Tesis sobre el tema "Nuclear disruption"

The sudden loss of the plasma magnetic confinement, known as disruption, is one of the major issue in a nuclear fusion machine as JET (Joint European Torus), Disruptions pose very serious problems to the safety of the machine. The energy stored in the plasma is released to the machine structure in few milliseconds resulting in forces that at JET reach several Mega Newtons. The problem is even more severe in the nuclear fusion power station where the forces are in the order of one hundred Mega Newtons. The events that occur during a disruption are still not well understood even if some mechanisms that can lead to a disruption have been identified and can be used to predict them. Unfortunately it is always a combination of these events that generates a disruption and therefore it is not possible to use simple algorithms to predict it. This thesis analyses the possibility of using neural network algorithms to predict plasma disruptions in real time. This involves the determination of plasma parameters every few milliseconds. A plasma boundary reconstruction algorithm, XLOC, has been developed in collaboration with Dr. D. Ollrien and Dr. J. Ellis capable of determining the plasma wall/distance every 2 milliseconds. The XLOC output has been used to develop a multilayer perceptron network to determine plasma parameters as ?i and q? with which a machine operational space has been experimentally defined. If the limits of this operational space are breached the disruption probability increases considerably. Another approach for prediction disruptions is to use neural network classification methods to define the JET operational space. Two methods have been studied. The first method uses a multilayer perceptron network with softmax activation function for the output layer. This method can be used for classifying the input patterns in various classes. In this case the plasma input patterns have been divided between disrupting and safe patterns, giving the possibility of assigning a disruption probability to every plasma input pattern. The second method determines the novelty of an input pattern by calculating the probability density distribution of successful plasma patterns that have been run at JET. The density distribution is represented as a mixture distribution, and its parameters arc determined using the Expectation-Maximisation method. If the dataset, used to determine the distribution parameters, covers sufficiently well the machine operational space. Then, the patterns flagged as novel can be regarded as patterns belonging to a disrupting plasma. Together with these methods, a network has been designed to predict the vertical forces, that a disruption can cause, in order to avoid that too dangerous plasma configurations are run. This network can be run before the pulse using the pre-programmed plasma configuration or on line becoming a tool that allows to stop dangerous plasma configuration. All these methods have been implemented in real time on a dual Pentium Pro based machine. The Disruption Prediction and Prevention System has shown that internal plasma parameters can be determined on-line with a good accuracy. Also the disruption detection algorithms showed promising results considering the fact that JET is an experimental machine where always new plasma configurations are tested trying to improve its performances.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Nuclear Science and Engineering, 2008.
Includes bibliographical references (p. 41).
Disruptions are one of the largest problems facing tokamaks. In a large-scale experiment such as ITER, disruptions may cause crippling damage and severe setbacks in experimentation. One method for disruption mitigation involves the use of a gas jet which has been tested on both stable plasmas and vertical displacement events (VDEs) on Alcator C-Mod. In both cases, the jet was successful in mitigating disruption effects. The gas jet has not yet been tested on other types of disruptions. Locked-mode disruptions are easily created in C-Mod and could be used to test the effectiveness of the gas jet as a mitigation method if the jet could be fired early enough. It has been empirically observed that the electron cyclotron emissions (ECE) signal displays a flattening of the normally-present sawteeth before the current quench occurs in certain locked-mode disruptions. A procedure has been written which detects the ECE sawtooth suppression by calculating changes in the standard deviation of the signal over a moving time-window. This procedure has been programmed into the digital plasma control system (DPCS) for real-time testing. The procedure successfully located the locked modes present during a run.
by Sarah Martha Angelini.
S.M.

During herpes simplex virus 1 (HSV-1) replication, newly constructed capsids escape the nucleus to undergo maturation in the cytoplasm via a process termed nuclear egress. Capsids perform nuclear egress through localized disruption of the nuclear lamina, envelopment of the inner nuclear membrane to create a perinuclear enveloped virion, and de-envelopment of the outer nuclear membrane for capsid release into the cytoplasm. Critical virial factors for this process are viral proteins pUL31 and pUL34 that interact to form heterodimers. These heterodimers form larger hexameric arrays to drive membrane budding. Through the characterization of phenotypes of UL34 point mutants, we are able to further study the underlying mechanisms of nuclear lamina disruption and nuclear budding. One such mutant, UL34(Q163A), results in impaired virus production, cell-cell spread, and an inability to disrupt lamin A/C networks. Selection for extragenic suppression of UL34(Q163A) yielded the UL31(R229L) mutation, that partially rescued the growth and spread defects of UL34(Q163A), but was unable to regain the ability to disrupt lamin A/C networks. Through this study we concluded that disruption of lamin A/C networks was not required for efficient HSV-1 replication. In order to understand the underlying mechanisms of membrane budding, the previously characterized UL34(CL13) double mutant, which results in a 100-fold reduction in virus production, a severe impairment in cell-cell spread, and an accumulation of capsid-less perinuclear vesicles was further studied. Characterization of the single mutations of UL34(CL13), UL34(R158A) and UL34(R161A) revealed that neither single mutation was responsible for spread or growth defect, but that either single mutation resulted in a promiscuous budding phenotype. Through this study, we concluded that although individual steps of the nuclear egress pathway are tightly regulated, alteration of the regulation at a single step does not grossly impact HSV-1 replication.

Successful implementation of advanced electrical power generation technology into the global marketplace requires at least two fundamental ideals: cost effectiveness and the guarantee of public safety. These requirements can be met by thorough design and development of technologies in which safety is emphasized and demonstrated. A detailed understanding of the many physical processes and their synergistic effects in a complicated fusion energy system is necessary for a defensible safety analysis. One general area of concern for fusion devices is the production of particulate, often referred to as dust or aerosol, from material exposed to high energy density fusion plasma. This dust may be radiologically activated and/or chemically toxic, and, if released to the environment, could become a hazard to the public. The goal of this investigation was to provide insight into the production and transport of particulate generated during the event of extreme heat loads to surfaces directly exposed to high energy density plasma. A step towards achieving this goal was an experiment campaign carried out with the Surface InteRaction Experiment at North Carolina State (SIRENS), a facility used for high heat flux experiments. These experiments involved exposing various materials, including copper, stainless steel 316, tungsten, aluminum, graphite (carbon), and mixtures of carbon and metals, to the high energy density plasma of the SIRENS source section. Material mobilized as a result of this exposure was collected from a controlled expansion chamber and analyzed to determine physical characteristics important to safety analyses (e.g., particulate shape, size, chemical composition, and total mobilized mass). Key results from metal-only experiments were: the particles were generally spherical and solid with some agglomeration, greater numbers of particles were collected at increasing distances from the source section, and the count median diameter of the measured particle size distributions were of similar value at different positions in the expansion chamber, although the standard deviation was found to increase with increasing distances from the source section, and the average count median diameters were 0.75 micron for different metals. Important results from the carbon and carbon/metals tests were: particle size distributions for graphite tests were bi-modal (i.e. two distributions were present in the particle population), particles were generally smaller than those from metals-only tests (average of 0.3 micron), and the individual particles were found to contain both carbon and metal material. An associated step towards the goal involved development of an integrated mechanistic model to understand the role of different particulate phenomena in the overall behavior observed in the experiment. This required a detailed examination of plasma/fluid behavior in the plasma source section, fluid behavior in the expansion chamber, and mechanisms responsible for particulate generation and growth. The model developed in this work represents the first time integration of these phenomena and was used to simulate mobilization experiments in SIRENS. Comparison of simulation results with experiment observations provides an understanding of the physical mechanisms forming the particulate and indicates if mechanisms other than those in the model were present in the experiment. Key results from this comparison were: the predicted amount of mass mobilized from the source section was generally much lower than that measured, the calculated and measured particle count median diameters were similar at various locations in the expansion chamber, and the measured standard deviations were larger than those predicted by the model. These results implicate that other mechanisms (e.g., mobilization of melted material) in addition to ablation were responsible for mass removal in the source section, a large number of the measured particles were formed by modeled mechanisms of nucleation and growth, and, as indicated by the large measured standard deviations, the larger particles found in the measurement were from an aerosol source not included in the model. From this model, a detailed understanding of the production of primary particles from the interaction of a high energy density plasma and a solid material surface has been achieved. Enhancements to the existing model and improved/extended experimental tests will yield a more sophisticated mechanistic model for particulate production in a fusion reactor.

Les systèmes du cycle du combustible nucléaire, composés de réacteurs, de divers combustibles et de différentes installations de cycle, sont complexes et en constante évolution. Grâce à leur capacité à faire des projections de stratégies industrielles et à évaluer les impacts associés sur le système du cycle du combustible nucléaire, les scénarios électronucléaires sont considérés comme un outil puissant d'aide à la décision. Les études de scénarios aident les décideurs à identifier les forces et les faiblesses des différentes stratégies d'évolution d’un parc nucléaire et puis à proposer des trajectoires d'évolution possibles pour l'industrie nucléaire en fonction des contraintes de la physique, de l'économie, de l'industrie, etc.Cependant, les études de scénarios sont généralement soumises à différents types d'incertitudes, en particulier la soi-disant « incertitude profonde » (« deep uncertainty » en anglais). Ce concept fait référence à des « inconnues inconnues », auxquelles les résultats de l'étude de scénarios ne conviennent pas. En effet, sous l'impact des incertitudes profondes, c'est-à-dire les disruptions, les trajectoires proposées par les études de scénarios peuvent devenir invalides : elles ne satisfont plus aux contraintes du scénario.Afin de rendre les trajectoires valides à nouveau après une disruption due à l'incertitude, la première possibilité est d'étudier la stratégie de résistance. La stratégie de résistance consiste à trouver des trajectoires qui restent valides sous l'impact de l'incertitude sans réajustements exogènes des trajectoires. Cependant, les capacités de résistance des scénarios sont limitées : la résistance n'est adaptée qu'aux incertitudes à faible impact, alors que l'impact d'une incertitude profonde est généralement fort.Comme solution complémentaire à la stratégie de résistance, nous proposons d'utiliser les stratégies de résilience. Les stratégies de résilience consistent à utiliser des mesures préconçues, appelées « leviers », pour réajuster la trajectoire lorsque la stratégie de résistance est insuffisante. Nous cherchons à utiliser l'effet des réajustements exogènes des trajectoires, qui sont introduits à travers les leviers, pour contrebalancer l'impact de la disruption et garder la trajectoire valide. Pour évaluer la résilience des scénarios, nous avons développé un cadre d'analyse de résilience, basé sur l'algorithme SUR (Stepwise Uncertainty Reduction).Nous avons appliqué la stratégie de résilience développée à deux problèmes de scénario dans lesquels un parc nucléaire français simplifié avec une réduction de puissance incertaine est considéré. Pour définir la validité des trajectoires, nous avons imposé cinq contraintes sur le taux d'utilisation des usines de retraitement, la séparation du plutonium, la teneur de plutonium dans le combustible MOX et le stockage du combustible usé. Dans chaque problème, nous avons donné une trajectoire préalable supposée à la suite d'une étude de scénarios avec une hypothèse pour maintenir la puissance installée constante à l’avenir. Nous avons supposé qu'à la suite de la disruption du contexte de l'étude, la puissance électrique totale est disruptée et réduite à l’avenir. Les résultats ont montré que les trajectoires préalables dans les deux problèmes sont résilientes vis-à-vis des disruptions supposées : il est possible de maintenir les trajectoires préalables valides en réajustant le retraitement et les charges de combustible MOX dans les réacteurs. Ces résultats démontrent que les évolutions du parc nucléaire dans les trajectoires préalables sont flexibles face à la disruption de la puissance électrique totale
Nuclear fuel cycle systems, composed of reactors, various fuels, and different cycle facilities, are complex and in constant evolution. Thanks to their abilities to make projections of industrial strategies and to assess the associated impacts on nuclear fuel cycle systems, nuclear fuel cycle scenarios are considered as a powerful tool for decision-making analyses. Scenario studies assist decision-makers in identifying the strengths and weaknesses of different strategies for a nuclear fleet evolution and then proposing possible evolution trajectories for the nuclear industry according to constraints from physics, economics, industry, etc.However, scenario studies are usually subject to different kinds of uncertainties, especially the so-called “deep uncertainty.” This concept refers to “unknown unknowns,” which scenario study results are unsuited to address. Indeed, under the impact of deep uncertainty, i.e., disruptions, the trajectories proposed by the scenario studies can become invalid: they do not satisfy the scenario constraints anymore.In order to make the trajectories valid again after disruption due to uncertainty, the first possibility is to study the resistance strategy. The resistance strategy consists of finding scenario trajectories that remain valid under the impact of uncertainty without exogenous readjustments of trajectories. However, the resistance capabilities of scenarios are limited: resistance is only adapted to uncertainties with small impact, while the impact of deep uncertainty is usually strong.As a complementary solution to the resistance strategy, we propose using resilience strategies. The resilience strategies consist of using predesigned measures, called “levers,” to readjust the scenario trajectory when the resistance strategy is insufficient. We aim to use the effect of the exogenous readjustments of trajectories, which are introduced through the levers, to counterbalance the impact of disruption and remain the trajectory valid. To evaluate the resilience of scenarios, we developed a resilience analysis framework, based on the start-of-the-art SUR (Stepwise Uncertainty Reduction) algorithm.We applied the developed resilience strategy to two scenario problems in which a simplified French nuclear fleet with uncertain power reduction is considered. To define the validity of trajectory, we imposed five constraints about the reprocessing plant utilization ratio, plutonium separation, plutonium content in MOX fuel, and spent fuel storage. In each problem, we gave a prior trajectory supposed as a result of a scenario study with a hypothesis to keep the installed power constant in the future. We assumed that following the disruption of the study context, the total electricity power is disrupted and reduced in the future. The results showed that the prior trajectories in both problems are resilient for the assumed disruptions: it is possible to keep the prior trajectories valid by readjusting the reprocessing and the MOX fuel loadings in reactors. Such results demonstrate the evolutions of the nuclear fleet in the prior trajectories are flexible in front of the disruption of total electricity power

Mutations in the LMNA gene, which encodes the nuclear lamin A/C protein, are implicated in the pathogenesis of over ten phenotypically diverse diseases, including dilated cardiomyopathy (DCM). Two hypotheses have been proposed to explain the tissue specificity of LMNA mutations: 1) Nuclear fragility which may affect to a greater extent mechanically stressed tissues such as cardiac muscle (structural hypothesis); and 2) dysregulation of gene transcription. The objective of this study was to determine which hypothesis explains how LMNA mutations cause DCM. In our cohort of 25 DCM patients, 34% displayed cardiomyocytes with nuclear abnormalities, however, point mutations or large deletions of the lamin A/C gene were found in both patients with or without nuclear envelope damage. Moreover, some DCM patients with nuclear damage were free of lamin A/C gene somatic mutation. These results provide support for the structural hypothesis, but do not exclude the hypothesis of dysregulation of gene transcription. To test the latter hypothesis, we used cellular models expressing lamin A/C mutant proteins. We showed that, although the distributions of some proteins known to be involved in transcription regulation, such as SUMO1 or Ubc9 are disturbed, lamin A/C mutants have no significant effect on transcriptional activity of GATA-4 and MEF2C, two sumoylated transcription factors involved in cardiac gene transcription. In conclusion, we confirmed that structural hypothesis plays a role in DCM pathogenesis, however at this point, the gene-regulation hypothesis can not be excluded. We showed for the first time that a large deletion encompassing several exons of the LMNA gene and leading to a marked reduction of lamin A/C proteins in cardiac tissue, and thus to haploinsufficiency, is a new molecular mechanism associated with dilated cardiomyopathy.

Micrometeoroids and orbital debris (MMOD) is a growing issue with international importance. Micrometeoroids are naturally occurring fragments of rock and dusk that exist throughout the solar system. Orbital debris is human made material like rocket bodies, paint flakes, and the effluent of spacecraft collisions. Even small MMOD particles on the order of 1 cm in diameter have the potential to destroy critical spacecraft systems. Because of this, MMOD is a threat to all spacecraft in orbit. Even governments that most sternly oppose US international policy have a stake when it comes to minimizing MMOD flux. Space-based assets are essential to support the growing demand for high-capacity communications networks around the world. These networks support services that civilian and military users have grown accustomed to using on a daily basis: Global Positioning System (GPS), Satellite Radio, Internet Backhaul, Unmanned Areal Vehicles (UAVs), and Reconnaissance Satellites [Figure \ref{figure:skynet}]. A sudden loss of these services could degrade the warfighter's capabilities and cripple commercial enterprises that rely on these technologies. Manned space efforts like the International Space Station (ISS) could also suffer as a result of increased MMOD flux.

Historically, “offender”-focused discourses have dominated the field of criminology while overlooking those family members who become subject to justice intervention by virtue of their familial bonds. In this qualitative study, unstructured interviews were conducted with eight nuclear family members of criminalized persons in Ontario and Quebec. Participant accounts reveal that the interviewed family members self-imposed significant moral and legal responsibilities for their relatives following criminal justice intervention and simultaneously experienced negative role re-evaluation driven by feelings of guilt, failure, and self-blame. Participants’ inherent lack of control over their criminalized relatives’ behaviours and the criminal justice system’s decisions exacerbate negative impacts of criminalization on non-criminalized relatives’ self-concepts. This lack of control increases the stress of criminal justice intervention while straining family resources. When relatives are justice-involved for prolonged period, the family becomes stuck in a constant state of stress and uncertainty, which may have lasting consequences on the family if left unmanaged. Criminal justice intervention as a disruptive event then reconfigures the family in ways that often leave lasting impacts on nuclear family relationships. This thesis engages with Boss’ (1999, 2006) theory of Ambiguous Loss to analyze participants’ experiences and demonstrate the consequences of criminalization on various nuclear family members in a Canadian context. To mitigate certain limitations of Boss’ (1999, 2006) theory, criminal justice intervention is first defined as a disruptive event that transforms family members’ known realities into threatening and uncertain environments. This thesis then explores the stress and strain that justice intervention places upon the family and applies the theory of Ambiguous Loss to understand criminalization as a source of ambiguous loss. Further, this thesis expands the scope of Boss’ (1999) theory beyond the experiences of certain populations (i.e. children of incarcerated parents) and discovers the limitations of this theory in the context of criminological research. It also opens the door for future research to apply this theory to criminalized populations.

Endocrine Disruptors (EDCs) are a class of anthropogenic environmental contaminants that affect the endocrine systems of living organisms. The lack of knowledge of endocrine systems prevents the understanding of the effect of PEs in invertebrates. Accordingly, this thesis aims to address the issue of endocrine disruption in marine invertebrates by exploiting the larval development of the Mediterranean mussel Mytilus galloprovincialis. By applying the logic of the Adverse Outcome Pathway-AOP, the work in this thesis attempted to characterize the mechanism of action of the model EDCs BPA, TBBPA and TBT in mussel larvae and to identify plausible neuroendocrine pathways by which they induce their deleterious biological effect. The neuroendocrine elements regulating the biogenesis of the larval shell have been characterized and the morphogenetic process established as sensitive to neuroendocrine disruption. Plausible neuroendocrine AOPs that could be linked to the biological effects of the model EDCs have been identified. In addition, the possible AOP of TBT has established a plausible relationship between nuclear receptor signaling and the neuroendocrine system of mussel larvae. The results of this thesis represent essential evidence that will help and advance the current understanding of endocrine disruption mechanisms in invertebrate organisms.

Der Ursprung ultra-hochenergetischer kosmischer Strahlung (UHECRs) ist eine der wichtigsten offenen Fragen der Astrophysik. Gammastrahlenblitze (GRBs) galten als potentielle Quellen, da sie zu den energetischsten Ereignissen im Universum zählen. Konventionelle Szenarien sind jedoch durch Neutrinodaten stark eingeschränkt. Außerdem weisen Messungen der chemischen Zusammensetzung kosmischer Strahlen auf schwere Kerne hin, welche in zu dichten Strahlungsfeldern disintegrieren würden. Um dieses Dilemma zu umgehen deuten neue Studien auf versteckte Beschleuniger hin, welche schwer zu detektieren sind. In dieser Dissertation präsentieren wir neue Ansätze um nukleare Prozesse in astrophysikalischen Quellen effizient und selbstkonsistent zu berechnen. Wir quantifizieren diese Wechselwirkungen anhand der nuklearen Kaskade, welche die Disintegration schwerer Kerne in leichtere Fragmente beschreibt. Auch in umfassenden Modellen, wie sie in dieser Arbeit entwickelt werden, sind GRBs durch Neutrinodaten unter Druck. Dennoch zeigen wir, dass eine Population von GRBs niedriger Luminosität konsistent mit derzeitigen Messungen ist und zugleich auch das Spektrum und die Zusammensetzung kosmischer Strahlung über den Knöchel hinweg sowie Neutrinodaten beschreiben kann. Aus unserer Prozedur können wir zusätzlich weitere Quelleneigenschaften wie die baryonische Ladung oder die Ereignisrate bestimmen. Wir zeigen weiter, dass auch von schwarzen Löchern zerrissene Sterne mögliche Kandidaten eines gemeinsamen Ursprungs der gemessenen kosmischen Strahlung und PeV-Neutrinos sind. Sie können jedoch durch kosmogenische Neutrinos von LLGRBs abgegrenzt werden. Schließlich wenden wir unser Modell auf das Gravitationswellenereignis GW170817 an. Wir zeigen für verschiedene Jet-Szenarien, dass der erwartete Neutrinofluss weit unter der Sensitivität derzeitiger Instrumente liegt. Dennoch könnten verschmelzende Neutronensterne die kosmische Strahlung unterhalb des Knöchels erklären.
The origin of Ultra-High Energy Cosmic Rays (UHECRs) is still one of the most important open questions in astrophysics. Gamma-Ray Bursts (GRBs) were considered as potential sources as they are among the most energetic events known in the Universe. However, conventional GRB scenarios are strongly constrained by astrophysical neutrino data. In addition, cosmic ray composition measurements indicate the presence of heavy nuclei, which would disintegrate if the radiation fields in the source were too dense. In order to circumvent this dilemma, recent studies point towards hidden accelerators, which are intrinsically hard to detect. In this dissertation, we present novel approaches to efficiently and self-consistently calculate the nuclear processes in astrophysical sources. We quantify these interactions by means of the nuclear cascade, which describes the subsequent disintegration of heavy nuclei into lighter fragments. Even in sophisticated source-propagation models, as the ones developed in this thesis, conventional GRBs are in tension with neutrino data. However, we demonstrate that a population of low-luminosity GRBs is not only consistent with current constraints, but can even describe the UHECR spectrum and composition across the ankle as well as neutrino data simultaneously. From our fitting procedure we can further constrain certain source properties, such as the baryonic loading and the event rate. Furthermore, we show that stars disrupted by black holes are viable candidates for a simultaneous description of cosmic ray and PeV neutrino data too. However, they can be discriminated from LLGRBs by cosmogenic neutrinos. Finally, we apply our model to GW170817. We show for different jet scenarios that the expected neutrino flux is orders of magnitude below the sensitivity of current instruments. Nevertheless, binary neutron star mergers could in principle support cosmic rays below the ankle.

Fish are particularly vulnerable to the exposure of anthropogenic pollutants, with a vast array of endocrine disrupting chemicals (EDCs) introduced into the aquatic environment via sewage discharge, waste disposal and land runoff. Brominated flame retardants (BFRs) are halogenated flame retardants that are used to effectively inhibit the flammability of various materials including plastic products, electrical appliances, construction materials and textiles. BFRs are ubiquitous environmental contaminants and are known to disrupt thyroid hormone (TH) homeostasis in several vertebrate species, including fish. Given the vital role of THs in a wide range of developmental processes and physiological functions, assessing and identifying thyroid disrupting chemicals is crucial for safe guarding the long-term health of humans and wildlife. In fish the molecular mechanisms underlying TH disruption by BFRs and the effects on TH-sensitive tissues during early life stages remains unclear. This has been limited by the lack of fundamental knowledge on the TH system of fish and the difficulties associated with examining transcriptional changes in discrete embryonic-larval tissues. Here I have established the expression profiles of a suite of genes in the hypothalamic-pituitary-thyroid (HPT) axis of zebrafish (Danio rerio) during embryonic-larval stages and their regulation by the biologically active TH (3, 5, 3′- tri-iodothyronine; T3). Using molecular tools (whole mount is situ hybridisation and RT-PCR), I demonstrate that a number of genes display spatial and temporal expression profiles during embryo/larval development, and their regulation by T3 was tissue- and developmental stage-specific. I subsequently demonstrated that TBBPA and BDE-47, two important BFR compounds, disrupted TH homeostasis at multiple levels of the HPT axis of zebrafish embryo-larvae after short sub-acute exposures. These compounds altered the expression of genes associated with TH conjugation and clearance, thyroid follicle development and TH transport. In addition, we suggest that TH target genes in the brain, liver, pronephric ducts and craniofacial tissues of zebrafish embryo-larvae may be particularly vulnerable to TBBPA and BDE-47 exposure. It has been proposed that environmental pollutants can disrupt TH signalling in wildlife by disrupting the activity of thyroid receptors (TRs), ligand-binding transcription factors, which mediate the genomic actions of THs. The ability of BFRs to disrupt fish TRs has not yet been examined. Here I developed an in vitro reporter gene transcriptional assay for zebrafish thyroid hormone receptors (zfTRα and zfTRβ) in human embryonic kidney cells and investigated their interactions with several BFR compounds. The assays were optimised and validated using the natural TR agonist T3 in cells transiently transfected with two reporter vector constructs, pGL4.24-PAL and pGL4.24-DR4. None of the six brominated flame retardants tested, namely, tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD), 2,2′,4,4′-tetra-bromodiphenyl ether (BDE-47), 2,2′,4,4′,6-penta-bromodiphenyl ether (BDE-100), 2,2′,3,4,4′,5′,6-hepta-bromodiphenyl ether (BDE-183) and deca-bromodiphenyl ether (BDE-209) had an agonistic effect on zfTRα and zfTRβ activity. These results are consistent with our previous finding which suggests that altered TH homeostasis may be a result of increased metabolism and excretion of THs and/or changes in the production of TH by the thyroid follicles. In conclusion, this investigative work aids the understanding of fundamental TH processes in fish, such as gene expression and regulation, and increases our understanding of the mechanisms and potential targets of BFRs in fish.

Nuclear hormone receptors (NHRs) are ligand-activated transcriptions factors which transduce the effects of various hormones as well as nutritional and other environmental signals. They thus function to maintain physiological homeostasis by integrating the tissue expression of specific target genes to regulate a wealth of biological processes including reproduction, development, metabolism and environmental adaptation. Mounting evidence indicates NHRs are the target of endocrine disrupting compounds (EDCs), exogenous chemicals, often of anthropogenic origin, which disrupt NHRs and thus the processes under their control. EDCs can interfere with NHR signalling by activating receptors (agonists), by inhibiting the actions of the receptor (antagonists), or by disrupting endogenous hormone synthesis, secretion, transport or metabolism. Much of the focus to date has been on the risk of EDCs to reproductive functions, via estrogen and androgen NHRs in humans, and also in aquatic organisms. However environmental pollutants also have the potential to interact with other NHRs, particularly in aquatic environments, and cause dysregulation of other critical physiological processes, including energy homeostasis, immune functions and the stress response. To address this possibility a reporter gene assay was developed, allowing the high-throughput screening of pollutants for their interactions with piscine NHRs with critical roles in energy homeostasis, stress reponse and immune functions, namely the peroxisome proliferator-activated receptors (PPARs) and corticosteroid receptors (CRs) from European plaice (Pleuronectes platessa) and European flounder (Platichthys flesus), respectively. Complementary DNA (cDNA) sequences encoding the ligand-binding domains of PPARs and CRs, critical for receptor-ligand interactions and receptor activation, were ligated to the DNA-binding domain (DBD) of the yeast Gal4 transcription activator protein to create experimental expression plasmid constructs. Co-transfection of these expression plasmids into the fathead minnow (FHM) cell line with an upstream-activating sequence (UAS)-firefly luciferase reporter gene plasmid increased luciferase expression in the presence of known PPAR and CR ligands. Several aquatic pollutants including pharmaceuticals, industrial by-products and biocides were tested for their potential to disrupt PPAR and CR functions by interacting with these receptors in an agonistic or antagonistic manner. Several fibrates, a group of pharmaceutical compounds used to treat dyslipidemia in humans by targeting the PPARs, were able to activate plaice Gal4-PPARα and Gal4-PPARβ in the reporter gene assay, indicative of an interaction with PPAR receptors in non-target species. Fibrates which did not activate Gal4-PPARα were able to inhibit the activation of Gal4-PPARα by the PPARα-specific agonist, Wy14643, suggesting differential effects of fibrates on human and flounder PPARs. In addition some metabolites of widespread phthalate ester pollutants were also agonists of the Gal4-PPARα and Gal4-PPARβ constructs. The Gal4-PPARγ construct was unresponsive to almost all the compounds tested, including the mammalian PPARγ agonist, rosiglitazone. The exception to this was the phthalate metabolite monobenzylphthalate, which induced a small increase in firefly luciferase in Gal4-PPARγ transfected cells. All of the above effects required concentrations of at least 10 µM, which are unlikely to be encountered in the aquatic environment. In contrast bis(tributyltin) oxide (TBTO), a notorious environmental pollutant, inhibited Gal4-PPARα and Gal4-CR constructs at concentrations as low as 1 nM and 100 nM, respectively. These concentrations are lower than those reported in aquatic environments, or in fish tissues, making TBTO a candidate endocrine disruptor in fish by inhibiting PPARα and CR signalling. A European flounder cDNA microarray was used to investigate the trasnscriptional responses of flounder hepatocytes to TBTO (10 nM) exposure. Exposure to TBTO and Wy14643, both alone and in combination, indicated a TBTO-driven downregulation of several potential PPARα-target genes with functions in the immune system, the proteasome, and lipid metabolism, although, based on mammalian comparisons, some potential PPARα-target genes were also upregulated, indicating differences in mammalian and fish PPAR-target genes or reflecting the complexity of organisms at a higher organisational level than cell-based assay systems. However, the microarray-based approach was useful in formulating further hypotheses about the effects of TBTO on PPARα signalling. Overall, these results indicate that exogenous chemicals entering the aquatic environment can interfere with NHRs with functions in energy homeostasis, immune functions and stress, in non-target organisms. The cell-based reporter gene assay is a useful tool for identifying potential endocrine disruptors which target PPARs and CRs and would be a useful method in a first tier testing approach, limiting the use of live animal models and enabling investigation into specific receptors which are targets of endocrine disrupting compounds. Although more work is required to confirm the physiological consequences of TBTO inhibition of PPARα, the results presented here indicate that organisms inhabiting TBTO-polluted environments may experience suppression of the immune system, an increase in non-functional or misfolded proteins through suppression of genes involved in the ubiquitin/proteasome system and a disruption in lipid homeostasis.

Les Perturbateurs Endocriniens (PE) sont une classe de contaminants environnementaux d'origine anthropique qui affectent les organismes en perturbant leur système endocrinien. Notamment, le manque de compréhension détaillée des systèmes endocriniens et leur voie de signalisation empêche la compréhension de l’effet de PEs chez les invertébrés. Conformément, cette thèse vise à aborder la question de la perturbation endocrinienne chez les invertébrés marins en exploitant le développement larvaire de la moule méditerranéenne Mytilus galloprovincialis. En appliquant la logique de l’Adverse Outcome Pathway-AOP, le travail de cette thèse vise à caractériser le mécanisme d'action de PEs modèles BPA, TBBPA et TBT chez les larves de moule et à identifier de plausibles voies neuro-endocriniennes par lesquelles ils induisent leur effet biologique délétère. Les éléments neuroendocriniens régulant la biogenèse de la coquille larvaire ont été caractérisés et le processus morphogénétique établi comme sensible à la perturbation neuroendocrinienne. Les plausibles AOPs neuro-endocriniens qui pourraient être liées aux effets biologiques de PEs modèles ont été identifiées. En plus, l'AOP possible du TBT a établi une relation plausible entre la signalisation des Récepteurs Nucléaires et le système neuroendocrinien des larves de moule. Les résultats de cette thèse représentent des preuves essentielles qui aideront et feront avancer la compréhension actuelle des mécanismes de perturbation endocrinienne chez les organismes invertébrés
Endocrine Disruptors (EDCs) are a class of anthropogenic environmental contaminants that affect organisms by disrupting their endocrine system. Notably, the lack of detailed understanding of endocrine systems and their signaling pathway prevents understanding of the effect of PEs in invertebrates. Accordingly, this thesis aims to address the issue of endocrine disruption in marine invertebrates by exploiting the larval development of the Mediterranean mussel Mytilus galloprovincialis. By applying the logic of the Adverse Outcome Pathway-AOP, the work in this thesis attempted to characterize the mechanism of action of the model EDCs BPA, TBBPA and TBT in mussel larvae and to identify plausible neuroendocrine pathways by which they induce their deleterious biological effect. The neuroendocrine elements regulating the biogenesis of the larval shell have been characterized and the morphogenetic process established as sensitive to neuroendocrine disruption. Plausible neuroendocrine AOPs that could be linked to the biological effects of the model EDCs have been identified. In addition, the possible AOP of TBT has established a plausible relationship between nuclear receptor signaling and the neuroendocrine system of mussel larvae. The results of this thesis represent essential evidence that will help and advance the current understanding of endocrine disruption mechanisms in invertebrate organisms

Doutoramento em Biologia
A Directiva Quadro da Água (DQA) foi introduzida pela União Europeia (UE) em 2000 com o objetivo de proteger as águas de superfície (interiores, de transição e costeiras) e subterrâneas. Esta ambiciosa directiva lançou novos desafios, entre os quais a necessidade de desenvolver novas ferramentas ecotoxicológicas para a avaliação do estado ecológico das massas de água. O tributilestanho (TBT), um biocida amplamente utilizado em tintas antiincrustantes e identificado como agente causador do fenómeno de imposex / intersex em gastrópodes, está incluído na lista de substâncias prioritárias da DQA. No entanto, esta directiva não considera qualquer ferramenta de biomonitorização específica para avaliar os efeitos nocivos do TBT nos ecossistemas. O único objetivo concreto para esta substância é referido nas Normas de Qualidade Ambiental (NQA) e atingido a 0,2 ng TBT / L (NQA - Média Anual). Mais tarde, em 2008, a Directiva Quadro da Estratégia Marinha (DQEM) foi introduzida na UE com objectivos semelhantes à DQA, mas direcionada para águas marinhas. No entanto, uma vez mais, não foi proposta nenhuma ferramenta ecotoxicológica para a monitorização dos efeitos biológicos provocados por este poluente. Assim, de forma a integrar a biomonitorização da poluição por TBT ao abrigo destas directivas, esta tese tem como objetivo desenvolver e testar, sob diferentes cenários, ferramentas baseadas nos biomarcadores imposex/intersex para avaliar o estado ecológico de massas de água. Assim, é proposto um sistema de classificação baseado nos níveis de imposex/intersex de populações de gastrópodes para avaliar o estado ecológico de águas costeiras e de transição, relativamente à poluição por TBT. Utilizando a Ria de Aveiro (NW Portugal) como estudo de caso, três bioindicadores - os gastrópodes Nucella lapillus, Nassarius reticulatus e Littorina littorea - foram utilizados tendo em conta o elemento de qualidade biológica para invertebrados bentónicos da DQA, de forma a classificar o estado das massas de água desta região. O índice de sequência do vaso deferente (VDSI) e o índice de intersex (ISI) foram os parâmetros de imposex/intersex selecionados para avaliar o estado ecológico, relativamente à poluição por TBT. Os limites definidos pelos Rácios de Qualidade Ecológica (EQR) foram obtidos para cada espécie, a fim de definir as cinco classes de estado ecológico (Excelente, Bom, Razoável, Medíocre e Mau). A espécie N. lapillus é proposta como bioindicador chave na monitorização da poluição por TBT, no entanto, o uso combinado das outras espécies revela-se muito útil porque permite monitorizar uma área maior e uma maior diversidade de habitats. Esta ferramenta de monitorização multiespecífica demonstrou ser bastante útil para avaliar a evolução temporal do estado ecológico das massas de água da Ria de Aveiro entre 1998 e 2013, que demonstrou uma clara melhoria, alcançando um bom estado ecológico em 2013. Este trabalho propõe também uma outra ferramenta destinada a avaliar a qualidade dos sedimentos. Esta ferramenta consiste num bioensaio em que se expõem fêmeas de N. reticulatus a sedimentos recolhidos no ambiente para determinar se ocorre um aumento de imposex. Este bioensaio provou ser uma ferramenta complementar e com relevância ecológica na monitorização da poluição por TBT na medida em que fornece informação sobre a fracção biodisponível de TBT nos sedimentos e permite avaliar locais onde não existem bioindicadores disponíveis. Para testar o uso de ambas ferramentas em programas de monitorização da DQA, estas foram aplicadas em dois sistemas estuarinos - Minho e Lima - localizados no Norte de Portugal. A monitorização de imposex evidenciou um bom estado ecológico no Minho mas o estuário do Lima não conseguiu alcançar o objetivo de bom estado ecológico, apresentando locais com um estado "moderado" ou de qualidade inferior. Em conformidade, o bioensaio com sedimentos confirmou a presença de TBT biodisponível apenas nos sedimentos do estuário do Lima, já que se observou o aumento de alguns parâmetros de imposex com estes sedimentos. O uso combinado destas ferramentas permitiu aumentar a área de monitorização nestes sistemas estuarinos, uma vez que não foi possível encontrar bioindicadores em todas as estações de amostragem, sendo nesse caso obtidas apenas amostras de sedimento. O critério proposto para a monitorização de imposex na DQA foi também testado ao longo da costa portuguesa de forma a avaliar a evolução do estado ecológico de 2000 a 2014. Em 2014, as fêmeas de N. lapillus colhidas em praias ao longo da costa exibiram baixos níveis de imposex, exceto numa estação situada na Zambujeira do Mar (SW Portugal) onde foram encontradas fêmeas estéreis. Já as fêmeas de N. reticulatus mostraram baixos níveis de imposex na maioria das estações amostradas, porém elevados níveis foram observados em áreas sujeitas a um intenso tráfego naval. Comparando estes valores com os observados em anos anteriores, é possível perceber que ambas as espécies recuperaram de forma significativa ao longo dos últimos 15 anos. N. lapillus apresentou uma redução dos níveis de imposex mais pronunciada do que N. reticulatus, porém esta diminuição parece ter abrandado nos últimos anos (2011-2014), prevendo-se uma estabilização por algum tempo em níveis muito baixos. N. reticulatus também mostrou uma diminuição gradual, mas os valores de imposex indicam uma recuperação mais lenta em portos de pesca e marinas. Isto representa uma mudança das principais fontes de poluição por TBT, já que os níveis de imposex em grandes portos comerciais, outrora locais altamente poluídos, caíram rapidamente. Assim, na costa Portuguesa, o bom estado ecológico foi alcançado na maior parte das estações localizadas em águas costeiras, no entanto uma qualidade ecológica inferior foi observada em massas de água de transição, especialmente onde estão localizados portos de pesca e marinas. A classificação da qualidade ecológica da costa portuguesa segundo a DQA (proposta apresentada nesta tese) e a OSPAR, relativamente à poluição por TBT, é analisada no presente trabalho. Esta comparação é da maior importância, pois tanto as premissas da DQA, como as ferramentas de monitorização utilizadas pelas convenções regionais marinhas (por exemplo a OSPAR) devem ser integradas na aplicação da DQEM. As classificações obtidas segundo a DQA e a OSPAR apresentam um resultado global muito semelhante para 2014, e ambas denotam uma apreciável recuperação do estado ecológico ao longo dos últimos anos. A integração da monitorização de imposex/intersex na DQEM é aqui recomendada e discutida, tendo em conta possíveis divergências nos objetivos definidos por cada legislação e os meios para alcançá-los. Mesmo que a utilidade das ferramentas empregadas para monitorizar a poluição por TBT tenham sido comprovadas, a monitorização de imposex/intersex a ser aplicada na DQA e DQEM só será eficaz se: (i) houver uma boa escolha de bioindicadores; (ii) o imposex/intersex constituírem uma resposta específica para a poluição por TBT porque se outros contaminantes tiverem a capacidade de induzir imposex ou intersex, estes biomarcadores perdem grande parte do seu valor operacional. Por estas razões, esta tese apresenta ainda alguns resultados que devem ser considerados para a validação da utilização do imposex/intersex na monitorização da poluição por TBT. A Ria de Aveiro é uma área que tem sido intensamente estudada em relação à poluição por TBT e, conforme descrito anteriormente, apresentou uma importante melhoria do estado ecológico em relação a este poluente prioritário. No entanto, uma tendência contrária foi observada na evolução dos níveis de imposex no gastrópode Peringia ulvae. Esta espécie - considerada por vários autores como um bom bioindicador da poluição por TBT - não demonstrou uma recuperação dos níveis de imposex, apesar dos níveis de TBT e de imposex/intersex noutras espécies terem vindo consistentemente a diminuir nesta área. Em vez disso, houve um aumento global na percentagem de fêmeas afetadas por imposex e nos níveis de VDSI até 2012, sendo que os níveis de imposex em 2015 são ainda semelhantes aos encontrados em 1998. As possíveis razões para o padrão contracorrente observado nesta espécie podem estar relacionados com a interferência de outros factores que influenciam a expressão do imposex; por exemplo, a indução de imposex por parasitas - que são difíceis de controlar quando a monitorização é realizada com esta espécie - pode enviesar a interpretação dos resultados. É, portanto, necessária uma escolha criteriosa dos bioindicadores a utilizar no âmbito das directivas DQA e DQEM. O imposex não é apenas um efeito exclusivo do TBT, uma vez que já foi descrito que o trifenilestanho (TPT) pode também causar este fenómeno em algumas espécies de gastrópodes. Aqui, é descrita, pela primeira vez, a indução de imposex por TPT em N. lapillus, a espécie que foi proposta como bioindicador chave na DQA. Curiosamente, o desenvolvimento do imposex em fêmeas de N. lapillus injetadas com TPT é diferente das injectadas com TBT, uma vez que as primeiras desenvolvem uma via de imposex principalmente afálica (sem desenvolvimento do pénis). Estes resultados sugerem que o TPT e o TBT podem agir de forma diferente no processo de masculinização de fêmeas de gastrópodes, lançando novas perspectivas sobre as hipotéticas vias subjacentes ao desenvolvimento de imposex. É importante saber se existem outros contaminantes, para além dos organoestânicos, capazes de induzir (ou interferir com) o desenvolvimento de imposex ou intersex, com vista à validação das ferramentas de monitorização propostas nesta tese. Para tal, um ensaio com um gene repórter foi realizado em células transfectadas com recetores humanos, cujos ortólogos foram anteriormente identificados em gastrópodes (RXR, PPAR, ER e RAR). Sabendo que o imposex é causado pela ativação dos recetores nucleares RXR e PPARy por parte do TBT e TPT, é importante perceber se outros contaminantes são capazes de ativar estes recetores. Testaram-se vários contaminantes ambientais e mesmo que algumas substâncias tenham tido a capacidade de ativar ligeiramente o RXR e PPARy, nenhuma foi capaz de o fazer com uma potência semelhante ao TBT e TPT, sugerindo que estas são as únicas substâncias testadas capazes de induzir imposex para concentrações ambientalmente relevantes. Não obstante, todas as substâncias que foram capazes de ativar os recetores foram identificadas e o seu potencial para causar disrupção endócrina nos gastrópodes é discutido. Em conclusão, as ferramentas propostas nesta tese podem ser extremamente úteis na monitorização do ambiente aquático ao abrigo das recentes directivas da União Europeia. Em Portugal existem três peças legislativas que se sobrepõem espacialmente nas águas costeiras (DQA, DQEM e OSPAR) e águas de transição (DQA e OSPAR), e podem diferir nos seus objetivos específicos relativamente à poluição por TBT. Logo, existe uma necessidade urgente de encontrar objetivos comuns na UE, tanto químicos e biológicos, para este poluente. Para além disto, e num momento em que a busca de modelos biológicos não vertebrados está a aumentar em ecotoxicologia, esta tese realça o uso de gastrópodes como uma possível opção. O trabalho desenvolvido nesta tese mostra que a poluição por TBT tem vindo a diminuir em Portugal mas ainda existem alguns locais onde os níveis são altos e não atingem os objectivos definidos pela OSPAR e pela DQA (segundo a proposta aqui apresentada). As ferramentas propostas nesta tese podem ser úteis para acompanhar a evolução da situação em Portugal e na UE nos próximos anos e identificar os locais onde será necessário melhorar a qualidade ecológica das massas de água relativamente a esta substância prioritária, de acordo com as metas estabelecidas pela legislação vigente.
The Water Framework Directive (WFD) was introduced in the European Union (EU) in 2000 with the objective to protect the EU surface waters (inland, transitional and coastal) and ground waters. This ambitious directive raised new challenges, as the need to develop quick and low-cost effect-based monitoring tools, which are increasingly being recommended to perform a proper environmental assessment under this directive. Tributyltin (TBT), a biocide largely used in antifouling paints and identified as a causative agent of imposex/intersex in gastropods, is listed as a priority substance in WFD. However, this directive does not consider any particular biomonitoring tool to assess TBT deleterious effects on the ecosystems. The only explicit objective for TBT regards the Environmental Quality Standard (EQS – Annual Average = 0.2 ng TBT/L). Later on, in 2008, the Marine Strategy Framework Directive (MSFD) was introduced in the EU with similar objectives but specifically related to marine waters that again failed to propose an effect based tool to monitor this pollutant. Therefore, to integrate TBT pollution monitoring within these legislative frameworks, it is the aim of this thesis to develop effect based tools centred on the imposex/intersex biomarkers in order to assess the ecological quality status of EU water bodies regarding TBT pollution. This work proposes a scoring system based on imposex/intersex levels to assess the WFD ecological quality status regarding TBT pollution of transitional and coastal waters. Taking Ria de Aveiro (NW Portugal) as a case study, three bioindicators – the gastropods Nucella lapillus (dog-whelk), Nassarius reticulatus (netted-whelk) and Littorina littorea (periwinkle) - were used under the general WFD benthic invertebrate quality element to classify the ecological status of this area. The vas deferens sequence index (VDSI) and the intersex index (ISI) were selected as biomarkers to assess the condition of this quality element regarding the impact of TBT pollution. EQR boundaries were set for each species in order to define the five ecological status classes (High, Good, Moderate, Poor and Bad). N. lapillus is proposed as a key bioindicator species, however the combined use of further species is very useful to cover a wider monitoring area and a higher diversity of habitats. This multi-species monitoring tool was useful to assess the temporal evolution of the ecological status of Ria de Aveiro water bodies between 1998 and 2013, showing that all the surveyed area improved and reached a good ecological status in 2013. This work also proposes other imposex based tool that allows to evaluate the sediment quality, regarding TBT pollution. The use of this bioassay has proven to be a practical and ecological relevant tool as (i) it can give information for sites with no native populations of snails, (ii) it provides early identification of polluted sites anticipating future imposex levels or early identification of recovering, and (iii) it yields information on the bioavailable fraction of the TBT in the sediment. Therefore, this tool can also be of extreme usefulness under the scope of recent European legislative frameworks. To confirm the suitability of the above mentioned tools in WFD monitoring programs, they were both applied in two estuarine systems - Minho and Lima - located in NW Portugal. The imposex field monitoring evidenced a good ecological status in Minho while the Lima estuary fail to reach the WFD good ecological status objective by presenting sites with “Moderate” and “At Best Poor” ecological status. Accordingly, the sediment bioassay confirmed the presence of bioavailable TBT only in the Lima sediments. The combined use of these tools allowed to increase the monitoring area in these estuarine systems and enhanced the robustness of the assessment, as the bioindicators fail to exist at many sites of the study area. The proposed criteria for imposex monitoring under the WFD was tested along the Portuguese coast to assess the evolution of the ecological status from 2000 to 2014, using N. lapillus and N. reticulatus as bioindicators. Lately, the dogwhelks collected in coastal shores exhibited low imposex levels, except at one station in Zambujeira do Mar (SW Portugal) where sterile females were still found. Accordingly, the netted-whelk showed low levels of imposex at the majority of the sampled stations but high levels were still observed at areas subjected to intense naval traffic. When comparing these data regarding the most recent years with past imposex levels, it is perceived that both species recovered significantly over the last 15 years. N. lapillus presented a more pronounced decrease in imposex levels than N. reticulatus, however this declining trend decelerated from 2011 to 2014. N. reticulatus also showed a gradual decrease but the imposex values indicate a slower recovery at fishing harbours and marinas, which represents an apparent shift in the TBT hotspots as imposex levels in large commercial ports fell rapidly. Consequently, in the Portuguese coast a good ecological status was achieved in most of the stations located in coastal waters while a worse ecological quality was observed in transitional water bodies where fishing ports and marinas are located. A comparison between the proposed WFD imposex tool and the OSPAR Assessment Criteria for imposex shows that both classifications schemes present a general similar result for 2014 and point to a clear recovery of the quality status over the years. This comparison between two legislations is of foremost importance since both WFD premises and Regional Sea Conventions (e.g OSPAR) knowledge and monitoring tools should be integrated by member states on the application of the MSFD. Therefore the imposex/intersex monitoring integration within MSFD is discussed taking into account possible divergences between the objectives defined by each piece of legislation and the means to achieve it. Even if the usefulness of the employed tools to monitor TBT pollution has been proved, the WFD’s monitoring proposal will only be effective if: i) there is a good choice of the bioindicators species; and ii) imposex/intersex are specific responses to TBT pollution, i.e., if other contaminants induce imposex or intersex, then these biomarkers lose much of their operational value. For that reason this thesis presents novel information regarding these aspects in order to validate this methodology and better interpret the obtained results. Ria de Aveiro is an area that has been intensively studied regarding TBT pollution and, as previously reported, presented a major improvement of the ecological status regarding this priority pollutant. An opposite trend has been however observed in the imposex evolution in the gastropod Peringia ulvae. This species - regarded by some authors as a good bioindicator of TBT pollution - did not show an imposex recovery despite the fact that TBT levels have been consistently decreasing in this area. Instead, there was a global increase in the percentage of females affected by imposex and VDSI levels until 2012, and the imposex levels in 2015 were similar to those found in 1998. Possible reasons for this counter current pattern could be related with other factors that can influence imposex expression, such as parasitism, that probably cannot be totally controlled when monitoring is performed with this species. Hence, there is the need to carefully choose the right bioindicators to implement TBT monitoring assessments under WFD or MSFD, and P. ulvae is not recommended for this purpose. Imposex is not an exclusive response to TBT since triphenyltin (TPT), a related organotin, is already known to cause the same phenomenon in some gastropod species. This thesis describes, for the first time, that TPT also induces the development of imposex in N. lapillus, the species considered the key bioindicator in the WFD monitoring proposal. However, imposex development in TPT-injected females followed mostly an aphallic route (no penis development). These results suggest that TPT and TBT may act differently in the sequential process of female masculinization casting new insights about the hypothetical pathways underlying imposex development. Besides TBT and TPT, it is important to know if other environmental contaminants can induce or interfere with the imposex/intersex development to ensure that the proposed tools are specific to organotin pollution. With this purpose, a reporter gene assay was performed using the GAL4/UAS system in cells transfected with human receptors, which were already identified in gastropods: Retinoid X Receptor (RXR), Peroxisome Proliferator-Activated Receptor gamma (PPARγ), Retinoic Acid receptor (RAR) and Estrogenic Receptor (ER). It has been reported in the literature that imposex is caused by the activation of the nuclear receptors RXR and/or PPARγ by TBT or TPT, and therefore if other contaminants can bind these receptors they could have the potential to induce imposex/intersex as well. Even if there were other substances that slightly activated RXR and PPARγ, none was able to do it with similar potency than the organotins (TBT and TPT), suggesting so far that these are the only tested substances capable to induce imposex at realistic environmental concentrations. Nevertheless, all substances that were able to activate the reporter gene were identified and their potential to cause endocrine disruption in gastropods is discussed. Concluding, the tools proposed in this thesis can be of extreme utility to monitor the aquatic environment under the frame of the recent EU directives. Moreover, in Portugal there are three legislative pieces that are spatially overlapped regarding coastal waters (WFD, MSFD and OSPAR) and transitional waters (WFD and OSPAR), and they may differ in their specific objectives regarding TBT pollution. Therefore there is a need to find common EU objectives, both chemical and biological, for this pollutant. Furthermore, in a time where the search of non-vertebrate biological models in ecotoxicology is increasing, this thesis enhances the use of gastropods as an option. Also the work here developed shows that TBT pollution has been decreasing in Portugal but there are some sites where levels are still high. In this scenario, the tools here proposed can be useful to track the evolution of TBT pollution during the next years and to identify waters bodies that will require an action to improve their ecological status.

La principale partie de ma thèse s’intéresse aux principes d'organisation du génome de la bactérie Escherichia coli. Le génome des bactéries est contenu dans le nucléoïde, un espace fortement organisé par de nombreux facteurs tels que la transcription, le sur-enroulement de l'ADN ou la présence de protéines spécifiques. Toutefois, les mécanismes précis qui régissent l'organisation du nucléoïde restent vaguent. La protéine Ndd du phage T4 provoque une désorganisation radicale du nucléoïde quand elle est exprimée chez E. coli. J'ai utilisé cette protéine Ndd pour tenter de révéler des structures encore inconnues liées à l'organisation du nucléoïde. En utilisant un nouveau système d'expression de Ndd chez E. coli il a été possible de montrer que cette protéine impacte l'ensemble du génome d'E. coli, aussi bien structurellement que transcriptionnellement. Des études en Hi-C ont permis de révéler que la structuration locale du nucléoïde n'est pas affectée par la présence de Ndd. Toutefois, l'expression de Ndd provoque une altération du profil de réplication d'E. coli avec une possible surreplication de la région proximale de l'origine de réplication. Nous avons aussi observé que l'expression de Ndd depuis le chromosome menait à des conséquences très différentes de son expression depuis un plasmide. La recherche de partenaires de Ndd qui pourraient expliquer ces effets par un screen dCas9 n'a donné que peu de candidats qui sont en cours de validation. Nous montrons ici que Ndd semble agir sur un ou des aspects précis mais encore indéterminés de l'organisation du nucléoïde et potentiellement indépendamment de la participation de gènes d'E. coli
The main project of my PhD concerned the principles of organization of the genome of the bacterium Escherichia coli. Bacterial genomes are held in the nucleoid, a space highly organized by many factors such as transcription, DNA supercoiling or the presence of specific proteins. However, some of the precise mechanisms that govern its organization remain unclear. The Ndd protein from phage T4 causes a radical disorganization of the nucleoid when expressed in E. coli. I used this Ndd protein to try to reveal previously unknown structures related to nucleoid organization. Using a novel expression system for Ndd in E. coli, it was possible to show that this protein impacts the entire genome of E. coli, both structurally and transcriptionally. Hi-C studies revealed that the Ndd-disrupted nucleoid remains locally structured in a manner similar to a normal nucleoid. However, Ndd expression causes an alteration in the replication profile of E. coli with possible over-replication at the origin of replication. We also observed that expression of Ndd from the chromosome leads to very different consequences than its expression from a plasmid. The search for a partner of Ndd that could explain these effects by a dCas9 screen yielded few candidates that are currently undergoing validation. Here we show that Ndd appears to act on one or more specific aspects of nucleoid organization and potentially independently of the involvement of genes from E. coli

Introduction ------------ The physicist Andreevich Artsimovich in the 1970 wrote that "thermonuclear [fusion] energy will be ready when mankind needs it". Considering the actual world energy situation and the effect on the environment due to the present harnessing of the different sources of energy, the hope is that time for fusion is finally arrived. Background and Motivation ------------------------- The activities carried out in the framework of this thesis regarded the devel- opment, implementation and application of algorithms for classification and prediction of disruptions in Tokamaks. The balance of plasmas in a magnetic field can be described by the theory of magneto-hydro-dynamic (MHD). MHD instabilities are among the most serious factors that limit fusion devices operation in magnetic confinement configurations. When they occur on a large scale can degrade the perfor- mance of the plasma and lead to loss of confinement and control. A disruption is a sudden loss of stability or confinement of tokamak plasma; it is a critical event in which the plasma energy is lost within a time span of few milliseconds exposing the plasma facing components to se- vere thermo-mechanical stresses and conductors surrounding the vessel to huge electromagnetic forces. Therefore, it becomes of primary importance to avoid or mitigate disruptions in order to preserve the integrity of the ma- chine. This aspect and the understanding of disruptive phenomena play a key role in design and running of new experimental devices as ITER, cur- rently under construction in Cadarache (France), which will have the task of demonstrating the feasibility of fusion energy production from a technical and engineering point of view. These considerations motivate a strong interest in developing methods and techniques aimed to minimize both number and severity of disruptions. Furthermore when a disruption occurs it would be particularly important to be able to distinguish among its difierent types in order to improve avoidance and mitigation strategies. Since physical models able to reliably recognize and predict the occurrence of disruptions are currently not available, the re- search carried out fits in the broad framework of machine learning techniques that have been exploited as an alternative approach to disruption prediction and automatic classification. Promising approaches to prediction and classification are represented by the so-called "data-based" methods: to this purpose, existing systems have been applied and further developed and new approaches have been investi- gated. The mentioned activity has been carried out in collaboration with the University of Cagliari and European Research Centers for nuclear fusion, taking as case study some of the most important experimental machines such as JET and ASDEX Upgrade (AUG), with several months of research spent at the Culham Science Centre. Outline of the Thesis --------------------- In chapter 1 the perspectives of fusion in the world energy context as an almost unlimited source of energy for the future are discussed, with particu- lar reference to the role of magnetic confinement. Furthermore, the bases of fusion reactions have been introduced. In chapter 2 the main aspects of plasma stability in tokamaks configu- rations are described with the aim to provide an adequate reference for all the discussions of the following chapters. In particular, the main parameters related to plasma stability, which have been used for the construction of the databases, have been introduced. The chapter 3 is focused on the description of the operational limits with reference to the main quantities which should be maximized to im- prove plasma performance. Everything, also in the previous chapters, has been framed to introduce the key problems which this thesis has addressed: analysis, prediction and classification of disruptions. After the main consid- erations about the operational limits, the main phases, the causes and the consequences of disruptions have been discussed, trying to integrate the sta- bility concepts introduced in the previous chapter. The chapter 4 is finalized to provide an insight of the Machine Learn- ing methods which represent the starting point of all the analysis and algo- rithms implemented for disruption prediction and classification. Today the large amount of data available from fusion experiments and their character of high-dimensionality make particularly difficult handling, processing, un- derstanding and extracting properly what is really important among all the available information. Machine Learning allows to deal with the problem in efficient way. Therefore, a framework of all the techniques exploited for the analysis has been provided, with particular reference to the Manifold Learn- ing algorithms as Self Organizing Maps (SOMs) and Generative Topographic Mappings (GTMs). Also reference methods such as k-Nearest Neighbor (k- NN) or more recent methods such as Conformal Predictors, exploited for validation and reliability assessment purposes, have been described. In chapter 5 the state of the art of machine learning techniques ap- plied to disruption prediction and classification is presented, describing in particular the main applications with the widely employed Neural Networks, such Multi Layer Perceptrons (MLPs), Support Vector Machines (SVMs) and Self Organizing Maps (SOMs), and statistical methods such as Discrim- inant Analysis or Multiple Threshold technique. Strengths and weaknesses have also been discussed with reference to a possible solution to overcome the drawbacks of these methods: the multi-machine approach. Chapter 6 is dedicated to the description of the databases used for all the analysis presented in the following chapters. In particular, the statistical analysis and the data-reduction algorithms that have been needed to build a reliable and statistically representative database have been discussed in detail. The last three chapters contain all the analysis and all the algorithms im- plemented for the mapping of the operational space, disruption classification and prediction. In chapter 7 the mapping of the JET operational space is presented. The first sections deal with projections and data-visualization with linear projection methods such as Grand Tour (GT) and Principal Com- ponent Analysis (PCA). In the central part, the same aspects have been taken into account by exploiting nonlinear Manifold Learning techniques, SOM and GTM, on the base of which a detailed analysis of the operational space has been performed. Such analysis, showing the potentiality of the methods, has been performed, regarding GTM model, through the implementation of a dedicated tool. Finally, an outliers' analysis and performance indexes appo- sitely proposed have been considered for evaluating the overall performance of the mapping. In the chapter 8 the developed automatic disruption classification for JET has been described. The chapter is divided in two parts: the first one describes the classification of disruptions belonging to the Carbon Wall (CW) campaigns, whereas in the second part the classification of disruptions with the ITER-like Wall (ILW) is framed in the assessment of the suitability of the automatic classifier for real time applications, in conjunction with prediction systems working online at JET. The reliability of the results has been vali- dated by comparison with a k-NN based reference classifier and through the recent conformal predictors, with which is possible to provide, in addition to the prediction/classification, the related level of confidence. Chapter 9 is dedicated to the disruption prediction at ASDEX Upgrade. The first part is related to the description of the database and the data- reduction technique used to select a representative and balanced dataset. Self-Organizing Map and the Generative Topographic Mapping have been exploited to map ASDEX Upgrade operational space and to build a disrup- tion predictor, introducing at the same time their potentiality for disruptions classification. Furthermore, the use of this two methods combined with a Lo- gistic model has been proposed to realize a predictive system able to exploit the complementary behaviors of the two approaches, improving the overall performance in prediction.
Prefazione ---------- Il fisico Andreevich Artsimovich nel 1970 scrisse che "l'energia da fusione nucleare sarà disponibile quando l'umanità ne avrà bisogno". Considerando l'attuale scenario energetico mondiale e l'impatto sull'ambiente dovuto allo sfruttamento delle diverse risorse energetiche, la speranza è che quel momento sia finalmente arrivato. Background e Motivazione ------------------------ Le attività svolte nell'ambito di questa tesi hanno riguardato lo sviluppo, l'implementazione e l'applicazione di algoritmi per la classificazione e la predi- zione di disruzioni nei Tokamak. L'equilibrio dei plasmi nei campi magnetici può essere descritto dalla teo- ria magneto-idro-dinamica (MHD). Le instabilità MHD sono tra i fattori che limitano più seriamente le operazioni nelle macchine a fusione a confinamento magnetico. Una disruzione è un'improvvisa perdita di stabilità e di confinamento nei tokamak; è un evento critico durante il quale l'energia immagazzinata nel plasma viene persa nell'arco di pochi millisecondi, esponendo i componenti della parete interna della camera da vuoto a severi stress termo-meccanici, e i conduttori circostanti a enormi forze elettromagnetiche. Quindi diventa di primaria importanza l'avoidance e la mitigazione delle disruzioni al fine di preservare l'integrità della macchina. Questo aspetto e la comprensione dei fenomeni disruttivi giocano un ruolo chiave nel progetto e nel funzionamento delle nuove macchine sperimentali come ITER, attualmente in costruzione a Cadarache (Francia), la quale avrà la finalità di dimostrare la fattibilità tecnica ed ingegneristica della produzione di energia da fusione. Queste considerazioni motivano un forte interesse nello sviluppo di metodi e tecniche atti a minimizzare sia il numero che l'entità delle disruzioni. In- oltre, quando si verifica una disruzione, sarebbe veramente importante rius- cire a distinguere tra i diversi tipi di disruzione, al fine di migliorare le strate- gie di avoidance e mitigazione. Dal momento che ad oggi non esistono modelli fisici in grado di riconoscere e predire in maniera affidabile l'arrivo di una disruzione, la ricerca portata avanti in questi anni si integra nel più ampio contesto delle tecniche di Machine Learning, le quali sono state utilizzate come approccio alternativo alla predizione ed alla classificazione automatica delle disruzioni. Approcci promettenti alla predizione ed alla classificazione sono rapp- resentati dai cosidetti approcci "data-based": a questo proposito sono state applicate e ulteriormente sviluppate diverse tecniche, e si è indagato su nuovi approcci. Le attività citate sono state svolte in collaborazione con l'Università di Cagliari e importanti centri di ricerca europei sulla fusione, prendendo in esame alcune delle più importanti macchine sperimentali, quali il JET (Regno Unito) e ASDEX Upgrade (Germania), con diversi mesi trascorsi al Culham Science Centre (Abingdon, Regno Unito). Outline della Tesi ------------------ Nel capitolo 1 vengono discusse le prospettive della fusione nel contesto energetico mondiale come fonte quasi illimitata di energia per il futuro, con particolare riferimento al ruolo del confinamento magnetico. Inoltre, sono state introdotte le basi sulle reazioni di fusione. Nel capitolo 2 vengono descritti gli aspetti principali della stabilità del plasma nelle configurazioni tokamak, con l'obiettivo di fornire un riferimento adeguato per tutte le discussioni dei capitoli successivi. In particolare ven- gono introdotti i principali parametri relativi alla stabilità del plasma, che sono stati utilizzati per la costruzione dei database. Il capitolo 3 è incentrato sulla descrizione dei limiti operativi con riferi- mento ai principali parametri che dovrebbero essere ottimizzati per migliorare le performance del plasma. Tutto, anche nei capitoli precedenti, è contestuale all'introduzione dei principali problemi che questa tesi si pone l'obiettivo di affrontare: analisi, predizione e classificazione delle disruzioni. Dopo le con- siderazioni sui limiti operativi, vengono discusse le fasi principali, le cause e le conseguenze dei processi disruttivi, cercando di integrarvi i concetti sulla stabilità introdotti nel capitolo precedente. Il capitolo 4 è invece finalizzato a fornire una panoramica sui metodi di Machine Learning che rappresentano il punto di partenza per tutte le analisi e gli algoritmi implementati per la predizione e la classificazione delle dis- ruzioni. Oggi la grande quantità di dati disponibili dagli esperimenti sulla fusione e il loro carattere di alta dimensionalità, rendono particolarmente difficile la gestione, l'elaborazione, la comprensione e l'estrazione di quelle informazioni che sono veramente importanti tra tutte quelle disponibili. Il Machine Learning consente di affrontare il problema in modo efficiente. Viene quindi fornito un quadro generale di tutte le tecniche utilizzate per l'analisi, con particolare riferimento agli algoritmi di Manifold Learning come la Self Organizing Map (SOM) e la Generative Topographic Mapping (GTM). Vengono inoltre descritti metodi di riferimento come il k-Nearest Neighbor (k-NN) o metodi più recenti come i predittori conformali, utilizzati per scopi di validazione e valutazione dell'affidabilità. Nel capitolo 5 viene presentato lo stato dell'arte relativamente alle tec- niche di Machine Learning applicate alla predizione e alla classificazione di disruzioni, descrivendo in particolare le principali applicazioni con le ampia- mente utilizzate Reti Neurali, quali Multi Layer Perceptrons (MLP), Support Vector Machines (SVM) e Self Organizing Maps (SOM), e i metodi statistici come la Discriminant Analysis o la tecnica Multiple Threshold. Vantaggi e svantaggi vengono discussi anche con riferimento ad una possibile soluzione per superare gli svantaggi di questi metodi: l'approccio multi-machine. Il capitolo 6 è dedicato alla descrizione dei database utilizzati per tutte le analisi che verranno presentate nei capitoli seguenti. In particolare vengono discussi in dettaglio l'analisi statistica e gli algoritmi di data-reduction che si sono resi necessari per costruire un database affidabile e statisticamente rappresentativo. Gli ultimi tre capitoli contengono le analisi e gli algoritmi implementati per il mapping degli spazi operativi, la classificazione e la predizione delle disruzioni. Nel capitolo 7 viene descritto il mapping dello spazio oper- ativo di JET. Le prime sezioni si occupano di proiezione e visualizzazione dei dati con metodi di proiezione lineari come Grand Tour (GT) e Principal Component Analysis (PCA). Nella parte centrale sono stati trattati gli stessi aspetti sfruttando tecniche non lineari di Manifold Learning, SOM e GTM, sulla base delle quali è stata effettuata una dettagliata analisi dello spazio op- erativo. Tale analisi, mostrando la potenzialità dei metodi, è stata eseguita, per quanto riguarda il modello GTM, mediante la realizzazione di un tool dedicato. Infine, le performance nel mapping sono state valutate attraverso l'analisi degli outlier e di indici di performance appositamente proposti. Nel capitolo 8 viene descritta la classificazione automatica implementata per le disruzioni al JET. Il capitolo è diviso in due parti: la prima descrive la classificazione delle disruzioni appartenenti alle campagne con la parete in carbonio, mentre nella seconda parte è descritta la classificazione con la parete metallica (ILW) contestualmente alla valutazione della idoneità del classificatore automatico per applicazioni in tempo reale, unitamente ai sis- temi di predizione on-line al JET. L'affidabilità dei risultati è stata validata attraverso il confronto con un classificatore di riferimento basato sulla tec- nica k-NN, e attraverso i più recenti predittori conformali, con cui è possibile fornire in aggiunta alla predizione/classificazione il relativo livello di confi- denza. Il capitolo 9 invece è dedicato alla predizione delle disruzioni ad AS- DEX Upgrade. La prima parte è relativa alla descrizione del database e della tecnica di data-reduction utilizzata per selezionare un insieme di dati rappresentativo ed bilanciato. SOM e GTM sono stati utilizzate per map- pare lo spazio operativo di ASDEX Upgrade e per costruire un predittore di disruzioni, introducendo al stesso tempo le loro potenzialità in termini di classificazione. Inoltre è stato proposto l'uso combinato di questi due metodi con un regressore logistico al fine di realizzare un sistema predittivo in grado

We constrain the recent star formation histories of the host galaxies of eight optical/UV-detected tidal disruption events (TDEs). Six hosts had quick starbursts of <200 Myr duration that ended 10-1000 Myr ago, indicating that TDEs arise at different times in their hosts' post-starburst evolution. If the disrupted star formed in the burst or before, the post-burst age constrains its mass, generally excluding O, most B, and highly massive A stars. If the starburst arose from a galaxy merger, the time since the starburst began limits the coalescence timescale and thus the merger mass ratio to more equal than 12: 1 in most hosts. This uncommon ratio, if also that of the central supermassive black hole (SMBH) binary, disfavors the scenario in which the TDE rate is boosted by the binary but is insensitive to its mass ratio. The stellar mass fraction created in the burst is 0.5%-10% for most hosts, not enough to explain the observed 30-200x. boost in TDE rates, suggesting that the host's core stellar concentration is more important. TDE hosts have stellar masses 10(9.4)-10(10.3) M circle dot,consistent with the Sloan Digital Sky Survey volume-corrected, quiescent Balmer-strong comparison sample and implying SMBH masses of 10(5.5)-10(7.5) M circle dot, Subtracting the host absorption line spectrum, we uncover emission lines; at least five hosts have ionization sources inconsistent with star formation that instead may be related to circumnuclear gas, merger shocks, or post-AGB stars.

This thesis presents theoretical results on the tidal disruption of stars by supermassive black holes (SMBHs). The multiwavelength ares produced by tidal disruption events (TDEs) have supernova-like luminosities, and associated relativistic jets can be visible to cosmological distances. TDEs probe the demography of quiescent SMBHs, and are natural laboratories for jet launching mechanisms and super-Eddington accretion. The first chapter broadly surveys TDE physics. The second and third chapters estimate the TDE rate following gravitational wave (GW) recoil of a SMBH (after a SMBH binary merger). Immediately after GW recoil, the TDE rate increases, sometimes to \(~10^{-1}\) TDEs per year. This "burst" of TDE flares can provide an electromagnetic counterpart to low frequency GW signals, localizing sources and measuring cosmological parameters. Millions of years later, recoiled SMBHs wandering through their host galaxies will produce spatially offset TDEs at a rate which is likely detectable with the LSST. In the fourth chapter, we show that standard estimates for \(\Delta\epsilon\), the energy spread of TDE debris, are wrong, sometimes by orders of magnitude. Correcting this error reduces the observability of many TDEs. We introduce a new analytic model for tidal disruption, calculate \(\Delta\epsilon\)'s dependence on stellar spin, estimate general relativistic corrections to \(\Delta\epsilon\), and quantify the GW signal generated from tidal compression. The fifth chapter presents hydrodynamical simulations of TDE debris circularization, focusing on eccentric, rather than parabolic, orbits. General relativistic precession drives debris circularization, in contrast to past simulations using smaller black holes. In the sixth chapter, we show that TDE light curves can constrain or measure SMBH spins, as Lense-Thirring torques produce quasiperiodic variability in disk emission. Precession of a relativistic jet could also measure SMBH spin, and we apply our model to the relativistic Swift 1644+57 TDE. The seventh chapter considers the disruption of neutron stars (NSs) by stellar mass black holes (BHs) or other NSs. Jet precession in associated short-hard gamma ray bursts is uniquely possible for NS-BH (not NS-NS) mergers. We quantify typical precession amplitudes and periods, and calculate their time evolution. If disk viscosities are relatively low, electromagnetic observations alone could distinguish NS-BH from NS-NS mergers.
Astronomy

La tesi contiene i postprints di alcune pubblicazioni dell'Autore: Pomatto, V., Palermo, F., Mosconi, G., Cottone, E., Cocci, P., Nabissi, M., Borgio, L., Polzonetti-Magni, A.M., Franzoni, M.F. Xenoestrogens elicit a modulation of endocannabinoid system and estrogen receptors in 4NP treated goldfish, Carassius auratus, (2011) General and Comparative Endocrinology, 174 (1), pp. 30-35. Palermo, F.A., Cocci, P., Angeletti, M., Felici, A., Polzonetti-Magni, A.M., Mosconi, G., Dietary Aloe vera components' effects on cholesterol lowering and estrogenic responses in juvenile goldfish, Carassius auratus (2013) Fish Physiology and Biochemistry, 39 (4), pp. 851-861. Palermo, F.A., Cocci, P., Angeletti, M., Polzonetti-Magni, A., Mosconi, G. PCR-ELISA detection of estrogen receptor β mRNA expression and plasma vitellogenin induction in juvenile sole (Solea solea) exposed to waterborne 4-nonylphenol (2012) Chemosphere, 86 (9), pp. 919-925. Palermo, F.A., Cocci, P., Nabissi, M., Polzonetti-Magni, A., Mosconi, G., Cortisol response to waterborne 4-nonylphenol exposure leads to increased brain POMC and HSP70 mRNA expressions and reduced total antioxidant capacity in juvenile sole (Solea solea)(2012) Comparative Biochemistry and Physiology - C Toxicology and Pharmacology, 156 (3-4), pp. 135-139. Cocci, P., Mosconi, G., Palermo, F.A., Effects of 4-nonylphenol on hepatic gene expression of peroxisome proliferator-activated receptors and cytochrome P450 isoforms (CYP1A1 and CYP3A4) in juvenile sole (Solea solea) (2013) Chemosphere, 93 (6), pp. 1176-1181.

We report the detection of a significant infrared variability of the nearest tidal disruption event (TDE) ASASSN-14li using Wide-field Infrared Survey Explorer and newly released Near-Earth Object WISE Reactivation data. In comparison with the quiescent state, the infrared flux is brightened by 0.12 and 0.16 mag in the W1 (3.4 mu m) and W2 (4.6 mu m) bands at 36 days after the optical discovery (or similar to 110 days after the peak disruption date). The flux excess is still detectable similar to 170 days later. Assuming that the flare-like infrared emission is from the dust around the black hole, its blackbody temperature is estimated to be similar to 2.1 x 10(3) K, slightly higher than the dust sublimation temperature, indicating that the dust is likely located close to the dust sublimation radius. The equilibrium between the heating and radiation of the dust claims a bolometric luminosity of similar to 10(43) - 10(45) erg s(-1), comparable with the observed peak luminosity. This result has for the first time confirmed the detection of infrared emission from the dust echoes of TDEs.

Anions including nucleic acids and lipids have been found to promote amyloid formation in diseases including neurodegenerative conditions such as Alzheimer’s and Creutzfeldt-Jakob disease. However, the direct effects of these close charge-based interactions are not well understood. It is unclear what effect amyloidogenic peptides would have on nucleic acid integrity. Similarly, the direct effects of amyloidogenic polypeptides on liposomes are not well understood. Here I have used a simplified system of short basic peptides with alternating hydrophobic and hydrophilic amino acid residues to study their interactions with polyanionic nucleic acids and fatty acid liposomes. Employing biophysical techniques including X-ray fibre diffraction, circular dichroism spectroscopy and electron microscopy I showed that the polymerized charges of nucleic acids and pseudo-polymerised charges of lipid membranes concentrated and enhanced the formation of amyloid from short basic peptides, many of which would not otherwise form fibres under the conditions explored. In turn, the same peptides bound nucleic acids and promoted their hybridisation at concentrations below their solution Kd, as shown by time-resolved FRET studies. The mutual interactions between peptides and nucleic acids lead to the formation of amyloid nucleic acid (ANA) fibres, which in addition to their importance in disease might have a potential in nano-engineering of biomaterials.

Tous les organismes présentent des rythmes biologiques sous le contrôle d’horloges internes synchronisées sur le cycle jour-nuit. La perturbation des horloges (travail posté chez l’homme) conduit souvent à des troubles métaboliques. A l’inverse, obésité et diabète sont associés à des perturbations circadiennes. Mon but est de comprendre les mécanismes impliqués dans le lien réciproque entre perturbations circadiennes et troubles métaboliques. Une première partie révèle le rôle de la leptine dans les troubles circadiens de souris génétiquement obèses, au niveau central et périphérique. Dans une seconde partie, nous montrons que l’altération des cycles jour-nuit induit une désynchronisation circadienne chez un rongeur diurne, entraînant un état pré-diabétique et un vieillissement prématuré des cellules hépatiques. Nos résultats ouvrent la voie à des traitements préventifs visant à diminuer les troubles circadiens des patients obèses et les troubles métaboliques des travailleurs postés
Most organisms exhibit biological rhythms, generated endogenously by circadian clocks, which are synchronized on the light-dark cycle. Disrupting circadian clocks (e.g, shiftwork) lead in most cases to the occurrence of metabolic disorders. Conversely, obesity and diabetes are associated with circadian disruptions. The aim of my project is to provide new insights in the understanding of mechanisms underlying the reciprocal relationships between circadian disruptions and metabolic disorders. We show in a first part that leptin is involved in circadian disturbances of genetically obese mice, at both central and peripheral levels. In a second part, by altering the light-dark cycle, we induce the circadian desynchronization of a diurnal rodent, which leads in turn to a pre-diabetic state associated with accelerated aging of hepatic cells. Our results pave the road to preventive treatments aiming at reducing circadian disruptions in obese patients and metabolic disorders in shiftworkers

The sporadic accretion following the tidal disruption of a star by a super-massive black hole (TDE) leads to a bright. UV and soft X-ray flare in the galactic nucleus. The gas and dust surrounding the black hole responses to such a flare with an echo in emission lines and infrared emission. In this paper, we report the detection of long fading mid-IR emission lasting up to 14 years after the flare in four TDE candidates with transient coronal lines using the WISE public data release. We estimate that the reprocessed mid-IR luminosities are in the range between 4 x 10(42) and 2 x 10(43) erg s(-1) and dust temperature in the range of 570-800 K when WISE first detected these sources three to five years after the flare. Both luminosity and dust temperature decrease with time. We interpret the mid-IR emission as the infrared echo of the tidal disruption flare. We estimate the UV luminosity at the peak flare to be 1 to 30 times 10(44) erg s(-1) and that for. warm dust masses to be. in the range of 0.05-1.3 M-circle dot within a few parsecs. Our results suggest that the. mid-infrared echo is a general signature of TDE in the gas-rich environment.

The rate of tidal disruption events (TDEs), R-TDE, is predicted to depend on stellar conditions near the super-massive black hole (SMBH), which are on difficult-to-measure sub-parsec scales. We test whether R-TDE depends on kpc-scale global galaxy properties, which are observable. We concentrate on stellar surface mass density, Sigma M-*, and velocity dispersion, sigma(nu), which correlate with the stellar density and velocity dispersion of the stars around the SMBH. We consider 35 TDE candidates, with and without known X-ray emission. The hosts range from star-forming to quiescent to quiescent with strong Balmer absorption lines. The last (often with post-starburst spectra) are overrepresented in our sample by a factor of 35(-17)(+21) or 18(-7)(+8), depending on the strength of the H delta absorption line. For a subsample of hosts with homogeneous measurements, Sigma M-* = 10(9)-10(10) M-circle dot/kpc(2), higher on average than for a volume-weighted control sample of Sloan Digital Sky Survey galaxies with similar redshifts and stellar masses. This is because (1) most of the TDE hosts here are quiescent galaxies, which tend to have higher Sigma M-* than the star-forming galaxies that dominate the control, and (2) the star-forming hosts have higher average Sigma M-* than the star-forming control. There is also a weak suggestion that TDE hosts have lower sigma(nu) than for the quiescent control. Assuming that R-TDE infinity Sigma M-*(alpha) x sigma(beta)(nu), and applying a statistical model to the TDE hosts and control sample, we estimate (alpha) over cap = 0.9 +/- 0.2 and (beta) over cap = -1.0 +/- 0.6. This is broadly consistent with RTDE being tied to the dynamical relaxation of stars surrounding the SMBH.

The overall objective of this dissertation was to examine exposures to the estrogenic endocrine disrupting chemicals (EEDCs), phthalates, bisphenol-A (BPA), and the metalloestrogens cadmium (Cd), arsenic (As), and manganese (Mn) in an older geriatric aged-population and examine associations with brain health. Given the evidence that EEDCs affect brain health and play a role in the development of cognitive dysfunction and neurodegenerative disease, and the constant environmental exposure through foods and everyday products has led this to becoming a great public health concern. Using a bioinformatic approach to find nuclear respiratory factor 1 (NRF1) gene targets involved in mitochondrial dysfunction, that are both estrogen and EEDC-sensitive, we found several genes involved in the gene pathways of Alzheimer’s disease (AD): APBB2, EIF2S1, ENO1, MAPT, and PAXIP1. Using the Center for Disease Control and Prevention (CDC), National Health and Nutrition Examination Survey (NHANES) 2011-2014 datasets to assess EEDC bioburden and associations with surrogate indicators of brain health, which include cognitive scores, memory questions, and taste and smell data, we found phthalate bioburden to be significantly higher in those with adverse brain health vii and significantly higher in females. In our logistic regression model when controlling for all known and suspected covariates in AD, in females, the phthalates in females ECP, MBP, MOH, MZP, and MIB in males and the phthalates COP, ECP, MBP, MC1, MEP, MHH, MOH, and MIB were significantly associated with poor cognitive test scores, poor memory, and taste and smell dysfunction. Among the metalloestrogens, Cd bioburden was higher in those with poor cognitive performance, poor memory, and taste and smell dysfunction, with the trend more significant in males. Among oral contraceptive (OC) and HRT (hormone replacement therapy) use, in our logistic regression model when controlling for all known and suspected covariates in AD, past OC and HRT use was associated with better cognitive test scores. The study provides further evidence of the complex role EEDCs play in overall brain health through other biological mechanisms and fills a gap in knowledge that demonstrates EEDCs effects on brain health in a geriatric age population.

Marine environments are under constant pressure from anthropogenic pollution. Chemical pollutants are introduced into the aquatic environment through waste disposal, sewage, land runoff and environmental exploitation (harbours, fisheries, tourism) leading to disastrous effects on the marine wildlife. Developmental malformations, reproduction failure including sex changes and high death rates are commonly observed in aquatic animal populations around the world. Unfortunately, the underlying molecular mechanisms of these pollution effects, in particular for marine invertebrate species, are often unknown. One proposed mechanism through which environmental pollution affects wildlife, is the disruption of nuclear receptors (NRs), ligand-binding transcription factors in animals. Environmental pollutants can directly interact with nuclear receptors, inducing incorrect signals for gene expression and subsequently disrupt developmental and physiological processes. Elucidation of the exact mechanism in invertebrates, however, is sparse due to limited understanding of invertebrate endocrinology and molecular regulatory mechanisms. Here, I have investigated the presence, expression and function of NRs in the Pacific oyster, Crassostrea gigas, and explored their interrelation with known environmental pollutants. Using a suite of molecular techniques and bioinformatics tools I demonstrate that the Pacific oyster possesses a large variety of NR homologs (43 NRs), which display individual expression profiles during embryo/larval development and supposedly fulfil distinct functions in developmental and physiological processes. Functional studies on a small subset of oyster NRs provided evidence for their ability to regulate gene expression, including interactions with DNA, other NRs or small molecules (ligand-binding). Oyster receptors also show a high likeliness to be disrupted by environmental pollutants. Computational docking showed that the retinoid X receptor ortholog, CgRXR, is able to bind and be activated by 9-cis retinoic acid and by the well-known environmental contaminant tributyltin. A potential interaction between tributyltin and the peroxisome proliferator-activated receptor ortholog CgPPAR has also been found. In addition, exposure of oyster embryos to retinoic acids and tributyltin resulted in shell deformations and developmental failure. In contrast, computer modelling of another putative target for pollutants, the retinoic acid receptor ortholog CgRAR, did not indicate interactions with common retinoic acids, supporting a recently developed theory of loss of retinoid binding in molluscan RARs. Sequence analyses revealed six residues in the receptor sequence, which prevent the successful interaction with retinoid ligands. In conclusion, this investigative work aids the understanding of fundamental processes in invertebrates, such as gene expression and endocrinology, as well as further understanding and prediction of effects of environmental pollutants on marine invertebrates.

Cell-free protein synthesis is an emerging technology that has many applications. The open nature of this system makes it a compelling technology that can be manipulated to answer many needs that are unavailable in other systems. This dissertation reports on engineering this technology for: 1) sense codon emancipation for incorporation of multiple unnatural amino acids; 2) expressing a hard-to-express anticancer biotherapeutic and introducing a just-add-water system; 3) a biosensing ligand that interacts with nuclear hormone receptors. Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A promising approach to sense codon emancipation is the targeted in vitro removal of native tRNA. Here we introduce a new in-vitro or "cell-free" approach to emancipate sense codons via efficient and affordable degradation of endogenous tRNA using RNase-coated superparamagnetic beads. The presented method removes greater than 99% of tRNA in cell lysates, while preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. Here we introduce a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The biosensor is based on an engineered, allosterically-activated fusion protein, which contains the ligand binding domain from a target NHR. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.

CAR (Constitutive Androstane Receptor, NR1I3) et PXR (Pregnane X Receptor, NR1I2) sont deux récepteurs nucléaires dédiés à la reconna issance et à l'élimination de molécules lipophiles potentiellement toxiques pour l'organisme. Ces facteurs de transcription peuvent être activés par des ligands d'origines et de structures diverses (médicaments, polluants environnementaux, produits de l'alimentation et de phytothérapies). L'activation de ces récepteurs entraîne l'expression des gènes majeurs de la fonction de détoxication entéro-hépatique (CYP450, transférases, transporteurs) permettant l'élimination de ces toxiques. Dans ce travail, nous avons dans un premier temps 1) montré que CAR contrôle l'expression de Spot14, une protéine pro-lipogénique, et 2) nous avons identifié une nouvelle isoforme de PXR (PXR-small) codant uniquement pour le domaine de liaison des ligands de PXR. Nous avons pu déterminer les origines de transcription par 5'-RACE PCR et montrer que PXR-small représente environ 10% de l'ensemble des transcrits de PXR dans le tissu hépatique sain par une approche de PCR qua ntitative. Nous avons pu détecter sa présence par western-blot sur des extraits de protéines nucléaires issus de tissus hépatiques et de lignées cellulaires hépatiques. Par des expériences de gel retard, nous avons observé que cette nouvelle isoforme tronquée, qui ne code que pour le LBD de PXR, ne peut pas se lier à l'ADN. Des expériences de gènes rapporteurs suggèrent que cette isoforme se comporte comme un dominant négatif de PXR. Enfin, la présence d'un ilot CpG situé juste en amont de PXR-small suggère que cette nouvelle isoforme pourrait être régulée épigénétiquement par méthylation, notamment dans les cellules tumorales
CAR (Constitutive Androstane Receptor, NR1I3) and PXR (Pregnane X Receptor, NR1I2) are two nuclear receptors devoted to the recognition and elimination of lipohilic molecules potentially toxic to the body.These transcription factors can be activated by ligands of different origins and structures (drugs, environmental pollutants, food products and herbal medicine...). The activation of these receptors leads to the expression of major genes of the detoxification process (CYP450, transferases, transporters) leading to the elimination of these toxics. In this work, we 1) showed that Spot14, a pro-lipogenic protein, is a target gene of CAR, then 2) we identified a novel isoform of PXR (PXR-small), coding only the ligand binding domain of PXR. By using 5'-RACE PXR, we established the origins of transcription of PXR-small and by quantitative PCR we observed that PXR-small represents about 10% of all PXR transcripts in human liver. By using western blo t, we detect its presence on nuclear protein extracts from liver tissues and hepatic cell lines. In Electromobility shift essays experiments, we observed that PXR-small cannot bind to DNA, while reporter essay experiments suggest that this isoform acts as a dominant negative of PXR. Finally, the presence of a CpG island just upstream of PXR-small suggests that this novel isoform might be regulated epigenetically by methylation, more particularly in tumor cells

Os transtornos do comportamento disruptivo (TDC), representados pelo transtorno de oposição desafiante e pelo transtorno de conduta, e o transtorno de déficit de atenção/hiperatividade (TDAH) são transtornos intimamente relacionados. Teorias e estudos sugerem o envolvimento de regiões relacionadas ao processamento de emoções e de recompensas, entre elas a amígdala e o núcleo accumbens (NAcc), em ambos os transtornos. Avaliar as conexões cerebrais do NAcc e da amígdala nos TCD e no TDAH pode contribuir para a elucidação da neurobiologia dos transtornos e de comportamentos relacionados. O objetivo do presente estudo foi caracterizar a conectividade funcional do NAcc e da amígdala em crianças com TDAH e com TCD e avaliar a relação entre a conectividade funcional destas regiões e comportamentos atípicos característicos e comuns a ambos os transtornos. Neste estudo, crianças (idade média = 11,28 anos) classificadas como apresentando TCD (n=22), TDAH (n=25) ou desenvolvimento típico (DT; n=236) foram submetidas a sessão de ressonância magnética funcional de repouso. Foi avaliada a conectividade funcional de repouso de 2 regiões de interesse (NAcc e amígdala) ao restante do cérebro. Em uma abordagem categórica, os mapas de conectividade foram comparados entre os grupos. Além disto, em uma abordagem dimensional, conectividade funcional do NAcc e da amígdala foi correlacionada a pontuações em 3 dimensões de comportamento: desatenção/hiperatividade, agressividade e problemas de conduta, gerando 3 mapas de correlação (conectividade x comportamento) para cada região. Nesta etapa toda a amostra foi incluída (n=283). Os resultados da abordagem categórica mostraram algumas conexões específicas do TCD e do TDAH. As conexões do NAcc à insula posterior e ao precuneus diferenciaram os TCD do DT e do TDAH. A conexão entre amígdala e giro lingual diferenciou TDAH de DT e de TCD. O TDAH também exibiu conectividade atípica da amígadala com o giro pré-central e com o lóbulo parietal inferior, comparado a crianças com DT. Não foi encontrada conectividade funcional alterada do NAcc em crianças com TDAH ou da amígdala em crianças com TCD, comparadas a crianças com DT. A abordagem dimensional demonstrou um padrão diferente de resultados. Pontuações de desatenção/hiperatividade e agressividade estiveram associadas a conectividade do NAcc ao giro fusiforme e ao córtex pré-frontal dorso-medial. Desatenção/ hiperatividade esteve correlacionada com conectividade da amígdala ao lóbulo parietal inferior, ao giro temporal médio e ao sulco pré-central superior. Agressividade esteve correlacionada com conectividade da amígdala ao precuneus e ao giro frontal superior. Problemas de conduta estiveram correlacionados com a conectividade NAcc-giro frontal superior e com a conectividade da amígdala ao giro cingulado posterior, ao precuneus, ao córtex pré-frontal medial e ao giro lingual. Os resultados indicam, portanto, que existem conexões funcionais do NAcc e da amígdala especificamente associadas aos TCD ou ao TDAH e que comportamentos atípicos comuns a ambos os transtornos estão relacionados a alterações na conectividade funcional do NAcc e da amígdala. Concluindo, a abordagem dimensional pode complementar a abordagem categórica na avaliação da neurobiologia dos TCD e do TDAH
Disruptive behavior disorders (DBD), represented by oppositional defiant disorder and conduct disorder, and attention deficit/hyperactivity disorder (ADHD) are intrinsically related disorders. Theories and studies suggest the involvement of regions related to emotional and reward processing, among them the amygdala and the nucleus accumbens (NAcc), in both disorders. Evaluating brain connections of the NAcc and of the amygdala in DBD and in ADHD may contribute to elucidate the neurobiology of the disorders and of related behaviors. The objective of this study was to characterize functional connectivity of the NAcc and of the amygdala in children with ADHD and with DBD, and to evaluated the relationship between functional connectivity of those regions and atypical behaviors characteristic and common to both disorders. In this study, children (mean age= 11.28 years) classified as DBD (n=22), ADHD (n=25), or typical development (TD; n=236) underwent resting-state functional magnetic resonance imaging session. Whole-brain resting-state functional connectivity of 2 regions of interest (NAcc and amygdala) was evaluated. In a categorical approach, connectivity maps were compared between groups. Furthermore, in a dimensional approach, functional connectivity of the NAcc and of the amygdala was correlated to scores in 3 behavior dimensions: inattention/hyperactivity, aggressiveness, and conduct problems, producing 3 correlation maps (connectivity vs. behavior) for each region. For this phase the entire sample was included (n=283). Results from the categorical approach showed some connections specific to DBD and to ADHD. NAcc connections to posterior insula and to precuneus differed DBD from TD and from ADHD. The connection between amygdala and lingual gyrus differed ADHD from TDC and from DBD. ADHD also exhibited atypical amygdala connectivity with precentral gyrus and with inferior parietal lobule, compared to children with TD. There was no altered NAcc functional connectivity in children with ADHD or altered amygdala functional connectivity in children with DBD, compared to children with TD. The dimensional approach showed a different pattern of results. Inattention/hyperactivity and aggression scores were associated with NAcc connectivity to fusiform gyrus and dorsomedial prefrontal cortex. Inattention/hyperactivity was correlated with amygdala connectivity to inferior parietal lobule, middle temporal gyrus, and superior precentral sulcus. Aggression was related with amygdala connectivity to precuneus and superior frontal gyrus. Conduct problems were correlated with NAcc-superior frontal gyrus connectivity, and with amygdala connectivity to posterior cingulate cortex, precuneus, medial prefrontal cortex, and lingual gyrus. Results indicate, therefore, that there are NAcc and amygdala functional connections specifically associated with DBD or with ADHD, and that atypical behaviors common to both disorders are related to changes in functional connectivity of the NAcc and of the amygdala. In conclusion, the dimensional approach may complement the categorical approach in evaluating the neurobiology of DBD and of ADHD

Les problèmes de développement induits par les perturbateurs endocriniens (PE) sont actuellement peu étudiés, alors qu’une exposition précoce peut entraîner plus tard des problèmes permanents. Les lignées transgéniques de poisson zèbre (Danio rerio) avec une expression tissu-spécifique de la GFP sont des outils utiles pour identifier les organes affectés par un composé donné. Nous avons utilisé 7 lignées transgéniques pour visualiser in vivo si 6 PEs connus et 3 médicaments pouvaient avoir des effets sur le développement du poisson zèbre. Ce crible a révélé que 4 produits chimiques ont des effets sur 4 organes différents. Le tétrabromobisphénol-A, ainsi que le diclofénac, la trichostatine A et l'acide valproïque) perturbent le développement du système vasculaire. De plus, les inhibiteurs de HDAC trichostatine A et acide valproïque inhibent le développement du pancréas et induisent des retards de développement dans le foie et dans les dents pharyngiennes.La médecine chinoise traditionnelle (TCM) est un élément de la médecine moderne. Cependant, nous savons peu de choses sur les activités biologiques des composés TCM au cours du développement. Nous avons étudié les effets de 3 plantes et de 5 composés sur l'embryogenèse du poisson zèbre. Des extraits aqueux de Astragalus membranaceus et Akebia quinata provoquent des retards du développement. Nous avons aussi constaté que le développement vasculaire a été affecté à différents niveaux par Salvia miltiorrhiza et 3 de ses composants principaux, soit utilisés seuls ou mélangés entre eux.Nos résultats montrent que les PEs et les TCM peuvent causer des problèmes lors de l'embryogenèse. Ils montrent également que le poisson zèbre est un outil puissant pour le criblage rapide in vivo de petites molécules et de leurs effets sur le développement. Ce travail nous permet d'établir un parallèle entre EDC et TCMs, qui peuvent agir sur des cibles similaires, tels que les récepteurs nucléaires
Development problems induced by endocrine disruptors (EDCs) are currently understudied. However, early exposure to EDCs may lead to deleterious and permanent problems in later lifetime. Zebrafish (Danio rerio) transgenic lines with tissue-specific expression of GFP are useful tools to identify the organs affected by a given compound. We have used 7 transgenic lines to visualize in vivo whether 6 known EDCs and 3 other pharmaceuticals can alter organogenesis during development of zebrafish. This screen revealed that 4 chemicals have effects on 4 different organs. The EDC tetrabromobisphenol-A, as well as the tested medicines (diclofenac, trichostatin A and valproic acid) disrupt vascular system development in zebrafish embryo. Moreover, HDAC inhibitors trichostatin A and valproic acid inhibit both endocrine and exocrine pancreas development. Developmental delays were also induced by trichostatin A and valproic acid in the liver and in the pharyngeal teeth. Traditional Chinese medicines (TCMs) are important components of modern medicine. However we know little about the biological activities of TCMs compounds during development. We used zebrafish embryos to study the effects of 3 plants and 5 of their major compounds on the development. We observed that zebrafish embryogenesis was delayed by water extracts from Astragalus membranaceus and Akebia quinata. We also found that the vascular development was affected at different levels by Salvia miltiorrhiza water extracts and by its 3 major components either used alone or mixed together.Our results show that EDCs and TCMs can cause problems during zebrafish embryogenesis. They also show that zebrafish is a powerful tool for rapid in vivo screening of small molecules and their effects on development. This work also enables us to draw a parallel between EDC and some TCMS, which may act on similar targets, such as nuclear receptors

博士
國立陽明大學
微生物及免疫學研究所
95
NuMA is a nuclear matrix protein in interphase and distributes to the spindle poles during mitosis. While the essential role of NuMA for mitotic spindle assembly is well established, a structural role of NuMA in interphase nucleus has also been proposed. Several observations suggest that the apoptotic degradation of NuMA may relate to chromatin condensation and micronucleation. Here, we demonstrate that four apoptotic cleavage sites are clustered at a junction between the globular tail and the central coiled coils domains of NuMA. Cleavage of a caspase-6-sensitive site at D1705 produced the R-form, a major tail-less product of NuMA during apoptosis. The other two cleavage sites were defined at D1726 and D1747 that were catalyzed respectively by caspase-3 and an unknown aspartase. A NuMA deletion mutant missing the entire cleavage region of residues 1701-1828 resisted degradation and protected cells from nuclear disruption upon apoptotic attack. Under such conditions, cytochrome c was released from mitochondria, but the subsequent apoptotic events such as caspase-3 activation, poly(ADP-ribose) polymerase degradation, and DNA fragmentation were attenuated. Conversely, the tail-less NuMA alone, a mutant mimicking the R-form, induced chromatin condensation and activated the cell death machinery. Furthermore, overexpression of a C-terminal half of the central aa-helical coiled-coils of NuMA generated an abnormal phenotype of which chromatin was squeezed into the nuclear edge. It seems that this NuMA fragment may compete with the endogenous NuMA for binding the same sites on the nuclear matrix. Overexpression of the globular tail of NuMA resulted in the increase of M-phase populations or induced mitotic arrest. Therefore, functions of the globular tail of NuMA may be involved in mitotic events. In conclusion, NuMA plays a structural role in maintaining nuclear integrity in interphase and also regulates mitotic progression.