OBJETIVOS: Comparar a expressão placentária de células natural killer deciduais (dNK), e a expressão sérica e placentária de suas citocinas reguladoras em gestações gemelares com pré-eclâmpsia (grupo pré-eclâmpsia, GPE) e sem comorbidades (grupo-controle, GC). MÉTODOS: Estudo transversal do tipo caso-controle, desenvolvido na Clínica Obstétrica do HC-FMUSP no período de julho de 2015 a junho de 2017. Foram obtidas amostras das regiões deciduais placentárias, para avaliação, por meio de técnica de imuno-histoquímica, da expressão de células dNK e suas interleucinas (IL) 10, 12 e 15, em pacientes que contemplaram os critérios de inclusão e concordaram em participar do estudo. Além disso, estas pacientes tiveram amostra sérica colhida no terceiro trimestre para dosagem de IL-10, IL-12 e IL-15 - por meio de kit comercial Milliplex®, que utiliza a tecnologia Luminex® xMAP®, da EMDMillipore (Merck Millipore Co., Alemanha) - e de fatores relacionados à angiogênese, como soluble fms-like tyrosine kinase-1 (sFlt-1) e placental growth factor (PlGF) - por meio de ensaio com imunoanalisador COBAS e411 (Roche Diagnostics, Alemanha). Os valores obtidos para as análises placentárias e séricas foram comparados entre o GPE e o GC, e a significância estatística estabelecida foi p < 0,05. RESULTADOS: Foram selecionadas 30 pacientes, sendo 20 no GC e 10 no GPE. Não se observaram diferenças significativas com relação às características maternas, gestacionais e de desfechos perinatais entre os dois grupos, exceto pela idade gestacional de início do pré-natal, menor no GPE (12,5 vs. 20,0 semanas, p = 0,015). Quanto à avaliação placentária, houve maior expressão de IL-15 no GPE (p = 0,001), e não houve diferença entre os grupos quanto à expressão placentária local de células dNK (p = 0,999), IL-10 (p = 0,063) e IL-12 (p = 0,135). Com relação às interleucinas séricas maternas, demonstrou-se aumento significativo nos níveis de IL-10 (22,7 vs. 11,9 pg/mL, p = 0,024) e IL-15 (15,9 vs. 7,4 pg/mL, p = 0,024) no GPE com relação ao GC, sem diferença entre os grupos para IL-12 (102,5 vs. 61,5 pg/mL, p = 0,373). A dosagem dos fatores relacionados à angiogênese demonstrou maiores níveis séricos maternos de sFlt-1 (15920 vs. 7978 pg/mL, p = 0,009) e da razão sFlt-1/PlGF (88,71 vs. 24,63, p = 0,002), e menores valores de PlGF (193,0 vs. 340,6 pg/mL, p = 0,036) no GPE. CONCLUSÃO: Observou-se maior concentração sérica materna tanto de fatores pró quanto anti-inflamatórios no GPE, quando comparado ao GC. Entretanto, não foram observadas diferenças entre os grupos quanto à expressão placentária de IL-10, importante fator anti-inflamatório. Estes achados podem sugerir que a tentativa sérica materna de equilibrar estas interleucinas não alcançou resposta localmente na placenta, contribuindo para o desenvolvimento da doença no GPE / OBJECTIVES: To compare the placental expression of decidual natural killer cells (dNK) and serum and placental expression of their regulatory cytokines in twin pregnancies with preeclampsia (preeclampsia group, PEG) and uncomplicated twin pregnancies (control group, CG). METHODS: This was a case-control study, developed in a tertiary referral center, from July 2015 to June 2017. Samples of the placental decidual region were obtained and analyzed by immunohistochemistry technique for the expression of dNK cells and interleukins (IL) 10, 12 and 15, in patients who met the inclusion criteria. In addition, maternal serum sample was collected in the third trimester for the dosage of IL-10, IL-12 and IL-15 - by means of a commercial Milliplex® kit using Luminex® xMAP® technology from EMDMillipore (Merck Millipore Co., Germany) - and angiogenesis factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) - by COBAS e411 immunoassay (Roche Diagnostics, Germany). The values obtained for the placental analyzes and maternal circulating factors were compared between PEG and CG and the statistical significance was set at p < 0.05. RESULTS: Thirty patients were selected, 20 in CG and 10 in PEG. There were no significant differences in maternal, gestational and perinatal outcomes between the two groups, except for the gestational age at the onset of prenatal care, which was lower in PEG (12.5 vs. 20.0 weeks, p = 0.015). PEG showed strong immunostaining for IL-15 (p = 0.001) when compared to CG, with no difference between the groups concerning the placental expression of dNK (p = 0.999), IL-10 (p = 0.063), and IL -12 (p = 0.135). Relating to maternal circulating interleukins, a significant increase in IL-10 (22.7 vs. 11.9 pg/mL, p = 0.024) and IL-15 (15.9 vs. 7.4 pg/mL, p = 0.024) was observed in PEG, with no difference between the groups for IL-12 (102.5 vs 61.5 pg/mL, p = 0.373). We also demonstrated higher maternal levels of sFlt-1 (15920 vs. 7978 pg/mL, p = 0.009) and sFlt-1/PlGF ratio (88.71 vs. 24.63, p = 0.002) and lower levels of PlGF (193 vs. 340.6 pg/mL, p = 0.036) in PEG. CONCLUSION: A higher maternal serum concentration of both pro- and anti-inflammatory factors was observed in the PEG. However, no difference was found between the groups regarding the placental expression of IL-10, an important anti-inflammatory factor. These findings may suggest that the maternal serum attempt to balance these interleukins did not reach local placental response, which contribute to the development of the disease in the PEG

Au cours de la réponse immunitaire antitumorale, l’instabilité génétique des tumeurs combinée à la pression de sélection du système immunitaire peut conduire, via l’immunoediting, à l’émergence de variants tumoraux résistants à la lyse par les effecteurs cytotoxiques. Une meilleure compréhension de ces mécanismes potentiellement impliqués dans la susceptibilité tumorale à la lyse naturelle et/ou spécifique pourrait permettre le développement de stratégies d’immunothérapie intégratives plus efficaces. Dans ce cadre nous avons étudié un modèle de résistance à la lyse spécifique impliquant un remaniement du cytosquelette d’actine (i). Nous avons pu mettre en évidence que l’inhibition conjointe de protéines interagissant avec l’actine (caldesmone, ézrine, radixine et moésine) générait une réduction de la susceptibilité des cellules tumorales à la lyse par les lymphocytes T cytotoxiques (CTLs). Parallèlement, nous avons identifié les microARNs différentiellement exprimés entre le variant résistant et la lignée parentale et étudié leur implication dans la susceptibilité tumorale à la lyse par les CTLs. Dans le but de déterminer le rôle d’une pression de sélection par les cellules tueuses naturelles (NK pour Natural Killer), de l’immunité innée, sur les cellules tumorales et l’émergence de variants résistants, nous avons aussi établi un modèle de coculture continue de cellules tumorales de mélanomes avec des cellules NK (ii). Les cellules tumorales obtenues sont résistantes à la lyse NK (mais toujours sensibles à la lyse spécifique par un clone lymphocytaire T cytotoxique) et établissent moins de contact et de synapse immunologique avec les cellules NK que la lignée parentale. L’analyse transcriptomique a révélé la baisse d'expression de B7-H6 (ligand d'un récepteur activateur des cellules NK) qui contribue partiellement au phénomène de résistance. De nombreux gènes impliqués dans les phénomènes de migration/invasion/adhérence sont également modulés et certaines propriétés cellulaires (croissance en milieu semi-solide, adhérence, migration) semblent refléter l’acquisition d’une agressivité tumorale accrue suite à la coculture. Nous avons finalement analysé l’impact sur la réponse antitumorale de la connexine-43, impliquée dans la formation des jonctions communicantes (GJ pour Gap Junction) (iii). Nous avons montré que sa présence à la synapse entre cellules tumorales et CTL n'exerce aucun impact sur la susceptibilité à la lyse. Néanmoins, les GJs sont impliquées dans l’émergence par stimulation antigénique de lymphocyte T CD8+ spécifique hautement réactif. / During antitumor immune response, cancer cells genetic instability combined with immune system selective pressure may drive to the emergence of tumor variant resistant to lysis by cytotoxic effector cells through a phenomenon called immunoediting. A better understanding of those mechanisms putatively involved in tumor susceptibility to natural and/or specific lysis would enable new integrative and more effective immunotherapeutic strategies. In this context, we studied a model of resistance to specific lysis linked to actin cytoskeleton remodeling (i). We showed that combined inhibition of actin interacting protein (caldesmone, ezrin, radixin and moesin) reduced tumor cells susceptibility to cytotoxic T lymphocytes (CTLs) lysis. Moreover, we identified microRNAs differentially expressed between parental cell line and resistant variant and are currently studying their impact on tumor susceptibility to CTLs lysis. In order to depict the role of innate immunity Natural Killer (NK) cells selective pressure, on tumor cells and on the emergence of resistant variants, we also established a maintained coculture model of melanoma cells with NK cells (ii). Selected cells obtained were resistant to NK cells-mediated lysis (but still susceptible to CTLs-mediated specific lysis) and formed less contact and immune synapse with NK cells than parental cell line. Transcriptomic analysis revealed the reduced expression of B7-H6 (ligand of an NK cells activating receptor) partially contributing to the resistance phenotype. The expression of several genes involved in migration/invasion/adhesion is also modulated and some cell characteristics (cell growth in semi-solid medium, adhesion, migration) tend to reflect the acquisition through coculture of an increased aggressiveness. Finally, we evaluated the impact of connexin-43 (Cx43), involved in the establishment of Gap Junctions (GJs), on antitumor response (iii). We showed that despite localization at the immune synapse between tumor target cell and CTL, Cx43 and GJs do not modulate susceptibility to CTL-mediated specific lysis. Nevertheless, GJs contribute to the emergence of highly reactive specific CD8+ T lymphocytes following antigen stimulation.

De nombreux prédateurs marins se nourrissent directement des prises des pêcheurs. Ces interactions, définies comme de la déprédation, engendrent des conséquences socio-économiques considérables pour les pêcheurs ainsi que des implications de conservation pour la faune sauvage. D’un côté, la déprédation endommage le matériel et augmente l’effort de pêche pour atteindre les quotas. D’un autre côté, la déprédation augmente le risque de mortalité des prédateurs marins (prise accidentelle ou rétorsion létale par les pécheurs). La pêcherie à la palangre est la plus impactée par la déprédation, principalement par les odontocètes, ce qui incite à trouver des solutions. La majorité des études se concentrant sur la déprédation s’est principalement basée sur des observations en surface, de ce fait la manière dont les prédateurs retirent les poissons sur les lignes reste confuse. Par ailleurs, l’impact de la déprédation sur le comportement des pêcheurs ainsi que les facteurs expliquant leur détectabilité n’ont reçu que peu d’intérêt. L’objectif de cette thèse est donc d’étudier ces problématiques par un suivi acoustique, une utilisation de balises et une approche en écologie comportementale humaine, en se concentrant sur la pêcherie palangrière française ciblant la légine australe (Dissostichus eleginoides) impactée par la déprédation des orques (Orcinus orca) et des cachalots (Physeter macrocephalus). Les capitaines ont été décrits comme recherchant leur ressource selon la théorie de « l’optimal foraging », mais avec des perceptions de la compétition et du succès de pêche qui divergent. Certains capitaines seraient ainsi plus enclins à remonter les palangres au plus proche et à rester sur une zone, même en présence de compétition, augmentant alors le risque d’interaction. L’acoustique des navires a révélé que certaines manoeuvres (marche arrière par exemple) propagent différemment sous l’eau. La manière dont les capitaines manoeuvrent leur palangrier influencerait ainsi leur détectabilité et donc leur risque d’interaction avec les prédateurs. D’autre part, l’utilisation de capteurs sur les palangres et les animaux a révélé que les orques et les cachalots sont capables de déprédater sur les palangres posées sur le fond marin. Ces observations laissent à penser que les odontocètes sont en mesure de localiser l’activité de pêche bien avant la remontée de la ligne, ce qui pourrait être expliqué par une signature acoustique spécifique du déploiement de la ligne. L’ensemble des résultats de cette thèse suggère que la déprédation sur les palangres démersales est très probablement sous-estimée. Cette thèse apporte également des éléments importants pour la lutte contre la déprédation, en montrant la nécessité de protéger les palangres dans l’intégralité du processus de pêche. / Many marine predator species feed on fish caught by fishers directly from the fishing gear. Known as depredation this interaction issue has substantial socio-economic consequences for fishermen and conservation implications for the wildlife. Costs for fishers include damages to the fishing gear and increased fishing effort to complete quotas. For marine predators, depredation increases risks of mortality (lethal retaliation from fishers or bycatch on the gear). Longline fisheries are the most impacted worldwide, primarily by odontocetes (toothed whales) depredation, urging the need for mitigation solutions to be developed. Most of studies assessing depredation have primarily relied on surface observation data, thus the way odontocetes interact with longlines underwater remains unclear. Besides, the way fishermen respond to depredation during fishing operations, or can influence their detectability to odontocetes, have been poorly investigated. This thesis therefore aimed at investigating these aspects through a passive acoustic monitoring, bio-logging and human ecology approaches, focusing on the French Patagonian toothfish (Dissostichus eleginoides) longline fisheries impacted by killer whales (Orcinus orca) and sperm whales (Physeter macrocephalus). Firstly, this thesis reveals that captains behave as optimal foragers but with different personal perception of competition and fishing fulfilment. Some captains would thus be more likely to stay within a patch or to haul closest longline even in presence of competition, suggesting these captains would show higher interaction rates. Additionally, the propagation of vessels’ acoustics varied depending on the type of manoeuvre (e.g. going backward vs. forward). The way captains use their vessels to navigate may therefore influence their detectability and so their depredation level. Secondly, loggers deployed on both the longlines (accelerometers) and odontocetes (GPS-TDR) revealed that killer whales and sperm whales are able to depredate on longlines while soaking on the seafloor. These observations suggest, therefore, that odontocetes can localise fishing activity before the hauling, which could be partially explained by specific acoustic signatures recorded during the setting process. Altogether, the results of the thesis suggest that depredation rates on demersal longlines are most likely underestimated. The thesis also brings some important insights for mitigation measures, suggesting that countermeasures should start from setting to hauling.

The killer whales that roam the northeastern Pacific Ocean have been the objects of studies since the 1970s, making them the most well-studied population of orcas in the world. Three distinct ecotypes of killer whales (Orcinus orca), known as residents, transients, and offshores, share these waters. The ecotypes are morphologically and behaviorally distinct to the extent that some scientists consider them separate species, with residents eating salmon, transients specializing on marine mammals, and offshores preferring Pacific sleeper sharks and Pacific halibut. Resident populations have endeared themselves to the region's locals with their striking black and white markings and their tendency to frolic in waters near the shore. However, both of the two resident populations on the coast of British Columbia and Washington State are at risk, with northern residents numbering some 300 and southern residents at just 74 individuals as of December 2018. Three deaths in the span of four months in spring and summer of 2018 brought widespread attention to the southern residents' plight. Live captures of killer whales for aquaria heavily impacted the population in the 1960s and 1970s, and today they face a combination of prey shortages, pollution, and disturbance from vessel traffic. If southern resident killer whales are to persist, federal, local, and state agencies need to quickly take mitigative action.

When Marineland of the Pacific opened in 1954 on the Palos Verdes Peninsula in greater Los Angeles, it was the second oceanarium in the world and the first on the West Coast. An initial investment of $3 million by Oceanarium Inc., owners of the popular Marine Studios park located near St. Augustine, Florida, ensured that Marineland was built with the same state of the art facilities needed to produce an authentic representation of the ocean floor on land. Building on Marine Studios' success exhibiting bottlenose dolphins (Tursiops truncatus), Marineland's central draw was its performing cetaceans. During the park's early years, its collectors pioneered the capture of Pacific dolphin species, such as the Common dolphin (Delphinus delphis) and the Pacific white-sided dolphin (Lagenorhynchus obliquidens), the short-finned pilot whale (Globicephala macrorhynchus), and were the first to capture a live killer whale (Orcinus orca) in 1961. By exposing audiences to previously unknown species through circus-like performances, Marineland played a central role in changing public perceptions of small cetaceans in the post-World War II era. However, with few prior studies to consult, Marineland curators experimented with their own methods of capture, husbandry, and veterinary care that often resulted in the harm or death of cetaceans under their care. Caretakers contended with animal aggression and sexual behavior, the refusal of animals to perform in show routines, and high mortality. Despite the difficulties posed by exhibiting cetaceans, advertisements, press interviews, and films advanced a contrary narrative that animals under Marineland's care enjoyed the conditions of captivity and performing for an audience. This thesis explores the tension between entertainment and animal care that defined the early years of cetacean captivity in North America.

Dexter Morgan is a serial killer, but he may not be blameworthy for his actions There are two possible explanations that could absolve Dexter of moral responsibility: (1) His inability to empathize with others makes it so that he cannot make appropriate moral decisions. Or (2) his serial killing tendencies are determined in nature, set off by events of which he had no control. I conclude that Dexter is, in fact, morally responsible for his actions because he is capable of second order desires

Some yeast strains under certain conditions secrete polypeptide toxins which are inhibitory to sensitive fungal cells into the medium. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer proteins are classified into 11 typical types (K1-K11). These toxins have different killing mechanisms on sensitive cells. Some of them hydrolyze major cell wall component, beta-1,3- glucans. As mammalian cells lack cell walls research and development of novel highly selective antifungals are mostly focused on the agents which target the components of the fungal cell wall. K5 type killer protein was characterized in our labarotory previously. This protein is an exo beta-1,3-glucanase which is stable at pH&amp / #8217 / s and temperatures appropriate for its biocontrol usage. Beta-1,3-glucan hydrolyzing activity of the K5 type killer protein highlighted the potential use of this protein as a selective antifungal agent. According to CLSI methodology, antifungal activity of the K5 type yeast killer protein was tested against 6 fungal strains causing postharvest spoilage in citrus fruits and found to be effective on Botrytis cinerea, Penicillium digitatum, Penicillium italicum whereas non effective on Colletotrichum gloeosporoides, Phythophythora citrophthora, Alternaria citri. The MIC values of the toxin for B.cinerea, P.digitatum, P.italicum were found to be 16 mikrogram/ml while IC 50 values of the toxin were 2.12, 3.31, 2.57 mikrogram/ml respectively. The results showed that K5 type yeast killer protein would be used as a novel and selective agent against B.cinerea, P.digitatum and P.italicum.

Les fièvres hémorragiques à virus Lassa (LASV) et Ebola (EBOV) représentent un important problème de santé publique en Afrique. Les réponses immunes et la pathogenèse associées à ces maladies sont peu connues. Les cellules NK ont un rôle important dans la réponse immune innée par leurs propriétés cytotoxiques, mais également dans l'induction des réponses adaptatives par leur production de cytokines et leurs interactions avec les cellules dendritiques (DC) et les macrophages. Ce projet s'attache à comprendre le rôle des cellules NK dans le contrôle de la réplication virale et dans l'induction des réponses immunitaires au cours de ces infections. Un modèle in vitro de coculture de cellules NK humaines avec des DC et macrophages autologues a été développé. L'activation des cellules NK et leurs fonctions ont été analysées après l'infection par LASV et EBOV. Par ailleurs, les réponses des cellules NK en réponse à LASV ont été comparées avec celles induites lors de l'infection par le virus Mopeia (MOPV), très proche de LASV mais non pathogène pour l'homme. Les macrophages, mais pas les DC, infectés par LASV ou MOPV induisent l'activation et l'augmentation des capacités cytotoxiques des cellules NK. Toutefois, les cellules NK ne sont pas capables de lyser les cellules infectées et ne produisent pas d'IFN-γ. Les cellules NK s'activent et sont capables de lyser les cellules infectées en présence de macrophages mais également de DC infectés par des LASV mutants. Cependant, les IFN de type I sécrétés en grande quantité en réponse à ces virus ne sont pas impliqués dans l'activation des cellules NK. L'infection par EBOV n'induit qu'une très faible activation des cellules NK en présence de DC ou macrophages et ne conduit pas à la sécrétion de cytokines, ni à la modification du potentiel cytotoxique.Ces résultats permettent d'améliorer la compréhension des réponses immunes et des mécanismes de pathogenèse mis en place lors des fièvres hémorragiques Lassa et Ebola.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production, which can eventually lead to immune complex (IC)-mediated organ damage. Due to the stimulation of plasmacytoid dendritic cells (pDC) by nucleic acid-containing ICs (DNA- or RNA-IC), patients with SLE have an ongoing interferon (IFN)-α production. IFN-α induces a general activation of the immune system that may initiate or propagate an autoimmune process if not properly regulated. Previous studies have shown that natural killer (NK) cells potently enhance the IFN-α production by pDCs. In study I, the mechanisms behind the NK cell-mediated increased IFN-α production by RNA-IC-stimulated pDCs were investigated. ICs triggered CD56dim NK cells via FcγRIIIA to the secretion of cytokines (e.g. MIP-1β) that promoted IFN-α production. Additionally, an LFA-1-dependent cell-cell interaction between pDCs and NK cells strongly contributed to the increased production of IFN-α. In study II, the RNA-IC-induced regulation of surface molecules on pDCs and NK cells was investigated. The expression of CD319 and CD229, which are two SLAM family receptors genetically associated with SLE, was induced on pDCs and NK cells by RNA-IC. IFN-α-producing pDCs displayed an increased expression of CD319 and CD229, whereas pDCs from patients with SLE had a decreased expression of CD319. In study III, we serendipitously identified an SLE patient harboring autoantibodies to the NK cell receptor CD94/NKG2A. In study IV, sera from 203 patients with SLE were analyzed for autoantibodies to the CD94/NKG2A, CD94/NKG2C and NKG2D receptors. Seven patients harbored anti-CD94/NKG2A autoantibodies, and two of these patient’s autoantibodies also reacted with CD94/NKG2C. Anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies both interfered with the HLA-E-mediated regulation of NK cell cytotoxicity, and facilitated the elimination of target cells expressing these receptors. Furthermore, these autoantibodies were found in a group of severely diseased SLE patients and their titers closely followed disease activity. In conclusion, this thesis provides insights to molecular mechanisms whereby NK cells regulate the IFN-α production, it further links the SLAM receptors to SLE, and it describes novel autoantibodies to receptors regulating NK cell cytotoxicity. Together these findings strengthen the assumption that NK cells are involved in the pathogenesis of SLE.

La kinase Aurora B appartient au complexe des protéines passagères. Ce complexe est impliqué dans la régulation de la condensation, constriction et ségrégation des chromosomes, ainsi que dans la cytokinèse. Son rôle est donc crucial pour prévenir la formation de cellules cancéreuses. Cependant, l'étude de la fonction précise d'Aurora B dans chacune des phases de la mitose est limitée par la durée de celle-ci, et par le manque de spécificité des inhibiteurs existants. Nous avons donc développé une stratégie basée sur la photo-inactivation de la kinase par le chromophore Killer-Red, fusionné à la protéine. L'émission locale de ROS après irradiation, permet alors la photo-inactivation spécifique et temporelle d'Aurora B. La photo-inactivation d'Aurora B avant anaphase aboutie soit à un arrêt de la mitose, soit à la régression du sillon de division, provoquée par l'entrée en anaphase en présence de chromosomes retardés. La photo-inactivation d'Aurora B en début d'anaphase a pour conséquence la régression du sillon de division en cytokinèse ; apportant la première indication directe de l'implication d'Aurora B dans le fuseau mitotique en cytodiérèse. De façon surprenante, la photo-inactivation de la kinase au niveau du corps résiduel, après constriction du sillon de division, n'affecte pas l'abscission. La photo-inactivation d'Aurora B n'affecte pas la localisation des autres membres du complexe des protéines passagères, indiquant que la kinase n'est pas impliquée dans la dynamique du complexe. Les résultats obtenus montrent sans aucun doute l'implication d'Aurora B dans chacune des phases de la mitose, suggérant que la phosphorylation par Aurora B de ses substrats permet le controle de la division cellulaire.