Tesis sobre el tema "Relais cellule"

The use of low cost fixed wireless relays has been proposed as a way to deploy high data-rate networks at an affordable cost. During the last decade, significant academic and industrial research has been dedicated to relays. Protocol architectures for cellular-relaying networks are currently considered for standardization as part of both IEEE 802.16 and 3GPP. Various relaying techniques have successfully been commercialized over the years. This dissertation concentrates on the particular case of large scale use of low cost relays, for which focus is put on signal processing and radio resource allocation, rather than on antenna and radio frequency (RF) design, or on network planning. A key question is how low relay cost is low enough for a relaying architecture to be viable from an economic point of view? We develop a framework for evaluating the viability of relaying solutions. The framework is based on a comparison between the relaying architectures and traditional single-hop cellular architectures. This comparative analysis is done from an operator perspective, and is formulated as a network-dimensioning problem. The associated investment decisions are based on financial measures (cost or profit) and taken under technical constraints (throughput, coverage, etc.).First, we consider a large number of traditional dimensioning scenarios, in which the radio network is design for a predefined traffic demand and target quality of service level. We show that the use of low cost relays can indeed be viable, but that the cost savings vary strongly from case to case and often are only modest. Due to the half-duplex nature of the low cost relays, these relays are best suited for providing coverage to guaranteed data-rates, at low end-to-end spectral efficiency, and in environments with strong shadow fading. The type of environment and the placement of relays are more important than the specific protocols and algorithms used in the network. Therefore, traditional network planning remains an essential and challenging task, which is unlikely to be replaced by large-scale (unplanned) use of relays.Second, we suggest a new direction of research in which the viability of relays is judged considering the entire life cycle of a radio network. We give several examples in which the temporary use of relays is economically viable, especially if the service uptake is slow or the uncertainty about the future demand is high. This is particularly relevant if the last-mile cost of a network is dominated by the backhaul transmission cost, and if relaying is implemented as a feature of an access point, rather than as a new device type.
QC 20100812

The rapid increase in use of smart phones and devices has introduced a growing demand in data traffic which the conventional homogeneous macro cells can no longer satisfy. A promising solution to this is by spectrum reuse through deploying various low-power small cells overlaid on the macro cell. This represents the heterogeneous cellular network that we see today. The change from homogeneous to heterogeneous environment requires new cellular network models to be developed. This thesis presents various novel approaches and models for both homogeneous and heterogeneous cellular networks. Cellular network models aim to provide insights into the performance of the network and its users. One of the most important metrics of network performance and user experience is the signal-to interference- plus-noise-ratio, or SINR. A network operator wanting to optimize the performance of a heterogeneous network needs to know the spatial distribution of the SINR. So, researchers need to carefully consider the network parameters and their spatial distributions while modeling cellular networks. Besides having low-power base stations deployed in hot spot regions, cooperative communication can also be used to improve user performance by reducing the effect of path loss and fading. Considering these facts, the proposed research considers the inclusion of cooperation while modeling cellular networks. Under the proposed scheme, a user in low coverage can use a nearby low-power base station via cooperation to communicate with the macro base station. The main contributions of this thesis can be summarized as below: • A simplified linear cellular network model is proposed which considers the average user distance to determine user performance under a uniform user distribution scenario. This model simplifies the linear grid based model while improving the conventional Wyner model with fading. The proposal is extended to incorporate Gaussian based non-uniform distribution of users within macro cells. The effect of cooperation on user performance is also included in the proposed model where a cell boundary user can use a nearby low-power base station as relay. Mathematical models for analytically evaluating both downlink and uplink transmissions are developed in a multi-cell scenario. The user distributions of interfering neighboring cells are also taken into account. • A two-dimensional heterogeneous cellular network model is developed which considers a realistic spatial distribution of both macro and low-power base stations. For avoiding the probability of severe interference caused by Poisson point process distributed macro base stations, the proposed model considers a grid based distribution for macro base stations and a random distribution for the low-power base stations. Cooperative communication is included in the proposed model where the low-power base stations can act as relays for macro users that are in low coverage. Mathematical models for analytically evaluating user outage and coverage performance is formulated. • A large number of the total cellular network users today are vehicular, i.e. traveling in public transports while using cellular connectivity. The signal strength inside vehicles are attenuated by vehicle penetration loss and hence, becomes weak. To solve this issue, recently researchers have proposed the use of moving relays (MRs) which are mounted on top of public transportation vehicles. However, the impact of having MRs in current heterogeneous cellular networks is not yet investigated. A network model is proposed which considers the presence of MRs on top of suburban trains and derives coverage performance of both vehicular and non-vehicular users for downlink. The effect of MRs in the overall network performance is also evaluated. • Cellular network modeling for uplink is generally known to be difficult since both signal and interference become user location dependent. Moreover, due to power control the transmission power of users varies across cell locations which complicate the analysis to a greater extent. A heterogeneous network model is proposed for uplink with the presence of MRs on suburban trains. Per-user fractional power control is considered and both vehicular and non-vehicular user outage performance is derived. • Cooperative communication via MRs is also included in the proposed heterogeneous network models for both uplink and downlink. According to the proposal, a train traveling in the macro cell with a MR mounted on top can be used as a relay for nearby non-vehicular users in low coverage. Different traveling directions for the train within macro cells are considered and the effect of cooperation via MR on user performance is analyzed for each case. Extensive simulations are carried out for evaluating network performance, which demonstrate the capability of the proposed models in substantially enhancing the performance. The performance evaluation is established with the inclusion of cooperation and MRs in the proposed heterogeneous cellular network models.

L'augmentation du trafic sur les réseaux sans fil ne permet plus de traiter les données en utilisant les protocoles standard des réseaux filaires, qui sont eux sans interférences. Ainsi, les nœuds des réseaux sans fil doivent coopérer en exploitant les corrélations inhérentes à la proximité des utilisateurs afin d'exploiter au mieux la capacité d'un tel réseau.Dans cette thèse, nous considérons tout d'abord le problème de codage de source avec information adjacente compressée. Le nœud coopératif, ayant accès à un signal corrélé avec celui de la source, peut en envoyer une version compressée au destinataire sur un lien indépendant, permettant d'économiser du débit sur le lien principal. En utilisant une caractérisation des cellules de Voronoi du quantificateur utilisé, nous avons pu améliorer un algorithme de décodage itératif basé sur des codes LDPC.La seconde partie de la thèse traite des problèmes de codage de canal, où les nœuds coopératifs sont des relais. L'exemple le plus simple d'une telle communication est le canal à relais, où un relais aide à la communication entre la source et la destination. Alors que dans le problème de codage de source, le canal de corrélation entre la source et le nœud coopératif est fixé, dans le codage de canal, la question est de savoir quelle opération effectuer au relais. Tout d'abord, nous considérons un problème quelque peu dual au problème de codage de source avec information adjacente compressée, en considérant des bruits corrélés au relais et la destination. Puis, nous étudions des bornes sur la capacité et des débits atteignables pour deux extensions du canal à relais, le canal à relais bidirectionnel avec des bruits corrélés au relais et aux destinations, où deux sources échangent leurs données avec l'aide d'un relais, et le canal multidirectionnel avec liens directs (qui modélisent la proximité des utilisateurs), où les utilisateurs sont regroupés dans des clusters et échangent leurs données localement au sein d'un même cluster avec l'aide d'un relais
The current wireless data traffic growth cannot be handled by classical multi-hop network protocols as in interference-free wired networks, thus it has been recognized that network nodes need to cooperate in order to take advantage of source and/or channel signal correlations, which is needed to achieve fundamental capacity limits.This thesis first considers a cooperative source coding problem, namely binary source coding with coded side information (CoSI): the helper node has access to a signal that is correlated with the source and may send a compressed version on a separate link to the destination, thus rate can be saved on the main source-destination link. Using a characterization of the Hamming-space Voronoi regions of the quantizer at the helper node, an improved practical scheme based on LDPC codes is proposed.The second part of the thesis considers cooperative channel coding, where helper nodes are relays. The simplest example of such a communication is the relay channel, in which a relay node helps the source to send its message to the destination. Whereas in the source coding problem, the correlation between source and side information is given, in channel coding, the main question is to find the best relaying operation. First, a somewhat dual problem to source coding with CoSI is studied, by considering correlated noises at the relay and destination. Then, various extensions of the relay channel are characterized using upper bounds on capacity and achievable rates: the two-way relay channel with correlated noises at the relay and destinations, where two sources wish to exchange their data with the help of a relay, and the multiway relay channel with direct links, where users, grouped into fully connected clusters (users in a cluster can overhear each others' messages), wish to exchange their messages locally within a cluster with the help of one relay

Relays can be used in cellular systems to increase coverage as well as reduce the total power consumed by mobiles in a cell. This latter benefit is particularly useful for mobiles operating on a depleted battery. The relay can be a mobile, a car or any other device with the appropriate communication capabilities. In thesis we analyze the impact of using relays under different situations. We first consider the problem of reducing total power consumed in the system by employing relays intelligently. We find that in a simulated, fully random, mobile cellular network for CDMA (Code Division Multiple Access), significant energy savings are possible ranging from 1.76 dB to 8.45 dB. In addition to reducing power needs, relays can increase the coverage area of a cell by enabling mobiles located in dead spots to place relayed calls. We note that use of relays can increase the useful service area by about 10% with real life scenarios. We observe that in heavy building density areas there is more need of relays as compared to low building density areas. However, the chance of finding relays is greater in low building density areas. Indeed, having more available idle nodes helps in choosing relays, so we conclude that unlike present day implementations of cellular networks, the base station should admit more mobiles (beyond the capacity of the cell) even if they are not placing calls since they can be used as relays. One constraint of using relays is the potential to add interference in the same cell and in neighboring cells. This is particularly true if the relays are not under power control. Based on our analysis, we conclude that in interference limited systems like CDMA the relays have to be under power control otherwise we will reduce the total capacity by creating more dead spots. Thus, we believe that either the base station should be responsible to allocate relays or relays should be provided with enough intelligence to do power control of the downlink. Finally, we show how utility of data services can be increased by use of relays.

L'hétérogénéité et l’irrégularité croissante des déploiements des réseaux sans fil de nouvelles générations soulèvent des défis importants dans l’évaluation de performances de ces réseaux. Les modèles classiques s’appuyant sur des modèles hexagonaux pour décrire les emplacements géographiques des nœuds de transmission sont difficilement adaptables à ces réseaux. Dans ce contexte, il a été proposé un nouveau paradigme de modélisation des réseaux sans fil qui s’appuie sur les processus ponctuels de Poisson (PPP), et de manière générale sur la géométrie stochastique. L'analyse, au travers de ces outils mathématiques, présente une complexité indépendante de la taille du réseau, et permet d’estimer avec précision des quantités pratiques liées aux performances des réseaux cellulaires. Cette thèse a porté sur la faisabilité mathématique de l'approche fondée sur les PPP en proposant de nouvelles méthodes mathématiques d’approximations justes incorporant des modèles de propagation du canal radio. Dans un premier temps, un nouveau cadre mathématique, considéré comme une approche Equivalent-in-Distribution (EiD), a été proposée pour le calcul exact de la probabilité d'erreur dans les réseaux cellulaires. L'approche proposée, s’appuyant donc sur la géométrie aléatoire et des modèles spatiaux, montre une complexité faible en terme d’évaluation numérique et est applicable à un grand nombre de configurations MIMO pour lesquelles nous considérons différentes techniques de modulation et techniques de récupération du signal. Dans un deuxième temps, nous étudions les performances des réseaux cellulaires en présence de relais, où trois processus ponctuels de Poisson modélisent respectivement les nœuds relais, les stations de base, et les terminaux mobiles. Pour ce modèle, nous avons considéré des critères souples d'association. Le cadre mathématique proposé et les résultats associés ont montré que les performances dépendent fortement des exposants des fonctions d’atténuation sur les deux premiers sauts sans fil. Nous montrons aussi qu’une mauvaise configuration du réseau peut amener à des gains négligeables de l’utilisation de cette technique. Enfin, nous considérons la modélisation des réseaux cellulaires au travers d’un PPP et d’un modèle unifié d'atténuation de signal généralisée qui prend en compte deux types de liaisons physiques : line-of-sight (LOS) et non-line-of-sight (NLOS). Un modèle de complexité réduite décrivant les propriétés de la liaison radio a aussi été proposée et permet de prendre en compte dans nos calculs un grand nombre de modèle radio proposés dans la littérature. Les résultats montrent, entre autres, qu’une densité optimale pour le déploiement des BS existe lorsque les liens LOS/NLOS sont classés en fonction de leur charge. Nous comparons nos résultats, s’appuyant donc sur un PPP pour modéliser la position des stations de bases et notre modèle de canal radio, avec des simulations de Monte Carlo décrivant des déploiements réels de stations de bases et un modèle de type blocages de construction empiriques. Une bonne correspondance est observée
The increasing heterogeneity and irregular deployment of the emerging wireless networks give enormous challenges to the conventional hexagonal model for abstracting the geographical locations of wireless transmission nodes. Against this backdrop, a new network paradigm by modeling the wireless nodes as a Poisson Point Process (PPP), leveraging on the mathematical tools of stochastic geometry for tractable mathematical analysis, has been proposed with the capability of fairly accurately estimating the performance of practical cellular networks. This dissertation investigated the mathematical tractability of the PPP-based approach by proposing new mathematical methodologies, fair approximations incorporating practical channel propagation models. First, a new mathematical framework, which is referred to as an Equivalent-in-Distribution (EiD)-based approach, has been proposed for computing exact error probability of cellular networks based on random spatial networks. The proposed approach is easy to compute and is shown to be applicable to a bunch of MIMO setups where the modulation techniques and signal recovery techniques are explicitly considered. Second, the performance of relay-aided cooperative cellular networks, where the relay nodes, the base stations, and the mobile terminals are modeled according to three independent PPPs, has been analyzed by assuming flexible cell association criteria. It is shown from the mathematical framework that the performance highly depends on the path-loss exponents of one-hop and two-hop links, and the relays provide negligible gains on the performance if the system is not adequately designed. Third, the PPP modeling of cellular networks with unified signal attenuation model is generalized by taking into account the effect of line-of-sight (LOS) and non-line-of-sight (NLOS) channel propagation. A tractable yet accurate link state model has been proposed to estimate other models available in the literature. It is shown that an optimal density for the BSs deployment exists when the LOS/NLOS links are classified in saturate load cellular networks. In addition, the Monte Carlo simulation results of the real BSs deployments with empirical building blockages are compared with those with PPP distributed BSs with the proposed link state approximation at the end of this dissertation as supplementary material. In general, a good matching is observed

Le corps genouille lateral dorsal (cgld est le relais thalamique de la voie visuelle retinocorticale. Les cellules relais du cgld, comme l'ensemble des cellules relais thalamiques, presentent deux modes de fonctionnement distincts: la modalite de transfert qui correspond a la fonction du cgld en tant que relais visuel et la modalite oscillatoire qui est observe durant l'assouplissement et le sommeil a ondes lentes. Le travail presente dans ce memoire s'interesse a ces deux aspects et analyse les mecanismes cellulaires qui les soutendent. Les experiences ont ete realisees sur des tranches maintenues in vitro au moyen d'enregistrements intracellulaires par la technique de courant impose. Dans la premiere partie de notre travail, nous avons etudie le role des circuits locaux inhibiteurs dans l'integration de l'information retinienne. Nos resultats montrent que le potentiel postsynaptique d'excitation declenche par la stimulation electrique du tractus optique est suivi de deux potentiels postsynaptiques d'inhibition. Le premier est de nature chlore et est du a l'action du gaba sur des recepteurs gabaa tandis que le deuxieme est potassique et est du a l'action du gaba sur des recepteurs gabab. Dans la deuxieme partie nous avons analyse les mecanismes cellulaires a la base du comportement oscillatoire des cellules relais du thalamus. Nos resultats montrent que des modifications ioniques du milieu extracellulaire induisent in vitro des comportements oscillatoires stables: un comportement autooscillant rythmique, un comportement non rythmique necessitant l'activation des recepteurs nmda et un comportement autooscillant en bouffees

The two regulatory subunits (R1 and R2) of PKA are differentially expressed in several cancer cell lines and studies indicate distinct roles for these subunits in growth control. In recent years, mutations of genes involved in cAMP signalling and resulting in the constitutive activation of cAMP formation have been identified as cause of endocrine neoplasia. In particular, activating mutations of the ? subunit of the stimulatory G protein gene have been found in about 30-40% of GH secreting pituitary adenomas and in subsets of thyroid, adrenocortical, ovarian and testicular Leydig cell tumors, while mutations of the PKA regulatory subunit 1A gene (PRKAR1A) have been identified in patients with Carney complex, a familial multiple endocrine neoplasia syndrome. This thesis evaluated the expression of the different PKA regulatory subunits (R1A, R2A, R2B) in human pituitary adenomas, cortisol-secreting adrenocortical tumors and melanomas, as well as the effects of selective subunit activation on cell proliferation. Our data indicate that altered PKA R1/R2 ratio characterize most endocrine tumors, when compared with the normal counterpart, and that pharmacological and genetic manipulations able to revert this unbalanced expression are able to induce significant anti-proliferative and pro-apoptotic effectis in human tumoral cells.

La récidive tumorale est l'un des principaux obstacles à surmonter à l'avenir pour améliorer la survie globale des patients atteints de cancer du côlon (CCR). Les échecs thérapeutiques observés chez les patients sont compatibles avec une accumulation de cellules souches cancéreuses (CSCs) résistantes aux médicaments. Dans cette étude, nous démontrons que le récepteur nucléaire PXR (NR1I2) agit comme un régulateur important de la chimiorésistance des CSCs coliques et de leur capacité à initier la rechute tumorale après traitement. Nous avons d'abord montré que l'expression de PXR augmente avec celle de certains marqueurs des CSCs dans des cellules cancéreuses de patients CCR traitées par chimiothérapies. Nous avons constaté que PXR est préférentiellement exprimé dans les CSCs coliques et qu'il contribue à l'enrichissement des CSCs après chimiothérapies in vitro et in vivo. Par des approches de transcriptomiques, nous avons observé qu'au sein des CSCs coliques, PXR contrôle l'expression d'un large réseau de gènes marqueurs des CSCs coliques, ainsi que des gènes impliqués dans la résistance aux médicaments ou à l'apoptose, ou impliqués dans la dissémination métastatique. Enfin, l'inhibition de PXR par interférence à ARN diminue la survie et auto-renouvèlement des cellules souches cancéreuses du côlon in vitro, ainsi que leur capacité à résister à la chimiothérapie après xénogreffes, conduisant à des retards importants de rechute tumorale après traitements par chimiothérapies in vivo. Cette étude suggère fortement que l'inhibition ciblée de PXR peut représenter une stratégie de traitement néo-adjuvant afin de diminuer la résistance aux médicaments et la récidive des patients CCR via la sensibilisation des cellules souches cancéreuses aux chimiothérapies classiques
Tumor recurrence is one of the major obstacles to overcome in the future to improve overall survival of patients with colon cancer. High rates and patterns of therapeutic failure seen in patients are consistent with a steady accumulation of drug-resistant cancer stem cells (CSCs). Here, we demonstrate that the nuclear receptor PXR (NR1I2) acts as a key regulator of colon CSC chemoresistance and of their ability to generate post-treatment tumor relapse. We first determined that the enrichment of PXR paralleled that of CSC markers upon treatment of colon cancer cells with standard of care chemotherapy. We found that PXR was highly expressed in colorectal cancer cells displaying CSC markers and function and that it was instrumental for the emergence of CSCs following chemotherapy in vitro and in vivo. mRNA profiling experiments in colon CSCs indicated that PXR transcriptionally controls a large network of genes including markers of stemness, genes involved in resistance to drug/apoptosis or migration/invasion. Finally, PXR down-regulation altered the survival and self-renewal of colon CSCs in vitro and hampered their capacity to resist chemotherapy in vivo, leading to significant delays of post-chemotherapy tumor relapse. This study strongly suggests that targeting PXR may represent a novel treatment strategy to prevent drug resistance and recurrence through the sensitization of CSCs to standard chemotherapy. Taken together, our data strongly suggest that PXR plays an instrumental role in the so-called "intrinsic" pan-resistance of CSCs against therapy

Cell Biology
Ph.D.
Opioids are used for the clinical treatment of pain, but can lead to tolerance and addiction. In this project we examined the role of the serotonin (5-HT) system originating from the dorsal raphe nucleus (DRN) during morphine exposure, withdrawal, abstinence and following an acute stressor capable of initiating behavioral relapse. Following four days of morphine exposure rats showed a preference for the morphine paired side of the conditioned place preference (CPP) chamber. After four days of morphine abstinence, rats showed no net preference for the morphine paired side. The next day rats were exposed to forced swim stress and returned to the CPP chamber where they demonstrated stress-induced reinstatement. Utilizing whole-cell patch-clamp we demonstrated an increase in the amplitude of inhibitory post-synaptic currents (IPSCs) in 5-HT DRN neurons, but not non 5-HT DRN neurons of morphine-conditioned subjects. Next the stress neurohormone corticotrophin releasing factor (CRF) was administered in vitro instead of forced swim. We found an increase in CRF-R2-mediated inward current of 5-HT DRN neurons in animals with a morphine history. From this experiment we concluded that morphine history sensitizes 5-HT DRN neurons to the GABAergic inhibitory effects of stress and to some of the effects of CRF. In the next series of experiments we surgically implanted either morphine or placebo pellets in rats for 72 hours to create physical dependence. The pellets were subsequently removed, and animals experienced up to seven days of abstinence with and without forced swim stress exposure. Real time quantitative PCR was used to measure the mRNA levels of genes at multiple points across this timeline. We examined genes involved in trophic support, stress responses and 5-HT regulation. We determined that mRNA levels for brain-derived neurotrophic factor (BDNF) and the BDNF receptor TrkB were downregulated after opiate exposure, and again following seven days of abstinence. Following seven days of abstinence there was a decrease in mRNA levels of the CRF-R1 receptor and an increase in mRNA levels of the CRF-R2 receptor. During acute opiate exposure there was a decrease in mRNA levels for the autoregulatory 5-HT1A receptor. Finally following forced swim, there was an increase in mRNA levels of the 5-HT synthesis enzyme TPH2. Collectively these results indicate that a morphine history in abstinent subjects may produce hypofunctioning of the 5-HT DRN system induced by multiple neurochemical mechanisms and this dysregulation may enhance vulnerability to stress-induced relapse.
Temple University--Theses

The abuse of and addiction to drugs of abuse, such as tobacco, alcohol, opioids, and illicit drugs, are growing global problems that affect the welfare of individuals and societies worldwide. The National Institute of Drug Abuse estimates the annual cost of substance abuse to be over $740 billion in costs related to drug intoxication, withdrawal and relapse. A primary challenge in the treatment of substance abuse is the tendency of users to relapse following acute or extended periods of abstinence; on average over 60% of substance abusers will return to drug use within a year of receiving treatment, many relapsing following stressful life events. Central to the successful treatment of drug addiction is understanding the cellular mechanisms by which relapse episodes occur. Current data suggest that the activation of pituitary adenylate cyclase activating peptide (PACAP) systems in the bed nucleus of the stria terminalis (BNST) is an important event underlying stress-induced reinstatement to drug-seeking in a rodent model of stress-induced relapse. In conjunction with immunohistochemistry and pharmacological treatments, we used this behavioral model of stress-induced relapse to evaluate PACAP and PACAP type-1 receptor (PAC1-R) signaling in stress-induced reinstatement to cocaine seeking. Activation of the PAC1 receptor appears to be critical to stress-induced reinstatement, as the selective PAC1-R agonist, maxadilan, produced reinstatement behaviors in the absence of stress. Moreover, BNST pretreatment with either mitogen activated protein kinase-ERK (MEK) or endocytosis inhibitors to block extracellular signal-related kinase (ERK) signaling attenuated stress-induced reinstatement. Furthermore, BNST phosphorylated ERK (pERK) expression, mediated by PAC1-R activation, is substantially potentiated in cocaine-experienced animals after stressor exposure, in a manner that is dependent on endosomal signaling and MEK activity. These data suggest that the activation of a PAC1 signaling cascade is a key event underlying stress-induced reinstatement. Furthermore, this data may suggest a permanent change in the BNST PACAP system (sensitization) following cocaine exposure.

Mon projet de thèse visait à caractériser de nouveaux aspects physiologiques relatifs à la biologie des ARN de transfert (ARNt) chez les plantes. Il se composait de deux chapitres. Le premier chapitre avait pour objectif de décrire d’un point de vue évolutif l’organisation génomique des gènes d’ARNt nucléaires (ADNt) de plantes, et caractériser les pendants moléculaires régulant leur expression. Par rapport aux autres organismes vivants, ce travail a permis de révéler une haute organisation des ADNt sur les chromosomes de plantes se caractérisant par : des déserts géniques au niveau des centromères, des distances inter-ADNt homogènes, et la présence de clusters d’ADNt répétés. Aussi, ce travail a permis de décrire les premières évidences d’un silencing épigénétique de l’expression d’ADNt de plantes organisés en clusters de répétitions. La conjonction de ces résultats suggère un rôle potentiel des ADNt dans l’orchestration des génomes tridimensionnels de plantes. Le deuxième chapitre avait pour objectif de caractériser la biogenèse et les fonctions de petits ARN non-codants dérivant d’ARNt, les tRF, chez A. thaliana. Ce travail a permis de montrer que les RNases T2 sont les principaux acteurs de leur biogenèse
My thesis project aimed at figuring out new physiological aspects related to transfer RNA (tRNAs) biology in plants. It was divided in two parts. The first one consisted in describing from an evolutionarily point of view the genomic layout of plant tRNA genes (tDNAs) onto nuclear chromosomes. Contrasting with non-plant organisms, plant nuclear tDNAs exhibit a higher order in their chromosomal scattering. This is due to centromeric regions exclusion combined to highly conserved inter-tDNA distances and tDNA repeat clusters occurrences. Moreover, that part consisted in deciphering elements of nuclear tDNA transcriptional regulation. We brought evidences of epigenetic silencing mechanisms at clustered tDNA loci. As a perspective, we propose tDNAs might play a role in three-dimensional genomes orchestration. The second aspect referred to characterizing the biogenesis and functions of small non-coding RNAs deriving from tRNAs: tRFs. The results obtained demonstrate that RNases T2 as the predominant molecular actors for their biogenesis

Notre groupe a montré que l’hypoxie sévère (0.1% O2) induit un arrêt du cycle cellulaire en G0 des cellules humaines CD34+ et des cellules murines FDCP mix. Peu d’études ont exploré l’existence de Cellules Initiatrices de Leucémie (CIL) dans les LAL et leur rôle dans les rechutes. Notre projet s’est focalisé sur l’effet de l’hypoxie sévère sur la quiescence des CIL dans les LAL, qui pourrait être responsable d’un pourcentage de rechutes. En effet dans la niche hématopoïétique, ou sont localisées les Cellules souches hématopoïétiques et probablement les CIL, la concentration d’oxygène avoisinerait 0,1%.Nous avons utilisé la lignée de LAL NALM6 pour explorer les effets de l’hypoxie sévère sur leur survie, leur cycle cellulaire et leur chimiorésistance. Nos résultats ont mis en évidence qu’une culture à 0.1% O2 durant 7 jours de la lignée NALM6: - inhibe leur prolifération sans surmortalité, - révèle une population restreinte de CIL quiescentes et chimiorésistantes capables d’induire une leucémie dans des souris. Nous avons recherché les relations entre l’hypoxie sévère et quelques caractéristiques des cellules primaires de patients atteints de LAL : existence et rôle de CIL résistantes à l’hypoxie et aux agents thérapeutiques conventionnels des LAL ; localisation de ces cellules résiduelles dans la moelle osseuse des souris xénogreffées. Nos résultats suggèrent que certaines rechutes de LAL pourraient être dues à la persistance à long terme de « quiescent/dormant » CIL dans les niches hypoxiques de la moelle osseuse. Ce modèle est intéressant pour explorer les mécanismes in vitro et in vivo de chimiorésistance dans les LAL et le rôle de l’environnement dans ce phénomène
Our group showed that severe hypoxia (0.1% O2) induces G0 cell-cycle-arrest of human CD34+ cells and of murine FDCP-mix Cells. Few studies explored the existence of quiescent Leukemia Initiating Cells (LIC) in ALL and their role in primary chemoresistance and relapses. Our project is focused on the effect of very low O2 concentrations in the maintenance of quiescent LIC in ALL, that could be responsible of a percentage of relapses. Indeed in bone marrow niches, where hematopoietic stem cells and probably LIC are located, the O2 concentrations are below 0.1%.In the present study we used the NALM-6 ALL cell line to explore the effects of culture at 0.1% O2 on their survival, cell cycle and chemoresistance. Our results evidence that a 7 days culture of NALM-6 cells at 0.1% O2: - inhibits their proliferation without major cell death; - reveals a restricted LIC population of quiescent and chemoresistant LIC; - maintains quiescent chemoresistant LIC that induce leukemia when injected in immunodeficient mice. We investigated the relationships between severe hypoxia and some characteristics of ALL primary cells obtained from patients: existence and role of quiescent chemoresistant LICs in ALL relapses; location of these residual cells inside the bone marrow of engrafted mice. Our results suggest that some ALL relapses could be due to the long term persistence of “quiescent / dormant” LIC in hypoxic bone marrow niches. This model is of interest for exploring the in vitro and in vivo (xenograft) mechanisms of chemoresistance in ALL and the role of the bone marrow environment in this phenomenon

L’outil génomique a considérablement modifié notre connaissance des hémopathies malignes, que ce soit sur le plan physiopathologique, diagnostique, pronostique ou thérapeutique. Dans la première partie de ce travail, nous avons travaillé sur une grande cohorte d’anémies réfractaires sidéroblastiques avec thrombocytose (ARS-T). Nous avons démontré qu’il s’agissait d’une entité indépendante, avec une présentation moléculaire particulière associant (i) des mutations de SF3B1 dans plus de 85% des cas, expliquant son versant myélodysplasique et (ii) des anomalies de JAK2 dans plus de 50% des cas, expliquant son versant prolifératif. La perspective de cette première partie est d’identifier la ou les mutation(s) responsables du caractère myéloprolifératif dans les ARS-T JAK2WT. Les lymphomes B-diffus à grandes cellules (LBDGC) représentent les lymphomes malins non-Hodgkiniens les plus fréquents chez l’adulte. Dans la deuxième partie de ce travail, nous avons réalisé l’analyse par SNP-array d’une série homogène d’échantillons issus de la cohorte CORAL, une étude prospective internationale portant sur les LBDGC en rechute. Notre objectif était d’identifier les anomalies de nombre de copies (ANC) associées à chacun des deux types de rechutes, précoces ou tardives. Les rechutes précoces sont associées à une forte proportion d’anomalies affectant les régulateurs du cycle cellulaire, de l’apoptose et de la transcription. Les rechutes tardives sont associées à des anomalies affectant les régulateurs de l’immunité et de la prolifération cellulaire. Cette étude permet de mieux comprendre les déterminants de la rechute dans les LBDGC et ouvre de nouvelles perspectives thérapeutiques
Genomics provided new insights in our knowledge of pathophysiology, diagnostic approach, prognosis and therapeutic perspectives in hematological malignancies. In the first part of this work, we studied a large cohort of Refractory Anemia with Ring sideroblasts and marked Thrombocytosis (RARS-T). We demonstrated that RARS-T can be considered as an independent entity, with a specific molecular pattern, associating : (i) SF3B1 mutations in more than 85% of cases, accounting for its myelodysplastic aspect and (ii) JAK2 mutations, accounting for its myeloproliferative aspect in more than 50% of cases. Future prospects of the first part of this work is to identify (the) mutation(s) responsible for the myeloproliferative part of JAK2WT RARS-T. Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma in adults. In the second part of this work, we performed SNP-array analysis of a homogeneous series of samples from the CORAL cohort, an international prognostic study on relapsed DLBCLs. Our purpose was to identify Copy Number Variations (CNV) associated ER or LR. ER DLBCLs are associated with high rates of CNVs affecting regulators of cell cycle, apoptosis and transcription. In LR DLBCLs, CNVs are related to immune response and cell proliferation. This study provides new insights into the genetic aberrations in relapsed DLBCLs and open up new therapeutic perspectives

Les communications mobiles grand public, le téléchargement de vidéos et l’utilisation d’applications mobiles représentent l’essentiel de l’utilisation actuelle des ressources radio dans les réseaux 4G ; mais pour que le spectre des usages et la diversité des utilisateurs soient grandement élargis, de nombreux efforts de recherche et de nombreuses propositions commencent à émerger pour la mise en place d’un nouveau standard appelé 5G, qui vise des secteurs très variés et qui sont des piliers importants d’une société : l’énergie, la santé, les médias, l’industrie ou le transport.Pour répondre à ces défis, ce nouveau standard devra regrouper plusieurs technologies parmi lesquelles, la réalisation d’un réseau Ultra-Dense (UDN) pour obtenir une couverture plus dense, plus robuste aux obstacles et augmenter la capacité du réseau. En conséquence, le réseau cellulaire ultra-dense est en train de devenir l'une des principales caractéristiques des réseaux cellulaires 5G. L'idée de base est d'obtenir des nœuds d'accès aussi proches que possible des utilisateurs finaux. L’obtention de cette solution prometteuse est réalisée par le déploiement dense de petites cellules appelées « Small Cells » dans des hotspots où un trafic immense est généré, en utilisant les ondes millimétriques pour étendre la bande passante de transmission.Ces « smalls cells » doivent optimiser au mieux la réception du signal selon l’emplacement de l’utilisateur par rapport à l’antenne pour concentrer l’émission du signal dans les directions utiles, par l’utilisation de réseaux d’antennes reconfigurables en diagramme et à fort gain. Cette méthode évite ainsi d’utiliser toute la puissance disponible pour émettre « à l’aveugle » en espérant tomber sur le terminal.Les travaux menés dans le cadre de cette thèse s'inscrivent dans ce contexte. L’objectif donc consiste à concevoir et réaliser un petit réseau d’antennes ou « Small Cells » travaillant dans les bandes de fréquences millimétriques doté d’une capacité à changer la direction du faisceau selon les besoins des utilisateurs. Une technique de reconfiguration de diagramme de rayonnement avec changement de polarisation appliquée sur un réseau d’antennes planaires a été développée. Ce manuscrit se divise comme suit : après un rappel des objectifs de la 5G ainsi que ses exigences dans le chapitre 1, nous avons introduit dans le chapitre 2, les architectures et la théorie des différents réseaux d’antennes, ainsi que les différentes techniques de dépointage de faisceaux. Le troisième chapitre est consacré à la conception d’un élément rayonnant en forme de croix à double polarisation et diagramme de rayonnement reconfigurable par utilisation d’éléments parasites commutables. Cet élément rayonnant a ensuite été utilisé dans le chapitre 4, pour concevoir des sous-réseaux et réseaux d’antennes multi-ports, reconfigurables, à fort gain
Consumer mobile communications, video downloads and the use of mobile applications represent most of today's use of radio resources in 4G networks; but in order to broaden the spectrum of uses and the diversity of users, many research efforts and numerous proposals are beginning to emerge for the implementation of a new standard called 5G, which targets a wide range of sectors and is important pillars of a society: energy, health, media, industry or transportation. To fulfil these challenges, this new standard will have to combine several technologies, including the creation of an Ultra-Dense Network (UDN) to obtain a dense coverage, more robust to obstacles and increase the capacity of the network. This promising solution is obtained by the deployment of dense small cells in hotspots where huge traffic is generated, and by using millimeter waves to extend the transmission bandwidth.These smalls cells must optimize the received signal according to the location of the user, by using a high gain beam reconfigurable antennas array. This method avoids using all the available power to issue "blind" hoping to fall on the terminal.The Objectives of this thesis is therefore to design a small antennas array or "Small Cells" working in mmWave bands with an ability to change the direction of the beam according to the needs of users. A dual polarized beam reconfigurable technique applied on a planar antenna array has been developed.This manuscript is divided as follows: after a reminder of the objectives of the 5G and its requirements in chapter 1, we have introduced in chapter 2, the architectures and theory of the different antenna arrays, as well as the different techniques of beam steer. The third chapter is dedicated to the design of a cross patch antenna with a dual polarization and reconfigurable beam by using switchable parasitic elements. This radiating element was then used in Chapter 4 to design a high-gain reconfigurable, multiport sub-arrays and antenna arrays

Le cancer colorectal est la première pathologie cancéreuse de la sphère digestive, tant en terme de fréquence que de mortalité par an. Chaque année, 41 000 nouveaux cas sont diagnostiqués et 17 000 décès sont dus à ce cancer en France. Deux paramètres cliniques expliquent la mortalité de ce cancer; d'une part le fait qu'un patient sur deux est diagnostiqué au stade métastatique ou va présenter des lésions métastatiques durant l'histoire de sa pathologie, d'autre part le fait que les patients après traitement vont fréquemment présenter une récidive de leur pathologie. L'utilisation de régimes de chimiothérapies avant et après résection métastatique améliore la survie sans récidive à court terme, mais à 2 ans post chirurgie l'avantage apporté est perdu. Ainsi, la compréhension des mécanismes d'échappement à la chimiothérapie et régissant la croissance tumorale est d'intérêt pour tenter de limiter la récidive tumorale. L'objectif de ce travail de thèse a consisté en l'analyse de sous-populations obtenues sous pression de chimiothérapie au 5-Fluorouracile (5FU) dérivées de la lignée cancéreuse colique HT29, ainsi que les mécanismes moléculaires associés. Notre clone le plus chimiorésistant isolé, le modèle cellulaire 5F31, quitte le compartiment prolifératif sous traitement à fortes doses de 5FU, ceci étant associé à une perturbation de la voie de signalisation de la Src kinase c-Yes et de son partenaire, le co-activateur transcriptionnel YAP. Sous traitement, les cellules chimiorésistantes entrent en quiescence, le complexe protéique entre c-Yes et YAP est perdu et la quantité totale et nucléaire de YAP diminue de manière significative (Igoudjil, Touil, Corvaisier et al. 2014 Clinical Cancer Research). Dès lors, la suite des travaux a consisté en l'étude du rôle potentiel de YAP sur la balance quiescence/prolifération sous 5FU. L'inhibition pharmacologique ou l'inhibition transitoire de l'expression de YAP et de son paralogue, la protéine TAZ, dans plusieurs lignées cancéreuses coliques induit l'augmentation de la fraction de cellules quiescentes, associée au ralentissement significatif de la croissance tumorale. A l'inverse, la surexpression d'une forme constitutivement active de YAP demeurant nucléaire sous 5FU maintient les cellules 5F31 en prolifération et sensibilise les cellules à la chimiothérapie. Au niveau des effecteurs protéiques, l'induction de quiescence (par traitement à la chimiothérapie ou inhibition de YAP/TAZ) est associée à la perte d'expression de la Cycline E1 et du facteur de transcription c-Myc. A l'inverse, la surexpression du dominant constitutivement actif de YAP dans les cellules 5F31 conduit à l'expression soutenue de la Cycline E1 sous 5FU, expression nécessitant l'activation du facteur de transcription CREB. L'inhibition de la Cycline E1 permet d'induire la quiescence cellulaire, proposant cette protéine comme l'un des effecteurs des protéines YAP/TAZ dans la régulation entre la quiescence et la prolifération cellulaire (Corvaisier et al, Oncotarget, 2016). En conclusion, nos données montrent l'importance du rôle des protéines YAP/TAZ dans le maintien des cellules en prolifération via l'expression notamment de la Cycline E1. Nos résultats sur cohorte de patients atteints de métastases hépatiques de cancers colorectaux montrent que l'expression des co-activateurs YAP/TAZ est liée à un index prolifératif plus important, confortant nos données sur le rôle de ces protéines dans la croissance tumorale. De plus, l'expression élevée de YAP et TAZ est associée en analyses multivariées à une récidive plus précoce et à une survie globale plus faible. Ainsi, l'étude de l'expression et du niveau d'activation de ces acteurs serait un marqueur pronostic intéressant dans l'anticipation de la récidive métastatique ; ainsi que des cibles thérapeutiques intéressantes pour tenter de limiter la rechute tumorale
Colorectal cancer is the most frequent and lethal cancerous pathology from the digestive system. Each year in France, 41 000 new cases are diagnosed and 17 000 patients die due to this pathology. This high mortality is mainly due to the rate of patients with liver metastatic lesions and the early relapse of those metastases after treatment. The use of chemotherapy prior to surgery induces a decrease of early relapse, however 2 years after resection this advantage is lost. Thus, understanding the mechanisms underlying escape to treatment is required to try to delay or prevent tumor recurrence.The aim of this doctoral work was to analyze clonal chemoresistant subpopulations derived from the colorectal cancer cell line HT29 after chronic exposure to 5-Fluorouracil (5FU) and molecular mechanisms associated with chemoresistance. The most chemoresistant clonal subpopulation, 5F31, stops its proliferation after treatment with high dose of 5FU, this behavior being associated with the modulation of the c-Yes/YAP axis. After treatment, 5F31 cells enter quiescence, interaction between c-Yes and YAP is lost and total and nuclear YAP protein expression reduces significantly (Igoudjil, Touil, Corvaisier et al. 2014, Clinical Cancer Research). The next step was to study functions of YAP protein in this chemotherapy- induced quiescence.Pharmacological or transient inhibition of YAP and its homolog TAZ, induces quiescence and reduces cellular growth in several colorectal cancer cell lines. On the other hand, overexpression of a constitutively active form of YAP in 5F31 cells forces cells to remain proliferative under 5FU treatment, enhancing 5F31 cell chemosensitivity to 5FU.Regarding proteic effectors, quiescence (either induced by 5FU or YAP/TAZ inhibition) is associated with loss of expression of the transcription factor c-Myc and Cyclin E1. In 5F31 cells expressing the active mutant form of YAP, Cyclin E1 expression is sustained after 5FU treatment through the activation of the transcription factor CREB. Cyclin E1 inhibition is sufficient to induce quiescence, therefore introducing this protein as one of the final effectors of YAP/TAZ co-activators in the regulation of the proliferation/quiescence switch in colorectal cancer cells (Corvaisier et al. 2016, Oncotarget).To conclude, our work reveals the importance of YAP/TAZ proteins for the maintenance of colorectal cancer cells proliferation through Cyclin E1 expression. Our work on liver metastases from patients with colorectal cancer shows that high expression of YAP/TAZ is connected to a higher proliferative index in metastatic lesions. Moreover, high YAP/TAZ expression is associated with shorter patient progression-free survival and shorter overall survival. Studying the expression and level of YAP/TAZ activation could be an interesting prognosis marker to anticipate metastatic relapse and potent druggable target to delay tumoral recurrence

L'objectif de cette thèse est d'étudier certains aspects des réseaux coopératifs sans fils à l'aide de la géométrie stochastique. Ça permets de considérer la distribution spatiale aléatoire des utilisateurs et les effets adverses de leur interaction, comme l’interférence.Nous étudions la performance, évaluée par la probabilité d'outage, atteignable dans un canal de relai full-duplex quand les nœuds opèrent dans un grand réseau sans fils où les émetteurs interférants sont modelés avec un processus ponctuel de Poisson homogène. Nous trouvons la probabilité d'outage des protocoles décodez-et-renvoyez (decode-and-forward, DF), et comprimez-et-renvoyez (compress-and-forward) et nous faisons une comparaison avec une transmission point à point et un protocole DF half-duplex. Ensuite, nous étudions une situation plus générale dans laquelle les émetteurs qui causent l'interférence peuvent aussi utiliser un relai ou faire des transmissions point à point. Nous étudions la relation entre les avantages de la cooperation et l'interférence qu'elle même génère.Dans la deuxième partie nous étudions la performance des stratégies de partage de vidéos par communications entre dispositifs mobiles (device-to-device, D2D) hors de la bande des communications cellulaires. Nous étudions la fraction des demandes de vidéos qui peuvent être satisfaites par D2D, c’est-à-dire, par le biais des émissions locales, plutôt que par la station de base. Pour étudier ce problème, nous introduisons un modèle de processus ponctuel, qui considère la stratégie de stockage dans les utilisateurs, le problème de comment lier les utilisateurs et les problèmes de la transmission et coordination entre les utilisateurs
The main goal of this work is to study cooperative aspects of large wireless networks from the perspective of stochastic geometry. This allows the consideration of important effects such as the random spatial distribution of nodes, as well as the effects of interference and interference correlation at receivers, which are not possible when a single link is considered in isolation.First, some aspects of the performance of the relay channel in the context of a large wireless network are considered. Mainly, the performance, in terms of outage probability (OP), of a single full-duplex relay channel utilizing decode-and-forward (DF) or compress-and-forward, when the interference is generated by uniform spatial deployment of nodes, modeled as a Poisson point process. The OP performance of these two protocols is compared with a point-to-point transmission and with a half-duplex DF protocol. Afterwards, the case in which more than one transmitter in the network may use a relay is considered. The effects of cooperation versus interference are studied, when the users use either full-duplex DF, or point-to-point transmissions. In a second phase, this work explores the advantages that could be obtained through out-of-band device-to-device (D2D) video file exchanges in cellular networks. These advantages are measured in terms of the fraction of requests that can be served in a time-block through D2D, thus avoiding a downlink file transfer from the base station. For this, a stochastic geometry framework is introduced, in which the user file-caching policy, user pairing strategy, and link quality and scheduling issues are considered

La protéine prion cellulaire (PrPC) est une protéine majoritairement exprimée à la surface des neurones, dont la conversion transconformationnelle en prion pathogène PrPSc, est à l'origine des maladies à prions. Il est clairement établi que la neurodégénérescence induite par la PrPSc dépend de l'expression de la PrPC dans les neurones et résulte d'une déviation de la/des fonction(s) de la PrPC par la PrPSc. Identifier le rôle de la PrPC est donc un pré-requis pour aborder les mécanismes de neurodégénérescence dans les maladies à prions. Une partie de mes travaux de thèse a permis de montrer que la PrPC exerce un rôle cytoprotecteur vis-à-vis de la cytokine inflammatoire TNFalpha. L'extinction de la PrPC dans les neurones (neurones PrPnull) rend ces cellules hypersensibles au TNFalpha en raison de l'accumulation membranaire des récepteurs au TNFalpha (TNFR). Mes travaux démontrent que la perte de la fonction régulatrice de la PrPC sur l'agrégation et la signalisation des intégrines bêta 1 dans les neurones PrPnull provoque la suractivation de la kinase PDK1, l'internalisation subséquente de l'alpha-sécrétase TACE, et un découplage de TACE vis-à-vis de l'un de ses substrats, TNFR. Étant donné la proximité phénotypique entre les neurones PrPnull (Ezpeleta et al. 2017) et les neurones infectés par la PrPSc (Pietri et al. 2013 ; Alleaume-Butaux et al. 2015), mes travaux plaident en faveur d'une perte de fonction cytoprotectrice de la PrPC dans les maladies à prions. Concernant l'infection à prions, mes travaux montrent que TACE internalisée en réponse à la suractivation de PDK1 est découplée d'un autre substrat, la protéine précurseur des peptides amyloïdes (APP), ce qui mène à l'accumulation des peptides neurotoxiques Abêta 40 et Abêta 42 caractéristiques de la maladie d'Alzheimer. Dans un contexte « infection à prions », les peptides Abêta 40/42 sont présents majoritairement sous une forme monomérique, et de façon plus discrète sous forme trimérique et tétramérique. Par des approches in vitro et in vivo, nous montrons que les peptides Abêta générés par les cellules infectées par les prions ne modifient ni la réplication ni l'infectiosité des prions. Néanmoins, nous démontrons que les formes oligomérisées d'Abêta sont capables de se déposer sous forme de plaques amyloïdes dans le cerveau des souris transgéniques APP23 infectées par les prions. Dans ces souris, les dépôts d'Abêta accélèrent la pathogenèse des prions. Le dernier axe de mon travail de thèse concerne les nanoparticules, des matériaux de taille nanométrique couramment utilisés dans de nombreux produits et procédés industriels. Mes travaux mettent en évidence que, à l'instar de la PrPSc et d'Abêta, des assemblages de nanoparticules de dioxyde de titane ou de noir de carbone se lient à la PrPC exprimée à la surface des neurones et dévient sa fonction de signalisation. Cette interaction PrPC/nanoparticules provoque, entre autres, la suractivation de PDK1, l'internalisation de TACE, et l'accumulation membranaire de TNFR. Les cellules neuronales exposées aux nanoparticules deviennent alors hypersensibles au stress inflammatoire TNFalpha. Le découplage de TACE à APP induit par les nanoparticules augmente aussi la production de peptides Abêta par les neurones. Même si aucune donnée épidémiologique n'associe une exposition aux nanoparticules à la maladie d'Alzheimer, mes travaux suggèrent une implication causale des nanoparticules dans l'initiation voire l'amplification de cette maladie
The cellular prion protein (PrPC) is a protein mostly expressed at the plasma membrane of neurons. Its transconformation into the pathogenic prion PrPSc is at the root of prion diseases. It is clearly established that the PrPSc-induced neurodegeneration depends on the expression of PrPC in neurons and results from the corruption of PrPC function(s) by PrPSc. Unravelling the role of PrPC is thus a prerequisite to grasp neurodegeneration mechanisms in prion diseases. Part of my work shows that PrPC exerts a cytoprotective function against TNFalpha inflammatory cytokine. PrPC silencing in neurons (PrPnull-neurons) renders these cells highly sensitive to TNFalpha due to surface accumulation of TNFalpha receptor (TNFR). My work demonstrates that the loss of PrPC regulatory function on the clustering and signaling downstream of bêta 1 integrins in PrPnull neurons provokes the overactivation of the kinase PDK1, subsequent internalization of TACE alpha-secretase, and uncoupling of TACE from TNFR substrate. Because of the phenotypic proximity between PrPnull neurons (Ezpeleta et al. 2017) and PrPSc-infected neurons (Pietri et al. 2013; Alleaume-Butaux et al. 2015), my work supports the view of a loss of PrPC protective function in prion diseases. As concerns prion infection, my work shows that after PDK1 overactivation, internalized TACE is uncoupled from another substrate, the amyloid peptides precursor protein (APP), leading to the accumulation of neurotoxic peptides Abêta 40 and Abêta 42, hallmarks of Alzheimer's disease. Within a prion infectious context, Abêta 40/42 peptides are predominantly present as monomers, and to a lesser extent, as trimers and tetramers. By combining in vitro and in vivo approaches, we show that Abêta peptides produced by infected neurons do not alter replication nor the infectivity of prions. Nevertheless, we demonstrate that oligomerized Abêta is able to form amyloid plaques in the brain of transgenic APP23 mice infected by prions. In these mice, Abêta deposits accelerate prion pathogenesis. The last axis of my work deals with nanoparticles, that is, nanometric materials commonly found in manufactured products and industrial processes. My work shows that, as PrPSc and Abêta, titanium dioxide or carbon black assemblies interact with PrPC at the surface of neurons and deviate its signaling function, which leads, inter alia, to PDK1 overactivation, TACE internalization, TNFR accumulation at the plasma membrane, and neuronal cells hypersensitivity to TNFalpha inflammatory stress. We also found that nanoparticle-induced TACE uncoupling from APP increases Abêta peptide production by neurons. Even if no epidemiological study has demonstrated to date a link between nanoparticle exposure and Alzheimer's disease, my work suggests an causal implication of nanoparticles in the initiation or amplification of this disease

Acute Lymphoblastic Leukemia (ALL) represents the most frequent cancer in childhood. Currently, more than 80% of children with ALL can be cured through intensive and risk-adapted chemotherapy protocols, but unfortunately, the remaining 20% ultimately relapse. Allogeneic hematopoietic stem cell transplantation (HSCT) is considered beneficial for approximately 10% of patients who are at high risk (HR) at frontline therapy according to the AIEOP-BFM protocol criteria, and for the majority of patients after ALL relapse. However, also after HSCT, relapse remains the leading cause of treatment failure in pediatric ALL. The strongest prognostic factor in childhood ALL is the monitoring of Minimal Residual Disease (MRD). MRD is defined as the persistence, in bone marrow (BM), of leukemic cells not identifiable through cyto-morphological methods. MRD diagnostics has been implemented into major frontline treatment protocols for pediatric ALL, in which it is routinely used to stratify patients into different risk classes: standard risk (SR), medium risk (MR) or high risk (HR) of relapse. The aim of MRD-based stratification is to refine therapy based on risk-class, maximizing cure and minimizing toxicities. Also for relapsed ALL patients and in patients undergoing HSCT, MRD assessment has been identified as one of the most relevant predictors of prognosis, useful to identify good and poor responders to the therapy. Nevertheless, the clinical significance of MRD in pediatric ALL patients given allogeneic HSCT has not yet been fully validated. The most widely used approach to detect MRD is represented by real-time quantitative PCR (RQ-PCR), a very sensitive and specific molecular assay. RQ-PCR is based on the patient-specific junctional regions of Immunoglobulin (Ig) and T-cell Receptor (TCR) genes rearrangements, detected on BM aspirates collected at diagnosis (or relapse) of ALL patient. In the first project of my PhD training (described in Chapter 1) we quantify MRD by RQ-PCR immediately before HSCT, in order to assess its clinical significance and impact on transplant outcome in a large cohort (119) of pediatric ALL patients in first, second or subsequent complete remission (respectively 1CR, 2CR or others CR). In addition, we consecutively analyzed MRD by RQ-PCR in 98/119 and 59/119 ALL patients, respectively during the first (post-HSCT1) and third (post-HSCT3) trimester after HSCT. The aim of these analyses was to address the question of whether MRD evaluation could provide further information to predict the risk of post-transplant leukemia recurrence. The overall 10-year event-free survival probability (EFSp) for patients with any level of positive MRD pre-HSCT was lower (39% for MRD < 1x10-3 and 18% for MRD ≥ 1x10-3) as compared with negative MRD patients (EFSp = 73%). When patients were analyzed according to the number of CR at HSCT, we observed that different levels of positivity had a different impact on EFSp: low-level MRD positivity had a negative impact only in patients transplanted in second or higher CR; while in first CR, only a high MRD positivity increased the risk of relapse. So pre-transplant MRD assessment confirmed to be a strong predictor of outcome and its effect was consistent throughout the different disease remissions. We also evaluated the EFSp according to the MRD assessment at post-HSCT1 and post-HSCT3. MRD negativity at early post-transplant was associated with a good EFSp (63%), that was even better when negativity was confirmed also at 3th trimester post-HSCT (pEFS = 84%). Also the variations of MRD levels over time were important. In particular the change between 1st and 3th trimester allowed to identify 2 categories of patients, with a dramatically different outcome: a group of patients with very poor prognosis (patients with an MRD increasing from post-HSCT1 to post-HSCT3) with an EFSp of only 8%, and a group of patients with very good prognosis (patients with unchanged negative MRD or decreasing to negative MRD and those with unchanged low-positive MRD) with an EFSp ≥ 80%. Overall, these results confirm that MRD assessment is important both before and after transplant, for early identification of patients with the highest risk of ALL recurrence and with a strong indication to a prompt immunological intervention and to adoption of new drugs. The second project (described in Chapter 2) was a preliminary study. We focused on a third generation PCR, the droplet digital PCR (ddPCR), that allows for an absolute quantification, with accurate concentration of target DNA. Instead, RQ-PCR allows for a relative quantification, since is based on the comparison with a calibration standard curve made with the diagnostic DNA of patient, for MRD level quantification in follow-up sample. Thus, availability of diagnostic sample can limit RQ-PCR assay. A broad spectrum of molecular markers has been yet interrogated using ddPCR for diagnostic purposes in various malignancies. Recently, the absolute method was evaluated for MRD quantification in lymphoproliferative disorders of adult, such as lymphomas and ALL; these reports showed a good correlation between quantitative PCR and ddPCR. However, there are still no studies in pediatric ALLs. In the light of this, we performed ddPCR analyses on BM samples of 65 pediatric ALL transplanted patients with the same primers and probes used for RQ-PCR and in the same reaction conditions. Comparing head-to-head the MRD results obtained with the two molecular approaches, we aimed to investigate the applicability of ddPCR for MRD assessment also in this context. First, we evaluated if positive but not-quantifiable (PNQ) MRD performed by RQ-PCR can be quantified by ddPCR; then we also evaluated the prognostic impact of pre-HSCT MRD levels assessed by ddPCR. A good level of concordance was found in results of both analyses (Pearson r = 0.98, P < 0.0001) and ddPCR was also able to quantify a various number of sample not-quantifiable by conventional RQ-PCR. Our results suggest that ddPCR has sensitivity, accuracy and reproducibility at least comparable with RQ-PCR. Statistical analyses have shown no significant differences in prognostic impact on outcome, if patients were stratified according to MRD levels detected by RQ-PCR and ddPCR, since EFSp of PNQ patients was very similar to that of MRD NEG by ddPCR (71% vs 68%, respectively). Despite this, the digital method was able to measure a positive and quantifiable value for 12 ALL patients who relapsed after HSCT, while RQ-PCR technique failed to identify relapse in advance. These preliminary data confirm that ddPCR may be an accurate and applicable tool for MRD evaluation also in the context of pediatric ALL clinical trials, but highlight the importance of extending the analysis on other retrospectively collected cases, to better define the role of ddPCR for prospective MRD evaluation in pediatric ALLs.
La Leucemia Linfoblastica Acuta (LLA) rappresenta la patologia tumorale più frequente in età pediatrica. Oltre l’80% dei bambini affetti da LLA viene trattato con successo grazie agli attuali protocolli di chemioterapia intensiva e basata sul rischio di ricaduta, ma sfortunatamente, il restante 20% ricade. Il trapianto di cellule staminali ematopoietiche (TCSE) ha un ruolo fondamentale nella guarigione di circa il 10% dei pazienti definiti ad alto rischio di ricaduta LLA in prima linea e per gran parte dei pazienti recidivati. Sfortunatamente, anche dopo il TCSE, la ricaduta si conferma come principale causa di fallimento terapeutico nelle LLA pediatriche. Il principale indicatore prognostico nelle LLA infantili è rappresentato dalla Malattia Residua Minima (MRM). La MRM è definita come la persistenza, all’interno del midollo osseo, di cellule leucemiche a livelli non identificabili attraverso esame citomorfologico. La valutazione della MRM è ormai parte integrante dei principali schemi terapeutici di prima linea, in cui viene usata per stratificare i pazienti in diverse classi di rischio di ricaduta (standard, intermedio o alto), con l'obiettivo di adattare la terapia al rischio individuale di ciascun paziente, ottimizzando le cure e riducendo al minimo la tossicità. Il monitoraggio della MRM è stato identificato come uno dei maggiori fattori predittivi di prognosi, anche per i pazienti ricaduti e per quelli trapiantati, in cui risulta ulteriormente vantaggioso per valutare la risposta alla terapia dei pazienti LLA. Ciononostante, il significato clinico della MRM nei pazienti sottoposti al TCSE non è ancora stato pienamente validato. L’approccio standard utilizzato per monitorare la MRM è attualmente rappresentato dalla real-time quantitative PCR (RQ-PCR), un saggio molecolare altamente sensibile e specifico, basato sulle regioni giunzionali dei riarrangiamenti dei geni delle immunoglobuline e del recettore dei linfociti T, identificati sugli aspirati midollari della diagnosi (o ricaduta) del paziente LLA. Nel primo progetto (descritto nel capitolo 1) del mio percorso di dottorato, abbiamo quantificato la MRM mediante PCR quantitativa immediatamente prima del TCSE, per valutare il suo significato clinico e l’impatto sull’outcome in una vasta coorte di pazienti pediatrici affetti da LLA (119), in prima remissione completa (1RC), seconda (2RC) o altre. Abbiamo poi analizzato MRM mediante RQ-PCR in 98/119 e 59/119 pazienti, rispettivamente durante il primo (post-TCSE1) e il terzo (post-TCSE3) trimestre dopo il trapianto. L’obiettivo di queste analisi è stato quello di capire se la valutazione MRM potesse fornire ulteriori informazioni, utili a identificare preventivamente i pazienti con maggior rischio di ricaduta leucemica dopo il trapianto. Dalle analisi di sopravvivenza in relazione ai livelli di MRM pre-TCSE nei pazienti LLA, qualsiasi livello di positività correla con un outcome sfavorevole (pEFS = 39% per MRM positiva < 1x10-3 e pEFS = 18% per MRM positiva ≥ 1x10-3), rispetto ai pazienti con MRM negativa (pEFS = 73%, P<0.0001). Inoltre, analizzando i pazienti in base al tipo di remissione al TCSE, livelli diversi di positività MRM correlano con un diverso impatto sulla pEFS: bassi livelli di positività MRM indicano infatti, una prognosi sfavorevole solo in pazienti trapiantati in seconda o altre RC, mentre in prima RC solo una positività alta si associa ad un aumentato rischio di ricaduta. Pertanto la MRM pre-TCSE si conferma come importante fattore predittivo di outcome e il suo effetto varia col variare del tipo di remissione al trapianto. È stata valutata, inoltre, la pEFS dei pazienti in base ai livelli di MRM post-TCSE1 e post-TCSE3; MRM negativa post-TCSE correla significativamente con un outcome favorevole, sia al 1° trimestre (pEFS = 63%), che ancor più se riscontrata al 3° trimestre (pEFS = 84%). Anche la valutazione prospettica del cambiamento di MRM è risultata significativa. In particolare, valutando la variazione di MRM dal 1° al 3° trimestre post-TCSE, i pazienti con MRM crescente hanno una prognosi sfavorevole (pEFS = 8%), mentre tutti gli altri gruppi correlano con una buona prognosi (pEFS ≥ 80%). Questi risultati confermano l’importanza del monitoraggio della MRM sia nel periodo precedente che successivo al TCSE, nell’identificare preventivamente pazienti ad alto rischio di ricaduta, possibili beneficiari di interventi immunologici preventivi. Il secondo progetto trattato (descritto nel capitolo 2) è stato uno studio preliminare, focalizzato su una PCR di terza generazione, la Droplet Digital PCR (ddPCR). Essa consente una quantifica di tipo assoluto, con un’accurata concentrazione del DNA target. La RQ-PCR fornisce, invece, una quantifica di tipo relativo, basata su una curva standard di calibrazione fatta con il DNA della diagnosi del paziente, per la quantificazione dei livelli di MRM di ciascun follow-up. Per cui, la PCR quantitativa può essere limitata dalla disponibilità di materiale diagnostico. Un ampio spettro di marcatori molecolari è già stato indagato mediante ddPCR per scopi diagnostici in varie patologie tumorali. Studi recenti hanno valutato l’applicabilità della ddPCR nell’ambito delle malattie linfoproliferative dell’adulto, come i linfomi e le LLA, mostrando una buona correlazione dei risultati fra le due metodiche in entrambi gli ambiti. Tuttavia, non sono ancora disponibili lavori che valutino questa correlazione nel campo delle leucemie pediatriche. Alla luce di questo, abbiamo eseguito analisi ddPCR sugli aspirati midollari di 65 pazienti pediatrici sottoposti a TCSE, utilizzando stessi primer e stesse sonde fluorescenti usati negli esperimenti RQ-PCR, nelle medesime condizioni di reazione. Mettendo a confronto i livelli di MRM emersi coi due approcci molecolari, si è investigata l’applicabilità della metodica assoluta per il monitoraggio della MRM anche in questo contesto. Inizialmente, sono stati valutati campioni risultati, mediante RQ-PCR, positivi ma non quantificabili (PNQ), per verificare se invece si potessero quantificare mediante ddPCR. Successivamente, è stato valutato anche l’impatto prognostico dei livelli MRM pre-TCSE ottenuti tramite ddPCR. Un buon livello di concordanza è emerso dai risultati ottenuti con entrambe le metodiche (Pearson r = 0.98, P < 0.0001); la ddPCR ha permesso, inoltre, di quantificare numerosi campioni risultati non quantificabili tramite RQ-PCR. I risultati suggeriscono che il metodo assoluto possieda sensibilità, accuratezza e riproducibilità almeno paragonabili alla PCR quantitativa convenzionale. I pazienti LLA analizzati sono stati stratificati sulla base dei livelli di MRD ottenuti con le due tecniche molecolari, ma nelle analisi di sopravvivenza non sono emerse differenze significative sulla prognosi. Infatti le pEFS dei pazienti con MRM negativa e positiva quantificabile per i due metodi risultano molto simili (rispettivamente 71% e 68%). Ciononostante, dal presente studio è emerso che col metodo digital sia stato possibile misurare un valore di MRM positivo e quantificabile per almeno 12 pazienti LLA che, in seguito al trapianto, hanno presentato una recidiva; viceversa, la RQ-PCR non era stata in grado di identificare anticipatamente la ricaduta di questi pazienti. Questi dati preliminari mostrano che la ddPCR possa essere un valido strumento per il monitoraggio della MRM e applicabile anche nel contesto dei trials clinici per pazienti LLA pediatrici. Tuttavia una prosecuzione dello studio ddPCR, con estensione della casistica analizzata, potrebbe essere utile a definire con precisione la significatività delle misurazioni con questa recente metodica.

Les cellules tueuses naturelles (NK) sont régulées par des récepteurs activateurs et inhibiteurs. La plupart des récepteurs inhibiteurs reconnaisse des molécules du complexe majeur d'histocompatibilité (CMH) de classe I, et protège les cellules saines des phénomènes d'auto-immunité médiés par les cellules NK. Cependant, certains récepteurs activateurs, incluant le récepteur killer cell Ig-like receptor (KIR) 2DS1, reconnaissent aussi des ligands CMH de classe I. Cela pose la question de savoir comment les cellules NK qui expriment des récepteurs activateurs deviennent tolérantes au soi. Nous avons cherché à déterminer si la présence de HLA-C2, le ligand du récepteurs 2DS1, peut induire les cellules NK qui expriment le 2DS1 à développer un état de tolérance au soi. Indépendamment de la présence ou de l'absence du ligand HLA-C2 dans le donneur, une activité anti-HLA-C2 a été identifiée in vitro dans certains clones NK 2DS1-positifs. La fréquence des clones NK avec réactivité anti-HLA-C2 était élevée parmi les donneurs homozygotes pour HLA-C1. De façon étonnante, nous n'avons pas constaté de différence statistiquement significative dans la fréquence de cytotoxicité anti-HLA-C2 entre les donneurs HLA-C2 hétérozygotes et les donneurs sans ligand HLA-C2. Par contre, les donneurs HLA-C2 homozygotes montrent une fréquence réduite de clones NK avec réactivité anti-HLA-C2 par rapport aux autres donneurs. Clones 2DS1-positifs qui co-expriment des KIR inhibiteurs spécifiques des molécules HLA de classe I du soi n’étaient pas communément cytotoxiques, et la cytotoxicité anti-HLA-C2 était limité presque exclusivement à des clones positifs seulement pour 2DS1 (« single positive » 2DS1 clones). Nous avons aussi identifié des clones 2DS1 « single positive » avec réactivité anti-HLA-C2 dans des patients recevant une greffe de cellules souches hématopoïétiques à partir de donneurs 2DS1. Ces résultats montrent que plusieurs cellules NK avec réactivité anti-HLA-C2 sont présentes dans des donneurs 2DS1 soit hétérozygotes soit homozygotes pour HLA-C1. En revanche, les clones 2DS1-positifs obtenus par des donneurs homozygotes pour HLA-C2 sont fréquemment tolérants aux antigènes HLA-C2
NK cells are regulated by inhibiting and activating cell surface receptors. Most inhibitory receptors recognize MHC-class I antigens, and protect healthy cells from NK cell-mediated auto-aggression. However, certain activating receptors, including the human killer cell Ig-like receptor (KIR) 2DS1, also recognize MHC-class I. This raises the question of how NK cells expressing such activating receptors are tolerized to host tissues. We investigated whether the presence of HLA-C2, the cognate ligand for 2DS1, induces tolerance in 2DS1-expressing NK cells. Anti-HLA-C2 activity could be detected in vitro in some 2DS1 positive NK clones irrespective of presence or absence of HLA-C2 ligand in the donor. The frequency of anti-HLA-C2 reactivity was high in donors homozygous for HLA-C1. Surprisingly, there was no significant difference in frequency of anti-HLA-C2 cytotoxicity in donors heterozygous for HLA-C2 and donors without HLA-C2 ligand. However, donors homozygous for HLA-C2 had significantly reduced frequency of anti-HLA-C2 reactive clones as compared to all other donors. 2DS1 positive clones that express inhibitory KIR for self-HLA class I were commonly non-cytotoxic, and anti-HLA-C2 cytotoxicity was nearly exclusively restricted to 2DS1 single positive clones lacking inhibitory KIR. 2DS1 single positive NK clones with anti-HLA-C2 reactivity were also present post-transplantation in HLA-C2 positive recipients of hematopoietic stem cell transplants from 2DS1 positive donors. These results demonstrate that many NK cells with anti-HLA-C2 reactivity are present in HLA-C1 homozygous and heterozygous donors with 2DS1. In contrast, 2DS1 positive clones from HLA-C2 homozygous donors are frequently tolerant to HLA-C2

Cette thèse étudie les communications directes entre les mobiles, appelées communications D2D, en tant que technique prometteuse pour améliorer les futurs réseaux cellulaires. Cette technologie permet une communication directe entre deux terminaux mobiles sans passer par la station de base. La modélisation, l'évaluation et l'optimisation des différents aspects des communications D2D constituent les objectifs fondamentaux de cette thèse et sont réalisés principalement à l'aide des outils mathématiques suivants: la théorie des files d'attente, l'optimisation de Lyapunov et les processus de décision markovien partiellement observable POMDP. Les résultats de cette étude sont présentés en trois parties. Dans la première partie, nous étudions un schéma de sélection entre mode cellulaire et mode D2D. Nous dérivons les régions de stabilité des scénarios suivants: réseaux cellulaires purs et réseaux cellulaires où les communications D2D sont activées. Une comparaison entre ces deux scénarios conduit à l'élaboration d'un algorithme de sélection entre le mode cellulaire et le mode D2D qui permet d'améliorer la capacité du réseau. Dans la deuxième partie, nous développons un algorithme d'allocation de ressources des communications D2D. Les utilisateurs D2D sont en mesure d'estimer leur propre qualité de canal, cependant la station de base a besoin de recevoir des messages de signalisation pour acquérir cette information. Sur la base de cette connaissance disponibles au niveau des utilisateurs D2D, une approche d'allocation des ressources est proposée afin d'améliorer l'efficacité énergétique des communications D2D. La version distribuée de cet algorithme s'avère plus performante que celle centralisée. Dans le schéma distribué des collisions peuvent se produire durant la transmission de l'état des canaux D2D ; ainsi un algorithme de réduction des collisions est élaboré. En outre, la mise en œuvre des algorithmes centralisé et distribué dans un réseau cellulaire, type LTE, est décrite en détails. Dans la troisième partie, nous étudions une politique de sélection des relais D2D mobiles. La mobilité des relais représente un des principaux défis que rencontre toute stratégie de sélection de relais. Le problème est modélisé par un processus contraint de décision markovien partiellement observable qui prend en compte le dynamisme des relais et vise à trouver la politique de sélection de relais qui optimise la performance du réseau cellulaire sous des contraintes de coût
This thesis considers Device-to-Device (D2D) communications as a promising technique for enhancing future cellular networks. Modeling, evaluating and optimizing D2D features are the fundamental goals of this thesis and are mainly achieved using the following mathematical tools: queuing theory, Lyapunov optimization and Partially Observed Markov Decision Process (POMDP). The findings of this study are presented in three parts. In the first part, we investigate a D2D mode selection scheme. We derive the queuing stability regions of both scenarios: pure cellular networks and D2D-enabled cellular networks. Comparing both scenarios leads us to elaborate a D2D vs cellular mode selection design that improves the capacity of the network. In the second part, we develop a D2D resource allocation algorithm. We observe that D2D users are able to estimate their local Channel State Information (CSI), however the base station needs some signaling exchange to acquire this information. Based on the D2D users' knowledge of their local CSI, we provide an energy efficient resource allocation framework that shows how distributed scheduling outperforms centralized one. In the distributed approach, collisions may occur between the different CSI reporting; thus, we propose a collision reduction algorithm. Moreover, we give a detailed description on how both centralized and distributed algorithms can be implemented in practice. In the third part, we propose a mobile relay selection policy in a D2D relay-aided network. Relays' mobility appears as a crucial challenge for defining the strategy of selecting the optimal D2D relays. The problem is formulated as a constrained POMDP which captures the dynamism of the relays and aims to find the optimal relay selection policy that maximizes the performance of the network under cost constraints

A significant challenge for fourth generation cellular systems is the reliable delivery of high speed (up to 1 gigabit per second) data to mobile or nomadic users throughout a cluttered urban environment. The wireless channel is a difficult channel over which to achieve high rate reliable communications. The wireless channel suffers many impairments such as small-scale multipath fading, shadowing, high path loss, co-channel interference, and Doppler shift due to mobility of the terminals and mobility in the propagation environment. Since radio spectrum is a scarce resource it is necessary to build cellular networks with high spectral efficiency. Two promising methods to solve this problem are multihop (MH) relaying and multiple-input multiple-output (MIMO) antenna techniques. The most difficult mobile users to serve reliably are those close to cell edges and those shadowed by large objects such as buildings. With MH relaying, a number of simple and inexpensive wireless relays are deployed throughout the cell to relay transmissions around obstacles and to reduce the path loss to distant mobile users. Also, MH relaying enables the deployment of small subcells throughout the cell, increasing the system's area averaged spectral efficiency. Various MIMO techniques can be used in scattering channels to increase capacity and reliability of data links in a wireless network. MH relaying and MIMO are key inclusions in emerging cellular standards such as IEEE 802.16 and LTE-Advanced, so it is necessary to study how these may be used jointly in a cellular environment. We look at various techniques available in MH relaying and MIMO, and assess the benefits and difficulties of these techniques when used in cellular systems. We put together a realistic cellular system model, with typical cellular topologies and well-accepted propagation models, and assess the performance of a multihop MIMO system. We find that there are tradeoffs in using these techniques jointly since they provide gains by somewhat conflicting methods. MH relaying lowers path loss and mitigates scattering in the channel, while MIMO benefits from significant scattering. As a result, it is necessary to understand how to design a MH-MIMO network carefully in order to maximize the net benefit.
Communications

Il fumo di tabacco è la principale causa di morte prevenibile nel mondo industrializzato. L’effetto farmacologico della nicotina gioca un ruolo fondamentale nella dipendenza da fumo di tabacco. La nicotina ha proprietà di rinforzo positivo e negativo, e induce condizionamento operante (comportamento motivato al consumo di nicotina) durante la fase di acquisizione della dipendenza. Vari studi pre-clinici e clinici hanno dimostrato l’importanza di alcuni fattori non farmacologici, come gli stimoli ambientali, nel mantenimento della dipendenza da nicotina e nell’induzione della ricaduta. Questi stimoli inizialmente neutri, ripetutamente associati alla nicotina (es. accendino) assumono un nuovo valore condizionato alla nicotina (CS) attraverso il condizionamento Pavloviano, ed acquisiscono la capacità di indurre “craving” (voglia di fumare) in assenza della droga. Considerata l’importanza delle associazioni CS-nicotina consolidate nella memoria del fumatore nel fenomeno della ricaduta, è stato proposto che trattamenti atti a distruggere le memorie formate ed associate alla nicotina potrebbero agire da pro-astinenti e anti-ricaduta nel trattamento della dipendenza da fumo di tabacco. Dopo una fase di apprendimento le memorie sono immagazzinate tramite un processo chiamato consolidamento. Il condizionamento operante (anche detto strumentale) e il condizionamento pavloviano portano alla formazione di diversi tipi di memorie associate alla droga e responsabili della ricaduta dopo lunga astinenza. Evidenze provenienti da studi sull’animale e sull’uomo dimostrano che le memorie, una volta richiamate/riattivate, tornano in uno stato labile durante il quale possono venire aggiornate e reimmagazzinate oppure distrutte. Il richiamo delle memorie dunque le destabilizza ed innesca un fenomeno detto di riconsolidamento necessario affinchè la memoria venga mantenuta. Vi è una certa evidenza che le memorie pavloviane possano subire riattivazione e riconsolidamento ed è stato proposto che trattamenti che intervengono per distruggere il riconsolidamento di queste memorie possono rendersi utili nella inibizione delle memorie associate alla paura e anche delle memorie associate alle droghe. La distruzione delle memorie associate alle droghe è stato proposto come potenziale trattamento per prevenire la ricaduta al consumo di droga indotta dai CS nei tossicodipendenti. Molti studi condotti nell’animale da laboratorio hanno dimostrato che il riconsolidamento delle memorie associate alle droghe può essere prevenuto attraverso la somministrazione di farmaci amnesici, che agiscono a specifici livelli molecolari (es., sistema adrenergico e glutamatergico), prima o dopo la loro riattivazione. Ad oggi non è ancora chiaro se tutte le memorie, o solo alcune, possono essere riattivate e riconsolidate. Ad esempio la possibilità che la memoria strumentale possa essere riattivata e riconsolidata o distrutta è ancora molto discussa ed esperimenti comportamentali volti a studiare la pura memoria strumentale potrebbero chiarire questa questione. Lo scopo di questo lavoro di tesi era di studiare se fosse possibile distruggere le memorie pavloviane e strumentali associate alla nicotina somministrando propranololo, antagonista del recettore β-adrenergico, o MK-801, antagonista del recettore N-methyl-d-aspartato (NMDARs). Inoltre abbiamo verificato la possibilità di utilizzare la tecnica dell’immunoistochimica per determinare il livello di espressione di Zif268, un marker molecolare specificamente coinvolto nel riconsolidamento della memoria, dopo la riattivazione di memorie pavloviane associate alla nicotina nel ratto. E’ stato utilizzato il modello di laboratorio della autosomministrazione di nicotina, basato sul paradigma del condizionamento operante e pavloviano, alla nicotina e ai CS associati alla nicotina. Abbiamo condotto due studi nei quali il trattamento farmacologico (propranololo o MK-801) veniva somministrato prima o dopo la riattivazione delle memorie pavloviane o strumentali associate alla nicotina. Abbiamo quindi testato l’effetto del trattamento farmacologico sulla ricaduta al comportamento di ricerca di nicotina. La riattivazione della memoria pavloviana è consistita nella presentazione dei CS in assenza della nicotina. La riattivazione della memoria strumentale è consistita nel permettere all’animale di premere la leva precedentemente associata all’infusione di nicotina, senza che la nicotina venisse somministrata. Abbiamo inoltre effettuato un esperimento di immunoistochimica su fettine di cervello di ratto per determinare l’espressione di Zif268 in amigdala basolaterale, regione maggiormente coinvolta nel risonsolidamento della memoria, dopo la presentazione dei CS associati alla nicotina. I risultati hanno dimostrato che il propranololo somministrato dopo la riattivazione della memoria pavloviana associata alla nicotina (30 CS) non è stato in grado di prevenire la ricaduta al comportamento di ricerca di nicotina. Un’ipotesi plausibile potrebbe essere che la memoria strumentale ancora presente non possa essere riattivata e riconsolidata, e quindi non possa essere nemmeno distrutta. Per verificare questa ipotesi abbiamo testato l’effetto dell’MK-801, dimostrato essere più efficace nella distruzione del riconsolidamento di diversi tipi di memoria, somministrato 30 minuti prima della riattivazione della memoria strumentale. I risultati hanno dimostrato che la somministrazione di MK-801 prima della riattivazione non previene la ricaduta al comportamento di ricerca di nicotina. Questi dati suggeriscono una prevenzione della destabilizzazione della memoria strumentale, che la blocca in una fase stabile, piuttosto che una distruzione del riconsolidamento. Un altro studio in cui MK-801 è stato somministrato dopo che la destabilizzazione della memoria strumentale ha avuto luogo (es., somministrato dopo la sessione di riattivazione) ha dimostrato che MK-801 è stato in grado di prevenire la ricaduta al comportamento di ricerca di nicotina. Infine l’immunoistochimica ha consentito di individuare un aumento del livello di espresione di Zif268 in amigdala basolaterale nei ratti sottoposti a riattivazione della memoria pavloviana associata alla nicotina. Questi dati confermano la validità della tecnica nella determinazione dell’espressione di markers molecolari specificamente coinvolti nel riconsolidamento della memoria. In conclusione, i nostri dati suggeriscono che: i) propranololo non distrugge il riconsolidamento della memoria pavloviana associata alla nicotina nelle nostre condizioni, ii) MK-801 somministrato prima della riattivazione potrebbe prevenire la destabilizzazione della memoria strumentale associata alla nicotina ma non ne distrugge il riconsolidatemnto nelle nostre condizioni, iii) MK-801 somministrato dopo la riattivazione della memoria strumentale associata alla nicotina ne distrugge il riconsolidamento nelle nostre condizioni, iiii) l’immunoistochimica è una tecnica adatta ad investigare l’espressione di markers molecolari specifiamente coinvolti nel riconsolidamento della memoria come Zif268, e potrà dunque essere utilizzata per dimostrare direttamente il riconsolidamento della memoria e suppoortare i risultati degli esperimenti comportamentali. Questi dati suggeriscono che la memoria strumentale potrebbe essere responsabile della mancanza di effetto di alcuni trattamenti farmacologici anti-ricaduta e che comunque questo tipo di memoria può essere distrutta se contrastata opportunamente. Lo sviluppo di nuovi farmaci capaci di agire a livello dei diversi meccanismi molecolari che sottostanno ai differenti tipi di memorie associate alle droghe potrebbere consentire una terapia coadiuvante alle attuali terapie anti-fumo e anti-ricaduta in grado di garantire una astinenza a lungo termine agli ex fumatori.
Tobacco use through cigarette smoking is the leading preventable cause of death in the developed world. The pharmacological effect of nicotine plays a crucial role in tobacco addiction. Nicotine is positively and negatively reinforcing and leads to the development of “operant conditioning” (motivated behaviour to nicotine consumption) in smokers during the acquisition phase of addiction. Several preclinical and clinical studies have also underlined the importance of non-pharmacological factors, such as environmental stimuli, in maintaining smoking behaviour and promoting relapse. Initially neutral stimuli that are repeatedly paired with a reinforcing drug (e.g. lighter) acquire a new conditioned value (conditioned stimuli, CS) through “Pavlovian conditioning” and become able to elicit craving even in the absence of the drug. Given the importance of the learned association between stimuli and nicotine in the phenomenon of relapse to nicotine-seeking behaviour, it has been proposed that treatment that disrupts the nicotine-associated memories could act as a pro-abstinent and anti-relapse therapy. After learning experience, memories are stored by a process called consolidation. Operant conditioning (also called instrumental learning) and Pavlovian conditioning lead to different drug-related memories formation (instrumental memories and Pavlovian memories) responsible for the relapse even after prolonged abstinence. However converging evidence from animal and human studies have revealed that memories may return to a vulnerable phase during which they can be updated, maintained and even disrupted. The retrieval of a memory indeed may destabilize the consolidated memory that requires a new process to be maintained. This hypothetical process is called reconsolidation. There is strong evidence that Pavlovian fear memories undergo reconsolidation and it was proposed that interventions to disrupt reconsolidation may help for specific and selective inhibition of fear related memories and, similarly, appetitive memories (i.e., for drug addiction). The disruption of drug-related memories reconsolidation has been proposed as a potential therapeutic target to prevent the CS-induced relapse in ex drug-addicts. Several animal studies have shown that the reconsolidation of some drug-related memories can be disrupted by the administration of an amnestic drug contingently upon retrieval of the memory acting at specific molecular levels (i.e. adrenergic and glutamatergic systems). However it is not known if all memories or only certain kind of memories could be retrieved and reconsolidated or disrupted. To date reconsolidation of instrumental memories is still under discussion and behavioural experiments targeting the pure instrumental memory reconsolidation disruption are needed to clarify this issue. The main objective of the present thesis was to study if it is possible to disrupt Pavlovian and instrumental nicotine-related memories reconsolidation by β-adrenergic receptor antagonist propranolol, or N-methyl-d-aspartate receptors (NMDARs) antagonist MK-801 respectively. We also verified the feasibility and reliability of Zif268 (a specific marker of memory reconsolidation) expression assessment by immunohistochemistry after retrieval of Palovian memory in rodents. The experimental approach used to address this issue was the laboratory model of nicotine self-administration in rats, based on the paradigms of operant and Pavlovian conditioning to nicotine and nicotine-associated cues. We performed two studies in which the pharmacological treatment (propranolol or MK-801) was associated to retrieval of Pavlovian or instrumental nicotine-related memories. We therefore assessed the effect of these pharmacological treatments on relapse to nicotine seeking behaviour. Retrieval of Pavlovian memories consists in presenting the CS in the absence of US. Retrieval of instrumental memories consists in allowing the animal to press the lever previously paired to nicotine reinforcement, without nicotine infusion. We also performed an immunohistochemistry assay in which the Zif268 level of expression was determined in basolateral amygdala (the most important region involved in memory reconsolidation) after nicotine CS presentation. Results showed that propranolol given after retrieval of Pavlovian memories (30 CS presentations) did not reduce the relapse to nicotine seeking behaviour compared to control groups that received vehicle injection in both retrieved or no-retrieved groups. It could be possible that instrumental memories, still present, do not undergo reconsolidation and could not be disrupted. To address this issue we tested the effect of MK-801, known to be more effective against instrumental memory than propranolol, given 30 minutes before the retrieval of instrumental memories. Results showed that pre-retrieval MK-801 injection did not prevent the relapse to nicotine-seeking behaviour when compared to control groups. This effect suggests a potential role of MK-801 in inhibition of the memory destabilization process instead of reconsolidation disruption. Further experiments in which MK-801 was given after memory destabilization was engaged (i.e. given after memory retrieval) showed that MK-801 prevented the relapse to nicotine-seeking behaviour. Finally immunohistochemistry showed an increased level of Zif268 expression in basolateral amygdala after retrieval of Pavlovian nicotine-related memories. These data confirm the validity and feasibility of immunohistochemistry to assess the expression of molecular markers correlating reconsolidation such as Zif268 after memory retrieval. In conclusion, our findings suggest that: i) propranolol did not disrupt Pavlovian memory reconsolidation in our conditions, ii) MK-801 given prior to retrieval session could prevent instrumental memory destabilization, but did not disrupt memory reconsolidation in our conditions, iii) MK-801 given after retrieval session disrupted memory reconsolidation in our conditions, iiii) immunohistochemistry is a feasible technique to investigate the expression of molecular markers correlating reconsolidation such as Zif268, thus it can be used to support our future behavioural studies. These data suggest that instrumental memory could be responsible for the lack of effect of some anti-relapse pharmacological treatments and that this kind of memory can be disrupted. New and specific pharmacological intervention, acting at specific molecular mechanisms that underlies reconsolidation of different kind of memories (i.e. Pavlovian but also instrumental memories), could be used as a potential co-adjuvant to current therapeutic interventions for smoking cessation and abstinence maintenance.

Pre-clinical studies of new molecules which can be developed as possible therapeutic effectors: in particular, the study of the protective effects of the hormone relaxin, as well as scavenger molecules of oxygen reactive species on cardiac hypoxia/reoxygenation.

abstract: Relapse after tumor dormancy is one of the leading causes of cancer recurrence that ultimately leads to patient mortality. Upon relapse, cancer manifests as metastases that are linked to almost 90% cancer related deaths. Capture of the dormant and relapsed tumor phenotypes in high-throughput will allow for rapid targeted drug discovery, development and validation. Ablation of dormant cancer will not only completely remove the cancer disease, but also will prevent any future recurrence. A novel hydrogel, Amikagel, was developed by crosslinking of aminoglycoside amikacin with a polyethylene glycol crosslinker. Aminoglycosides contain abundant amount of easily conjugable groups such as amino and hydroxyl moieties that were crosslinked to generate the hydrogel. Cancer cells formed 3D spheroidal structures that underwent near complete dormancy on Amikagel high-throughput drug discovery platform. Due to their dormant status, conventional anticancer drugs such as mitoxantrone and docetaxel that target the actively dividing tumor phenotype were found to be ineffective. Hypothesis driven rational drug discovery approaches were used to identify novel pathways that could sensitize dormant cancer cells to death. Strategies were used to further accelerate the dormant cancer cell death to save time required for the therapeutic outcome. Amikagel’s properties were chemo-mechanically tunable and directly impacted the outcome of tumor dormancy or relapse. Exposure of dormant spheroids to weakly stiff and adhesive formulation of Amikagel resulted in significant relapse, mimicking the response to changes in extracellular matrix around dormant tumors. Relapsed cells showed significant differences in their metastatic potential compared to the cells that remained dormant after the induction of relapse. Further, the dissertation discusses the use of Amikagels as novel pDNA binding resins in microbead and monolithic formats for potential use in chromatographic purifications. High abundance of amino groups allowed their utilization as novel anion-exchange pDNA binding resins. This dissertation discusses Amikagel formulations for pDNA binding, metastatic cancer cell separation and novel drug discovery against tumor dormancy and relapse.
Dissertation/Thesis
Doctoral Dissertation Bioengineering 2016

Chez diverses espèces animales, les informations sensorielles peuvent déclencher la locomotion. Ceci nécessite l’intégration des informations sensorielles par le système nerveux central. Chez la lamproie, les réseaux locomoteurs spinaux sont activés et contrôlés par les cellules réticulospinales (RS), système descendant le plus important. Ces cellules reçoivent des informations variées provenant notamment de la périphérie. Une fois activées par une brève stimulation cutanée d’intensité suffisante, les cellules RS produisent des dépolarisations soutenues de durées variées impliquant des propriétés intrinsèques calcium-dépendantes et associées à l’induction de la nage de fuite. Au cours de ce doctorat, nous avons voulu savoir si les afférences synaptiques ont une influence sur la durée des dépolarisations soutenues et si l’ensemble des cellules RS partagent des propriétés d’intégration similaires, impliquant possiblement les réserves de calcium internes. Dans un premier temps, nous montrons pour la première fois qu’en plus de dépendre des propriétés intrinsèques des cellules réticulospinales, les dépolarisations soutenues dépendent des afférences excitatrices glutamatergiques, incluant les afférences spinales, pour perdurer pendant de longues périodes de temps. Les afférences cutanées ne participent pas au maintien des dépolarisations soutenues et les afférences inhibitrices glycinergique et GABAergiques ne sont pas suffisantes pour les arrêter. Dans un deuxième temps, nous montrons que suite à une stimulation cutanée, l’ensemble des cellules RS localisées dans les quatre noyaux réticulés possèdent un patron d’activation similaire et elles peuvent toutes produire des dépolarisations soutenues dont le maintien ne dépend pas des réserves de calcium internes. Enfin, les résultats obtenus durant ce doctorat ont permis de mieux comprendre les mécanismes cellulaires par lesquels l’ensemble des cellules RS intègrent une brève information sensorielle et la transforment en une réponse soutenue associée à une commande motrice.
In various animal species, sensory information can initiate locomotion. This relies on the integration of sensory inputs by the central nervous system. In lampreys, the spinal locomotor networks are activated and controlled by the reticulospinal cells (RS) which constitute the main descending system. In turn, RS cells receive information coming from various synaptic inputs such as the sensory afferents. Once activated by a brief cutaneous stimulation of sufficient strength, RS cells display sustained depolarizations of various durations that rely on calcium-dependant intrinsic properties and lead to the onset of escape swimming. During the course of this Ph.D, we aimed at determining whether synaptic inputs can modulate the duration of the sustained depolarizations and if the different populations of RS cells share the same integrative properties, possibly involving the internal calcium stores. First, our results show for the first time that excitatory glutamatergic inputs, including ascending spinal feedback, contribute to prolong the sustained depolarizations for long periods of time. Cutaneous inputs do not contribute to maintain the sustained depolarizations and inhibitory glycinergic and GABAergic inputs are not sufficient to stop them. Second, we show that in response to cutaneous stimulation, the RS located in the four reticular nuclei display a similar activation pattern and can all produce sustained depolarizations which do not depend on internal calcium release to be maintained. Finally, the results obtained during this Ph.D allowed us to better understand the cellular mechanisms by which the RS cells integrate and transform a brief sensory information into a sustained response associated with a motor command.