres contextes non-apoptotiques. Dans ce projet, nous avons étudié les signaux de survie émis par le résultat du clivage de RasGAP par la caspase-3. Exprimée de façon ubiquitaire, RasGAP est sensible à l'activité de caspase-3 et contrôle la décision de la cellule à entreprendre la mort ou la survie cellulaire. A un taux d'activité faible, la caspase-3 clive RasGAP, ce qui mène à la génération d'un fragment N-terminal, appelé Fragment N, qui induit des signaux de survie via l'activation de la cascade Ras/PI3K/Akt. Cependant, lorsque l'activité de la caspase-3 augmente, le fragment N est clivé, ce qui a pour effet d'éliminer ces signaux de survie. Dans ce travail, nous avons étudié le rôle du Fragment N dans la protection des organes en état de stress et dans le maintien de leur fonctionnalité. En réponse à certains stress, nous avons découvert que les organes de souris n'exprimant pas la caspase-3 ou alors incapables de cliver RasGAP (souris Kl), et de ce fait n'ayant pas la possibilité de générer le Fragment N, perdaient leur faculté d'activer la protéine Akt et démontraient un taux d'apoptose plus élevé que des organes de souris sauvages. Le fait que les organes et tissus de ces souris manifestaient de graves dommages et dysfonctions met en évidence l'importance du Fragment N dans l'activation des signaux de survie via la protéine Akt et dans la neutralisation de l'apoptose induite par la caspase-3. En parallèle, nous avons investigué le rôle du Fragment N dans la régulation de l'activation de NF-kB, un facteur de transcription clé dans l'inflammation. Une activation soutenue de NF-kB peut être délétère par activation directe de l'apoptose ou peut mener à une réponse inflammatoire persistante. Nous avons découvert que le Fragment N, en favorisant l'export de NF-kB depuis le noyau, était capable de l'inhiber très efficacement. Le Fragment N régule donc l'activité de NF-kB et contribue au maintien de l'homéostasie dans des cellules stressées. Ces découvertes aident, de façon importante, à la compréhension de comment l'activation de la caspase-3 agit comme senseur de stress et décide du sort de la cellule soit en initiant une protection par le biais du fragment N, ou en induisant un suicide cellulaire. Cette étude définit le Fragment Ν comme ayant un rôle de pivot dans la protection contre des dommages patho-physiologiques, et ouvre des perspectives de développement de thérapies qui cibleraient à augmenter la résistance à divers traitements.

Nitric oxide and the resolution of inflammation: implications for atherosclerosis

The ubiquitous free radical, nitric oxide (NO), plays an important role in many biological processes including the regulation of the inflammatory response. Alterations in NO synthesis by endogenous systems likely influence inflammatory processes occurring in a wide range of diseases including many in the cardiovascular system (e.g. atherosclerosis). Progression of inflammatory conditions depends not only upon the recruitment and activation of inflammatory cells but also upon their subsequent removal from the inflammatory milieu. Apoptosis, or programmed cell death, is a fundamental process regulating inflammatory cell survival and is critically involved in ensuring the successful resolution of an inflammatory response. Apoptosis results in shutdown of secretory pathways and renders effete, but potentially highly histotoxic, cells instantly recognisable for non-inflammatory clearance by phagocytes (e.g., macrophages). However, dysregulation of apoptosis and phagocytic clearance mechanisms can have drastic consequences for development and resolution of inflammatory processes. In this review we highlight the complexities of NO-mediated regulation of inflammatory cell apoptosis and clearance by phagocytes and discuss the molecular mechanisms controlling these NO mediated effects. We believe that manipulation of pathways involving NO may have previously unrecognised therapeutic potential for limiting or resolving inflammatory and cardiovascular disease.

Desenvolupament d'aplicació de consulta de cases rurals per Android

Aquest projecte final de carrera pretén realitzar el desenvolupament d'una aplicació per al sistema Android, programada amb el llenguatge Java, que consulta una base de dades de cases rurals i aprofita les característiques i funcionalitats dels dispositius mòbils actuals com el posicionament geogràfic per GPS o la connexió a Internet.

Role and regulation of islet-Brain 1 in pancreatic β-cells

Résumé : L'insuline est produite et sécrétée par la cellule ß-pancréatique. Son rôle est de régler le taux de sucre dans le sang. Si ces cellules meurent ou échouent à produire suffisamment de l'insuline, les sujets développent le diabète de type 2 (DT2), une des maladies les plus communes dans les pays développés. L'excès chronique des lipoprotéines LDL oxydés (oxLDL) et/ou des cytokines pro-inflammatoires comme l'interleukine-1ß (IL-1ß) participent au dérèglement et à la mort des cellules ß. Nous avons montré qu'une chute des niveaux d'expression de la protéine nommée «mitogen activated protein kinase 8 interacting protein 1» ou «islet brain 1 (IB 1)» est en partie responsable des effets provoqués par les oxLDL ou IL-1ß. IB1 régule l'expression de l'insuline et la survie cellulaire en inhibant la voie de signalisation « c-jun N-terminal Kinase (JNK)». La réduction des niveaux d'expression d'IB1 provoque l'activation de la voie JNK en réponse aux facteurs environnementaux, et ainsi initie la réduction de l'expression de l'insuline et l'induction du programme de mort cellulaire. Les mimétiques de l'hormone "Glucagon-like peptide 1", tel que l'exendin-4 (ex-4), sont une nouvelle classe d'agents hypoglycémiants utilisés dans le traitement du DT2. Les effets bénéfiques de l'ex-4 sont en partie accomplis en préservant l'expression de l'insuline et la survie des cellules ß contre les stress associés au DT2. La restauration des niveaux d'expression d'IB1 est un des mécanismes par lequel l'ex-4 prodigue son effet sur la cellule. En effet, cette molécule stimule l'activité du promoteur du gène et ainsi compense la réduction du contenu en IB1 causée par le stress. Outre ce rôle anti-apoptotique, dans ce travail de thèse nous avons mis en évidence une autre fonction d'IB1 dans la cellule ß. La réduction de l'activité ou des niveaux d'expression d'IB1 induisent une réduction importante de la sécrétion de l'insuline en réponse au glucose. Le mécanisme par lequel IB1 régule la sécrétion de l'insuline implique à la fois le métabolisme du glucose et éventuellement le transport vésiculaire en contrôlant l'expression de la protéine annexin A2. En résumé, IB 1 est une molécule clé à travers laquelle l'environnement du diabétique pourrait exercer un effet délétère sur la cellule ß. L'amélioration de l'activité d'IB1 et/ou de son expression devrait être considérée dans les approches thérapeutiques futures visant à limiter la perte des cellules ß dans le diabète. Abstract : ß-cells of the pancreatic islets of Langerhans produce and secrete insulin when blood glucose rises. In turn, insulin ensures that plasma glucose concentrations return within a relatively narrow physiological range. If ß-cells die or fail to produce enough insulin, individuals develop one of the most common diseases in Western countries, namely type 2 diabetes (T2D). Chronic excess of oxidized low density lipoproteins (oxLDL) and/or pro-inflammatory cytokines such as interleukin 1-ß (IL-1ß) contribute to decline of ß-cells and thereby are thought to accelerate progression of the disease overtime. We showed that profound reduction in the levels of the mitogen activated protein kinase 8 interacting protein 1 also called islet brain 1 (IB1) causes ß-cell failure accomplished by oxLDL or IL-1 ß. IB1 regulates insulin expression and cell survivals by inhibiting the c-Jun N-terminal Kinase pathway. Diminution in IB 1 levels leads to an increase in activation of the JNK pathway induced by environmental stressors, and thus initiates loss of insulin expression and programmed cell death. The mimetic agents of the glucoincretin glucagon-like peptide 1 such as exendin-4 (ex-4) are new class of hypoglycaemic medicines for treatment of T2D. The beneficial property is in part achieved by preserving insulin expression and ß-cell survival against stressors related to diabetes. Restored levels in IB 1 account for the cytoprotective effect of the ex-4. In fact, the latter molecule .stimulates the promoter activity of the gene and thus compensates loss of IB1 content triggered by stress. Beside of the anti-apoptotic role, an additional leading function for IB 1 in ß-cells was highlighted in this thesis. Impairment in IB1 activity or silencing of the gene in ß-cells revealed a major reduction in insulin secretion elicited by glucose. The mechanisms whereby IB 1 couples glucose to insulin release involve glucose metabolism and potentially, vesicles trafficking by maintaining the levels of annexin A2. IB 1 is therefore a key molecule through which environmental factors related to diabetes may exert harmful effects on ß-cells. Improvement in IB 1 activity and/or expression should be considered as a target for therapeutic purpose.

On AUV control architecture

This paper surveys control architectures proposed in the literature and describes a control architecture that is being developed for a semi-autonomous underwater vehicle for intervention missions (SAUVIM) at the University of Hawaii. Conceived as hybrid, this architecture has been organized in three layers: planning, control and execution. The mission is planned with a sequence of subgoals. Each subgoal has a related task supervisor responsible for arranging a set of pre-programmed task modules in order to achieve the subgoal. Task modules are the key concept of the architecture. They are the main building blocks and can be dynamically re-arranged by the task supervisor. In our architecture, deliberation takes place at the planning layer while reaction is dealt through the parallel execution of the task modules. Hence, the system presents both a hierarchical and an heterarchical decomposition, being able to show a predictable response while keeping rapid reactivity to the dynamic environment

A survey addressing the fundamental matrix estimation problem

Epipolar geometry is a key point in computer vision and the fundamental matrix estimation is the only way to compute it. This article surveys several methods of fundamental matrix estimation which have been classified into linear methods, iterative methods and robust methods. All of these methods have been programmed and their accuracy analysed using real images. A summary, accompanied with experimental results, is given

The outcome of acute schistosomiasis infection in adult mice with postnatal exposure to maternal malnutrition

Maternal malnutrition during the lactation period in early development may have long-term programming effects on adult offspring. We evaluated the combined effects of parasitological behaviour and histopathological features and malnutrition during lactation. Lactating mice and their pups were divided into a control group (fed a normal diet of 23% protein), a protein-restricted group (PR) (fed a diet containing 8% protein) and a caloric-restricted group (CR) (fed according to the PR group intake). At the age of 60 days, the offspring were infected with Schistosoma mansoni cercariae and killed at nine weeks post-infection. Food intake, body and liver masses, leptinaemia, corticosteronaemia, collagen morphometry and neogenesis and the cellular composition of liver granulomas were studied. PR offspring showed reduced weight gain and hypophagia, whereas CR offspring became overweight and developed hyperphagia. The pre-patent period was longer (45 days) in both programmed offspring as compared to controls (40 days). The PR-infected group had higher faecal and intestinal egg output and increased liver damage. The CR-infected group showed a lower number of liver granulomas, increased collagen neogenesis and a higher frequency of binucleate hepatocytes, suggesting a better modulation of the inflammatory response and increased liver regeneration. Taken together, our findings suggest that neonatal malnutrition of offspring during lactation affects the outcome of schistosomiasis in mice.

Continuidad de cuidados de enfermería a través de un protocolo asistencial de transferencia

The creation and establishment within the healthcare team of the Primary Healthcare Centre of Baena of a Protocol and Procedures for healthcare that provides a communication tool for nurses enabled the healthcare team to develop a dynamic circuit and at the same time communication with nurses of the Critical Care and Emergency Team (DCCU) assigned to that centre. The created work environment for healthcare included Case Management Nurse, Primary Healthcare Nurse and Critical Care and Emergency Nurse. Thus, nursing assessment and actions for programmed and urgent healthcare, provided the first contact with DCCU nurses and reoriented them for the proposed healthcare plan for the patients, in addition to communicate with the rest of the healthcare team. This article presents the results of continuous nurse healthcare over nine months, applying this protocol.

Differential expression of the costimulatory molecules CD86, CD28, CD152 and PD-1 correlates with the host-parasite outcome in leprosy

Leprosy is a spectral disease exhibiting two polar sides, namely, lepromatous leprosy (LL) characterised by impaired T-cell responses and tuberculoid leprosy in which T-cell responses are strong. Proper T-cell activation requires signalling through costimulatory molecules expressed by antigen presenting cells and their ligands on T-cells. We studied the influence of costimulatory molecules on the immune responses of subjects along the leprosy spectrum. The expression of the costimulatory molecules was evaluated in in vitro-stimulated peripheral blood mononuclear cells of lepromatous and tuberculoid patients and healthy exposed individuals (contacts). We show that LL patients have defective monocyte CD86 expression, which likely contributes to the impairment of the antigen presentation process and to patients anergy. Accordingly, CD86 but not CD80 blockade inhibited the lymphoproliferative response to Mycobacterium leprae. Consistent with the LL anergy, there was reduced expression of the positive signalling costimulatory molecules CD28 and CD86 on the T-cells in these patients. In contrast, tuberculoid leprosy patients displayed increased expression of the negative signalling molecules CD152 and programmed death-1 (PD-1), which represents a probable means of modulating an exacerbated immune response and avoiding immunopathology. Notably, the contacts exhibited proper CD86 and CD28 expression but not exacerbated CD152 or PD-1 expression, suggesting that they tend to develop a balanced immunity without requiring immunosuppressive costimulatory signalling.

Apoptotic markers in protozoan parasites.

ABSTRACT: The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms.In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities.

Study of drug tolerance mechanisms and of the calcineurin pathway in the opportunistic pathogen "Candida albicans"

ABSTRACT :Azole antifungal drugs possess fungistatic activity in Candida albicans making this human pathogen tolerant to these agents. The conversion of azoles into fungicidal agents is of interest since their fungistatic properties increase the ability of C. albicans to develop drug resistance. In C. albicans, the phosphatase calcineurin (calcineurin) is essential for antifungal drug tolerance. Up to now, the only known target of calcineurin is Crzl, which is a transcription factor (TF) involved in responses to ionic stress. Thus, most of the components of the calcineurin signaling remain to be identified in C. albicans.In this work, the calcineurin pathway was investigated in order to i) characterize the role of calcineurin in the biology of C. albicans, ii) identify putative targets of calcineurin and iii) characterize the phenomenon of tolerance to antifungal drugs. Towards these aims, four different approaches were used.First, using C. albicans microarrays, an attempt was made to identify a set of calcineurindependent genes (CDGs). Since CDGs were highly dependent upon the external stimulus used to activate calcineurin (Ca2+ or terbinafine), this stimulus bias was bypassed by the construction of strains expressing a truncated autoactive form of calcineurin (Cmp1tr) in a doxycyclinedependent manner. The characterization of Cmpltr was undertaken and results showed that it mimicked awild-type activated calcineurin for all tested phenotypes (i.e. Cnbl-dependence, inhibition by FK506, phosphatase 2B activity, ability to dephosphorylate Crzl and to regulate Crz1-and calcineurin-dependent genes, role in antifungal drug tolerance and susceptibility, role in colony formation on Spider agar). Cmp1tr was therefore considered as a valid tool to study the calcineurin signaling pathway. In silico analysis of CDGs allowed the identification of i) a significant overlap between CDGs and genes regulated by the Cyrl signalíng pathway, ii) putative interactions between calcineurin activation and cell wall reorganization and phospholipid transport, iii) a putative interactión between calcineurin and the regulation of translation and iv) a putative relation between calcineurin and proteasome regulation. Further in silico analyses of the promoters of Crz1-independent CDGs were performed to identify TFs (other than Crz1) that were likely to regulate CDGs and therefore to be a direct target of calcineurin. The analyses revealed that Rpn4 and Mnl1 were TFs likely to be regulated by calcineurin.Second, in order to better characterize azole tolerance, an attempt was made to i) confirm the role of Hsp90 in fluconazole tolerance with a doxycycline-dependent Hsp90 expression system and ii) assess its calcineurin-dependence. Hsp90 was found to be significantly involved in fluconazole tolerance. However, results were not in agreement with the hypothesis that Hsp90 mediates fluconazole tolerance by the only downstream effector calcineurin. Rather Hsp90 is interacting with numerous components for fluconazole tolerance.Third, a collection of C. albicans TFs mutants were screened for loss of tolerance to terbinafine and fluconazole in order to identify TFs involved in antifungal drug tolerance. Out of the 265 TFs mutants screened, only the upc2Δ/Δ mutant showed a loss of fluconazole and terbinafine tolerance. Interestingly, no relation between Upc2 and calcineurin activity was found. These results suggested that the tolerance to antifungal drugs must not be only considered as a calcineurin-dependent phenomenon in C. albicans.Fourth, using FRCS analyses, an attempt was made to identify putative signs of programmed cell death (PCD) in calcineurin mutant cells upon loss of tolerance to terbinafine. A high proportion of cells died from both RO5-dependent (which is a sign of PCD) and ROS-independent (which is a sign of loss of homeostasis) processes in the calcineurin mutant. While these results suggest that calcineurin represses both loss of homeostasis and PCD, the role of calcineurin in PCD is still an open question.In conclusion, this work allowed i) the identification of several putative calcineurin targets, ii) the discovery of several links between calcineurin and signaling pathways and important biological processes and iii) the identification of novel components of calcineurin-independent mechanisms that participate in tolerance to antifungal drugs in C. albicans.RÉSUME :Les azoles sont des antifongiques qui présentent une activité fongistatique contre Candida albicans et rendent cette levure tolérante à ces agents. La conversion des azoles en agents fongicides est d'intérêts car leurs propriétés fongistatiques favorisent le développement de résistance aux drogues chez C. albicans. La calcineurine (calcineurin) est une phosphatase essentielle pour la tolérance aux antifongiques chez C. albicans. La seule cible connue de la calcineurin est Crz1, un facteur de transcription (FT) impliqué dans la réponse aux stress ionique. Ainsi, la plupart des constituants de la voie de signalisation de la calcineurin restent encore à être identifiés chez C. albicans.Dans ce travail de thèse, la voie de signalisation de la calcineurin a été étudiée de sorte à i) caractériser le rôle de la calcineurin dans la biologie de C. albicans, ii) identifier de nouvelles cibles de la calcineurin et iii) caractériser le phénomène de tolérance aux antifongiques. A ce propos, quatre approches ont été entreprises.Premièrement, des puces à ADN de C. albicans ont été utilisées afin d'identifier les gènes dépendants de la calcineurin (GDCs). Les GDCs étant étroitement dépendants du stimulus utilisé pour activer la calcineurin, le biais «stimulus» a été évité via la construction d'une souche exprimant une forme tronquée et autoactive de la calcineurin (Cmp1tr), en présence de doxycycline. La caractérisation de Cmp1tr a été entreprise et les résultats ont montré qu'elle mimait une calcineurin sauvage et activée pour la plupart des phénotypes testés (i.e. dépendance à Cnb1, inhibition par le FK506, activité phosphatase 2B, déphosphorylation de Crz1 et régulation de gènes dépendant de la calcineurin, rôle dans la tolérance et la susceptibilité aux antifongiques, rôle dans la formation des colonies sur milieu Spider). Cmp1tr a donc été considéré comme un outil pertinent pour l'étude de la voie de signalisation de la calcineurin. Les analyses in silico des GDCs ont permis l'identification i) d'un chevauchement entre les GDCs èt les gènes régulés par la voie de signalisation de Cyrl, ii) d'une interaction entre la calcineurin et la réorganisation de la paroi cellulaire ainsi que le transport des phospholipides, iii) d'une interaction entre calcineurin et la régulation de la traduction et iv) une relation entre la calcineurin et la régulation du protéasome. De plus, une analyse in silico des promoteurs des GDCs avec une régulation indépendante de Crz1 a permis d'identifier deux FTs qui pourraient être des cibles directes de la calcineurin, Rpn4 et Mnll.Deuxièmement, afin de caractériser la tolérance aux azoles, il a été entrepris i) de confirmer le rôle de Hsp90 dans la tolérance au fluconazole en utilisant un système d'expression dépendant de la doxycycline et ii) de caractériser sa dépendance à la calcineurin. Hsp90 a été montré impliqué dans la tolérance aux azoles. Cependant, les résultats n'ont pas corroboré une hypothèse expliquant le rôle d'Hsp90 dans la tolérance aux antifongiques par son unique. interaction avec la calcineurin. Il a été proposé que le rôle d'Hsp90 dans la tolérance aux antifongiques soit dû à ces multiples interactions avec le protéome de C. albicans plutôt que par son interaction avec un partenaire unique.Troisièmement, une collection de mutant pour des FTs de C. albicans a été criblée pour une perte de tolérance au fluconazole ou à la terbinafine, de sorte à identifier les FTs impliqués dans la tolérance aux antifongiques. Sur les 265 FTs passés au crible, seul le mutant upc2Δ/Δ a montré une perte de tolérance au fluconazole et à la terbinafine. Aucune relation n'a été trouvée entre la calcineurin et l'activité d'Upc2. Ces résultats suggèrent que la perte de tolérance aux antifongiques ne doit pas être considérée comme un phénomène exclusivement lié à la voie de signalisation de la calcineurin.Quatrièmement, en utilisant la cytométrie de flux, la présence de signes de mort cellulaire programmée (MCP) a été recherchée lors de la perte de tolérance du mutant calcineurin incubé avec de la terbinafine. Une grande proportion de cellules mortes incluant ou non une production de ROS (un signe de MCP) a été détectée dans le mutant calcineurin. Ces résultats préliminaires suggèrent que la calcineurin réprime autant la perte d'homéostasie qu'elle régule l'entrée en MCP. Cependant d'autres analyses sont nécessaires pour démontrer clairement le rôle de la calcineurin dans la régulation de la MCP.En conclusion, ce travail de thèse a permis i) l'identification de plusieurs cibles possibles de la calcineurine, ii) la découverte de plusieurs interactions entre la calcineurine et d'autres voies de signalisation et processus biologiques importants et iii) de démontrer la présence de voies indépendantes de la calcineurine impliquées dans la tolérance aux antifongiques chez C. albicans.

The forgotten face of regular physical exercise: a 'natural' anti-atherogenic activity.

Humans are not programmed to be inactive. The combination of both accelerated sedentary lifestyle and constant food availability disturbs ancient metabolic processes leading to excessive storage of energy in tissue, dyslipidaemia and insulin resistance. As a consequence, the prevalence of Type 2 diabetes, obesity and the metabolic syndrome has increased significantly over the last 30 years. A low level of physical activity and decreased daily energy expenditure contribute to the increased risk of cardiovascular morbidity and mortality following atherosclerotic vascular damage. Physical inactivity leads to the accumulation of visceral fat and consequently the activation of the oxidative stress/inflammation cascade, which promotes the development of atherosclerosis. Considering physical activity as a 'natural' programmed state, it is assumed that it possesses atheroprotective properties. Exercise prevents plaque development and induces the regression of coronary stenosis. Furthermore, experimental studies have revealed that exercise prevents the conversion of plaques into a vulnerable phenotype, thus preventing the appearance of fatal lesions. Exercise promotes atheroprotection possibly by reducing or preventing oxidative stress and inflammation through at least two distinct pathways. Exercise, through laminar shear stress activation, down-regulates endothelial AT1R (angiotensin II type 1 receptor) expression, leading to decreases in NADPH oxidase activity and superoxide anion production, which in turn decreases ROS (reactive oxygen species) generation, and preserves endothelial NO bioavailability and its protective anti-atherogenic effects. Contracting skeletal muscle now emerges as a new organ that releases anti-inflammatory cytokines, such as IL-6 (interleukin-6). IL-6 inhibits TNF-α (tumour necrosis factor-α) production in adipose tissue and macrophages. The down-regulation of TNF-α induced by skeletal-muscle-derived IL-6 may also participate in mediating the atheroprotective effect of physical activity.

PD-1 is a regulator of NY-ESO-1-specific CD8+ T cell expansion in melanoma patients.

The programmed death 1 (PD-1) receptor is a negative regulator of activated T cells and is up-regulated on exhausted virus-specific CD8(+) T cells in chronically infected mice and humans. Programmed death ligand 1 (PD-L1) is expressed by multiple tumors, and its interaction with PD-1 resulted in tumor escape in experimental models. To investigate the role of PD-1 in impairing spontaneous tumor Ag-specific CD8(+) T cells in melanoma patients, we have examined the effect of PD-1 expression on ex vivo detectable CD8(+) T cells specific to the tumor Ag NY-ESO-1. In contrast to EBV, influenza, or Melan-A/MART-1-specific CD8(+) T cells, NY-ESO-1-specific CD8(+) T cells up-regulated PD-1 expression. PD-1 up-regulation on spontaneous NY-ESO-1-specific CD8(+) T cells occurs along with T cell activation and is not directly associated with an inability to produce cytokines. Importantly, blockade of the PD-1/PD-L1 pathway in combination with prolonged Ag stimulation with PD-L1(+) APCs or melanoma cells augmented the number of cytokine-producing, proliferating, and total NY-ESO-1-specific CD8(+) T cells. Collectively, our findings support the role of PD-1 as a regulator of NY-ESO-1-specific CD8(+) T cell expansion in the context of chronic Ag stimulation. They further support the use of PD-1/PD-L1 pathway blockade in cancer patients to partially restore NY-ESO-1-specific CD8(+) T cell numbers and functions, increasing the likelihood of tumor regression.

An essential role for the Leishmania major metacaspase in cell cycle progression.

Metacaspases (MCAs) are distant orthologues of caspases and have been proposed to play a role in programmed cell death in yeast and plants, but little is known about their function in parasitic protozoa. The MCA gene of Leishmania major (LmjMCA) is expressed in actively replicating amastigotes and procyclic promastigotes, but at a lower level in metacyclic promastigotes. LmjMCA has a punctate distribution throughout the cell in interphase cells, but becomes concentrated in the kinetoplast (mitochondrial DNA) at the time of the organelle's segregation. LmjMCA also translocates to the nucleus during mitosis, where it associates with the mitotic spindle. Overexpression of LmjMCA in promastigotes leads to a severe growth retardation and changes in ploidy, due to defects in kinetoplast segregation and nuclear division and an impairment of cytokinesis. LmjMCA null mutants could not be generated and following genetic manipulation to express LmjMCA from an episome, the only mutants that were viable were those expressing LmjMCA at physiological levels. Together these data suggest that in L. major active LmjMCA is essential for the correct segregation of the nucleus and kinetoplast, functions that could be independent of programmed cell death, and that the amount of LmjMCA is crucial. The absence of MCAs from mammals makes the enzyme a potential drug target against protozoan parasites.

Influence of the Paracoccidioides brasiliensis14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

The fungal strain Paracoccidioides brasiliensisremains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensismolecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensisuses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.