Em muitos mamíferos, é notado um influxo de linfócitos no início da gestação, que é acompanhado por um processo de proliferação, diferenciação e migração destas células. Em camundongos estes linfócitos se distribuírem na região mesometrial de cada sítio de implantação (SIE). Estudos imunocitoquímicos indicaram que estes linfócitos são células Natural Killer, porém, sua morfologia e comportamento peculiar sugerem que elas representam uma subpopulação de NK específica do útero durante a gestação, sendo portanto denominadas células Natural Killer uterinas (uNK). A função conhecida para estas células em camundongos é a liberação de citocinas como IFN- que leva à dilatação das artérias espiraladas deciduais e manutenção da integridade decidual, contribuindo, portanto, para o desenvolvimento normal da decídua e da placenta. A infecção intra-uterina foi reconhecida como a causa primária de partos prematuros e corrobora para o desenvolvimento de leucomalácia periventricular e displasia broncopulmonar. Estudo recente em camundongos prenhes demonstrou que a injeção intra-uterina de LPS de E.coli provoca aumento da expressão de Fosfodiesterase 4B (PDE4B) e da atividade fosfodiestrase na interface materno-fetal, aumento nas concentrações de TNF-α, IL-1β, IL-6 e IL-10 no líquido amniótico o que induz o parto prematuro e a diminuição da viabilidade fetal. É esperado que a administração de LPS promova o aumento sistêmico e central de citocinas próinflamatórias, e que o animal responda com alterações endócrinas, imunológicas e comportamentais com o fito de favorecer o restabelecimento da homeostasia. Nesse contexto, o presente trabalho buscou avaliar os efeitos comportamentais da administração de LPS em camundongos, bem como os efeitos no útero prenhe e nas células uNK desses animais por meio do estudo morfológico, citoquímico e estereológico. O estabelecimento do comportamento febril foi identificado a partir da segunda hora após a administração de LPS, e esse evento ocorreu concomitante ao estabelecimento do comportamento análogo à ansiedade. Foi possível identificar nos animais tratados o comportamento de apatia e redução na capacidade exploratória, que somados ao comportamento depressivo, estabelecido após 1 hora da aplicação do LPS, fazem parte das alterações que constituem o comportamento doentio. O tratamento provocou também alterações no ambiente uterino, de tal forma que foi possível identificar um significativo aumento de células uNK imaturas na região do miométrio dos SIE. Além disso, o tratamento levou à diminuição da quantidade de células uNK maduras e senescentes nas três regiões estudadas dos SIE concomitante ao aumento de células uNK que possuíam padrão alterado na expressão de N-actilgalactosamina nos seus grânulos e superfície. O período onde foram encontradas tais alterações acompanhou o período onde houve perda na marcação de perforina, sugerindo a ocorrência de degranulação. A imunocitoquímica demonstrou que o tratamento com LPS parece ter induzido a despolimerização dos filamentos contrateis de α-actina causando relaxamento nos vasos que irrigam o ambiente uterino. / In mammalian, there is a lymphocyte influx in early pregnancy, which is accompanied by proliferation, differentiation and migration of these cells. These lynphocites distribute in mesometrial region of each embryo implantation site (EIS). Immunocytochemical studies indicated that these cells are Natural Killer cells, but their peculiar morphology and behavior suggest that they represent a uterus specific NK subpopulation during pregnancy called uterine Natural Killer cells (uNK). The known function for these cells in mice is cytokines releasing such as IFN- leading to decidual spiral arteries dilation and maintaining decidual integrity, contributing to normal development of decidua and placenta. Intrauterine infection was recognized as primary cause of preterm labor and supports the periventricular leukomalacia and bronchopulmonary dysplasia development. Recent study in pregnant mice showed that E.coli LPS intra-uterine injection causes increased expression of Phosphodiesterase 4B (PDE4B) and fosfodiestrase activity in maternal-fetal interface, increased TNF-α, IL-1β, IL-6 and IL-10 concentrations in amniotic fluid which leads to premature delivery and reduced fetal viability. It is expected that LPS administration promotes systemic and central proinflammatory cytokines increasing, and that the animal responds with endocrine, immune and behavioral disorders with the aim of promoting homeostasis restoration. In this context, the present study evaluated the behavioral effects of LPS administration in mice and the effects on pregnant uterus and uNK cells of these animals through morphological, cytochemical and stereological study. The feverish behavior establishment was identified from second hour after LPS administration, and this event was concomitant with anxiety-like behavior establishment. It was possible to identify treated animals apathy behavior, reduction in exploratory capacity and depressive behavior drawn 1 hour after LPS application as part of sickness behavior. The treatment also caused changes in the intrauterine environment, so that we could identify a significant increase of immature uNK cells in myometrium region of EIS. Furthermore, the treatment decreased the amount of mature and senescent uNK cells in the three EIS studied regions, concomitantly with the increasing granules and surface N-actilegalactosamine expression altered uNK cells. These changes were accompanied by the perforin labeling loss and a reduction on blood vessel α-actin labeling, suggesting respectively the uNK cell citotoxicity (perforin releasing) and actin contractile filament despolymerization causing relaxation of blood vessels that supplies the decidua and embryo. / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

Uma das populações de células imunológicas mais abundantes no útero durante a gestação é a das células Natural Killer uterinas (uNK) que, apesar de apresentarem grânulos citoplasmáticos ricos em perforina e granzimas, não apresentam atividade citotóxica durante uma gestação normal. As células uNK são importantes produtoras de fatores angiogênicos que promovem a dilatação e o remodelamento das artérias uterinas resultando no aumento do fluxo sanguíneo para a unidade feto-placentária em desenvolvimento. A administração sistêmica de Lipopolissacarídeo (LPS) de E. coli em camundongos pode desencadear a ativação de cicloxigenase-2 (COX-2) levando a um aumento nos níveis de prostaglandinas bem como um desbalanço das citocinas pró-inflamatórias e anti-inflamatórias no útero gestante alterando a atividade normal das células imunológicas refletindo em uma alteração na homeostase uterina. O presente trabalho teve como o objetivo avaliar se a administração do inibidor seletivo de COX-2 pode interferir nos efeitos do LPS no útero gestante, nos vasos sanguíneos deciduais, nas células uNK e na quantidade e qualidade da prole; contribuindo assim para a elucidação dos mecanismos da SRIS durante a gestação. A administração de LPS causou excessivo influxo de células uNK imaturas no útero gravídico, além de causar diminuição do número de células uNK maduras concomitante ao aumento de células uNK alteradas. Tais efeitos do LPS foram prevenidos pelo pré-tratamento com Nimesulida. A inibição da COX-2 também impediu a liberação de perforina por essas células bem como a diminuição da expressão de α-actina em vasos sanguíneos uterinos causados pelo LPS. O número de filhotes nascidos de mães tratadas com nimesulida+LPS no período mediano da gestação foi ainda menor do que o número de filhotes que nasceram das mães que receberam apenas LPS no mesmo dia de gestação. A injeção apenas de nimesulida não causou nenhum efeito na prole, levando-nos a acreditar que as alterações fisiológicas após a inflamação induzida por LPS são de fundamental importância para o restabelecimento da homeostase do útero, a fim de manter a gestação a termo. Fator de crescimento placentário (PGF) é um fator de crescimento angiogênico pleiotrópico que se encontra elevado durante a gravidez. Embora os padrões normais e anormais de PGF plasmático materno já estejam descritos na literatura, o papel preciso do PGF na angiogênese útero-placentária são indefinidos. Muitas mulheres que expressam baixos níveis de PGF, sobretudo na metade da gravidez, apresentam progressão para síndromes hipertensivas como a pré-eclâmpsia. Esse tipo de complicação gestacional está relacionada com o aumento da incidência de doenças cardiovasculares (CV) pós-parto tanto para mães quanto para os filhos, entretanto, não se sabe se baixos níveis de PGF aumentam o risco de doenças CV. Para avaliar a hipótese de que a deficiência de PGF limita a adaptação CV durante o período gestacional tardio contribuindo para o aumento do risco de complicações cardiovasculares, foram analisadas a estrutura e função do sistema cardiovascular em camundongos C57BL/6 (Pgf^+/+) e Pgf^-/- no período gestacional. Além disso, estruturas vasculares cerebrais foram analisadas pela técnica de moldagem vascular por perfusão com polímero e os animais dos dois grupos experimentais foram submetidos a testes comportamentais. A avaliação hemodinâmica por radiotelemetria mostrou que as mães, no cruzamento Pgf^-/- x Pgf^-/-, tiveram um declínio maior e mais sustentado na pressão arterial média em meados de gravidez do que as mães do grupo controle, mas a recuperação após a implantação do transmissor era rara. A avaliação hemodinâmica por pletismografia de cauda mostrou que os animais Pgf^-/- apresentaram maior pressão arterial (PA) no início e final da gestação com um maior intervalo de queda da PA após o período da implantação do blastocisto. Menor débito cardíaco materno e volume sistólico foram observados após o meio da gestação dos animais knockout quando comparados com animais controle. Além disso, esses animais não apresentaram o aumento no peso do ventrículo esquerdo (VE) naturalmente observado em Pgf^+/+ no final da gestação. A expressão gênica de Nos3 e Nos2 no VE é maior nos animais Pgf^-/- durante o período mediano da gestação. Foram observadas anomalias estruturais glomerulares e hipertrofia renal em camundongos Pgf^-/- não gestantes, durante a gestação e pós-parto. Assim, a deficiência de PGF limita adaptações fisiológicas e estruturais do sistema CV e Renal durante a gestação de camundongos. Estes resultados podem ter paralelos na gravidez humana e se relacionam com o aumento do risco de complicações CV pós-parto, que são sequelas de gestações de pré-eclâmpsia. A técnica de moldagem vascular com polímero revelou que a estrutura vascular central do cérebro da prole Pgf^-/- é deficiente, e testes comportamentais mostraram que isso tem reflexo em déficit cognitivo. Assim, a deficiência gestacional PGF também prejudica a regulação da pressão arterial materna e desenvolvimento vascular em órgãos essenciais da progênie como o cérebro. / One of the most abundant immunologic cell types in early decidua is the uterine Natural Killer (uNK) cell that despite the presence of cytoplasmic granules rich in perforin and granzymes does not degranulate in normal pregnancy. UNK cells are important producers of angiogenic factors that permit normal dilation of uterine arteries to provide increased blood flow for the growing feto-placental unit. Gram-negative bacteria Lipopolysaccharide (LPS) administration can trigger cyclooxygenase-2 (COX-2) activation leading to an increase in prostaglandin level and an imbalance of pro-inflammatory and anti-inflammatory cytokines impairing the normal immune cells activity as well as uterine homeostasis. The present study aimed to evaluate if COX-2 inhibitor can alter the effect of LPS on the pregnant uterus, on blood vessels of the decidua, on uNK cells and on litter size; thus contributing to the elucidation of Systemic Inflammatory Syndrome mechanisms during pregnancy. The LPS administration caused excessive immature uNK cells influx in pregnant uterus as well as a decrease in mature uNK cells number concomitantly with altered uNK cells increasing. Such LPS effects could be prevented by the nimesulide pretreatment. The COX-2 inhibition has also prevented perforin releasing by these cells as well as the α-actin down-regulation in uterine blood vessels caused by LPS. The number of pups born from mothers treated with Nimesulide + LPS in mid-pregnancy was smaller than the number of pups born of mothers who received only LPS on the same gestation day. The injection of nimesulide itself showed no effect in the offspring, leading us to believe that the physiological changes after inflammation induced by LPS are of fundamental importance for the restoration of uterine homeostasis in order to maintain pregnancy to term. / Placental growth factor (PGF) is a pleiotropic angiogenic growth factor elevated during pregnancy. Although normal and abnormal patterns of maternal plasma PGF are well known, the precise roles of PGF in uteroplacental angiogenesis, blood pressure regulation, vasculogenesis, as well as cardiovascular (CV) and renal adaptation are undefined. Many women who express low levels of PGF over mid-pregnancy, progress to the acute, emergency, hypertensive syndrome of preeclampsia (PE). This kind of gestational complication elevate postpartum cardiovascular disease risks for both mothers and offspring. In many pre-eclamptic women, plasma placental growth factor concentrations are lower than normal, however, it is unknown if lower PGF elevates postpartum CV risk. We hypothesized that PGF deficiency limits maternal CV adaptations during mid-late pregnancy and contributes to postpartum maternal CV risk elevation. Cardiovascular function and structure were evaluated in Pgf^-/- and C57BL/6 (B6/Pgf^+/+) mice across pregnancy and postpartum. Furthermore, cerebral vascular structures were analyzed by polymer vascular casting technique and animals from both experimental groups were subjected to behavioral testing. Hemodynamic assessment by radiotelemetry showed Pgf^-/- x Pgf^-/- dams had a larger and more sustained mid-pregnancy decline in mean arterial pressure than controls but recovery post-transmitter implantation was rare. Hemodynamic assessment by tail cuff plethysmography showed that Pgf^-/- mice had higher blood pressure in early and late pregnancy with a longer interval of depressed systolic pressure after implantation. Lower maternal cardiac output and stroke volume were present after mid-gestation versus Pgf^+/+. Pgf^-/- left ventricular (LV) mass was less than in Pgf^+/+ and expected gestational increases in LV mass were observed only in Pgf^ +/+. Nos3 and Nos2 expression were elevated in midgestation Pgf^-/- LV. Structural anomalies were present in virgin Pgf^-/- glomeruli and renal hypertrophy was associated with gestation and postpartum. Thus, PGF deficiency limits physiological and structural adaptations of the mouse maternal CV and renal systems over late pregnancy. These results may have parallels in human pregnancy and relate to the postpartum gains in CV risk that are sequelae to pre-eclamptic gestations. Polymer vascular casting of offspring brains and behavioral testing revealed aberrant central vascular structure and major cognitive deficits in Pgf^-/-. Thus, gestational PGF deficiency additionally impairs regulation of maternal arterial pressure and vascular development in key organs of progeny such as the brain. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

Type 2 (Th2) immune responses are required for immune defence against helminths, but can also have pathogenic effects in allergic conditions. This thesis examined two factors which may influence Th2 immunity at a cellular and molecular level: cross-talk between Natural Killer (NK) cells and dendritic cells (DCs) and the cell surface organisation of DCs. Cross-talk between NK cells and DCs is well-established to impact Th1 responses against tumours and infection; however the influence of this interaction during Th2 inflammation is unknown. To investigate this, human monocyte-derived DCs were stimulated in vitro with different pathogen-associated molecules; LPS or Poly(I:C) which polarise a Th1 response, or soluble egg antigen (SEA) from the helminth worm Schistosoma mansoni, a potent Th2-inducing antigen. These cells were then combined with autologous NK cells. Confocal microscopy showed polarisation of the NK cell microtubule organising centre (MTOC) and accumulation of LFA-1 at contacts between NK cells and immature or Th2-polarising DCs, but not Th1-polarising DCs, indicative of the assembly of an activating immune synapse. NK cells did not lyse DCs treated with LPS or Poly(I:C), but degranulated to and lysed both immature DCs and Th2 polarising DCs. Antibody blockade of NK cell activating receptors NKp30 and DNAM-1 prevented this lysis. Furthermore, depletion of NK cells in mice which were then transferred with Th2 polarising DCs led to an enhanced Th2 recall response. Thus, these data indicate a previously unrecognised role of NK cell cytotoxicity in restricting the pool of DCs involved in Th2 immune responses. Secondly, this thesis investigated the nanoscale organisation of MHC-II on the surface of Th1 and Th2 polarising DCs using ground state depletion super-resolution microscopy. MHC-II was relatively homogenously distributed across the membrane with no significant changes in clustering between immature, Th1 and Th2 polarising DCs. In contrast, imaging CD74, which can mediate internalisation of MHC-II, revealed increased expression and a more homogenous distribution of this receptor on the surface of Th2-polarising DCs compared to Th1-polarising DCs. These data suggest that changes in the clustering of CD74 could modulate MHC-II surface expression during Th2 responses. Overall, the results in this thesis indicate that both molecular and cellular level modulation of DC function contribute to the development of Th2 responses.

The media is one of the strongest influences on how society views the criminal justice system and all actors therein. This is especially true for offenders of violent crime. Notably, women who kill are rare. However, when women do murder someone, the media tends to over expose them and portray them in different ways. The current study is intended to examine how the media portrays women murderers. In particular, this research is focused on how fictional and true crime programs portray female killers. Do they portray them in a positive or negative light? Do they portray them realistically? Are true crime shows more realistic than fictional crime shows? Each of these questions was explored and it was found that true crime programs, even though not wholly realistic, do portray women much more realistically than fictional shows. It is important to study these portrayals in order to understand how women killers are portrayed, how society views and interprets these particular criminals, and what are the steps necessary in order to prevent and change the way media process this crime.

Marine mammals are in a precarious conservation position because of anthropogenic impacts and historic perceptions that they are a consumable commodity. In light of changing abiotic conditions, further evaluation is needed on the habitat use, behavior, and interactions among marine mammals. Conservation legislation has helped protect species, but the greatest ground swelling may be the advent of the commercial whale watching industry. The feeding grounds in Alaskan waters have made this area a prime tourism location, and these nutrient-rich waters have resulted in a confluence of marine mammal species, including the appealing and abundant humpback whale (Megaptera novaeangliae) that may associate with three ecotypes of killer whales (Orcinus orca). These species are interesting because they may travel together to feed on prey or be adversaries in a predator-prey relationship. Using whale watching as a platform, this study evaluated the effects of the presence of these two species separately and together, and of the type of interaction between them, on human perception. Data were collected via opportunistic observations and a retrospective pre- and post-survey instrument. Differences in humpback whale distribution and group size patterns were found relative to killer whale occurrence, although humpback whale behavioral states were unchanged. Changes in passenger conservation attitudes could not be attributed to species and behaviors but they were important determinates to whale watching satisfaction. Overall, more positive conservation attitudes and an increase in knowledge about marine mammals were reported after whale watching. These tours provide an opportunity for collecting meaningful scientific data and providing more in-depth education such as enhancing the appreciation for ecosystem services provided by marine mammals.

The purpose of this study was to investigate natural killer cell activity and the possible role of beta- endorphin in a natural model of autoimmune orchitis, the dark mink. An assay was developed to study natural killer cell activity in the mink and base-line levels of activity in this specie were determined. Natural killer cell activity was assessed in fertile mutation mink, primary infertile dark mink and secondary infertile Utah dark mink with autoimmune orchitis. The study included three sampling times: November, March and April. Natural killer cell activity was significantly lower in mink studied in April than it was in. mink studied in March or November, and there was a significant correlation of activity to fertility. The addition of beta-endorphin to natural killer cell cultures had no effect on activity. A preliminary study was done to determine concentration levels of beta-endorphin in mink plasma, pituitary, hypothalamus and testes. Measurable amounts were found in all tissue types studied. Although the number of samples was too limited to draw significant conclusions, there appears to be a trend toward lower beta-endorphin concentration, in pituitary tissue and plasma samples, of mink with autoimmune orchitis. Beta-endorphin levels did not appear to correlate with natural killer cell activity in the mink. Two assays, the natural killer assay and the blasto-genesis assay, were used to insure that the beta-endorphin preparation used in this study were active. Natural killer cell activity had been reported to be enhanced by the addition of beta-endorphin. In the present study natural killer cell activity of human peripheral blood mononuclear cells (PBMC) was enhanced in the presence of betaendorphin, suggesting that the peptide was active. In addition, the blastogenic response of human PBMC was enhanced by the addition of beta-endorphin to cultures in the present study.

Surgery precipitates a hypercoagulable state and has been shown to increase the development of cancer metastases in animal models, however mechanism(s) responsible for this are largely unknown. We hypothesize that the prometastatic effect of surgery may be secondary to postoperative hypercoagulable state. Surgical stress was induced in mice by partial hepatectomy or nephrectomy, preceded by intravenous injection of CT26-LacZ or B16F10-LacZ cells to establish pulmonary metastases with or without perioperative anticoagulation and their lung tumor cell emboli (TCE) were quantified. Fibrinogen and platelets were fluorescently labeled prior to surgical stress to evaluate TCE-associated fibrin and platelet clots. Surgery significantly increased metastases while anticoagulation with five different agents attenuated this effect. Fibrin and platelet clots were associated with TCE significantly more frequently in surgically stressed mice. Surgery promotes the formation of fibrin and platelet clots around TCE and this appears to be the mechanism for the increase in metastases seen following surgery.

Natural killer (NK) cells are lymphocytes of the innate immune system that play a major role in the destruction of both tumours and virally-infected cells. The cytotoxicity of NK cells is tightly controlled by signals received through activating and inhibitory receptors. NK cells express a variety of inhibitory receptors such as Ly49 receptors. Ly49 receptors bind to class I MHC molecules that expressed on normal cells. Using Ly49-deficient (NKCKD) mice we show that Ly49-KD NK cells successfully recognize and kill influenza virus-infected cells and that NKCKD mice exhibit better survival than wild-type mice. Moreover, influenza virus infection has a propensity to upregulate cell surface expression of MHC-I on murine lung epithelial cells in vivo. Significantly, we demonstrate increased lung damage of WT-mice versus NKCKD mice after influenza virus infection as determined by histological analyses. This data indicated that absence of Ly49 inhibitory NK receptors greatly enhances survival of infected mice.

Surgery precipitates a hypercoagulable state and has been shown to increase the development of cancer metastases in animal models, however mechanism(s) responsible for this are largely unknown. We hypothesize that the prometastatic effect of surgery may be secondary to postoperative hypercoagulable state. Surgical stress was induced in mice by partial hepatectomy or nephrectomy, preceded by intravenous injection of CT26-LacZ or B16F10-LacZ cells to establish pulmonary metastases with or without perioperative anticoagulation and their lung tumor cell emboli (TCE) were quantified. Fibrinogen and platelets were fluorescently labeled prior to surgical stress to evaluate TCE-associated fibrin and platelet clots. Surgery significantly increased metastases while anticoagulation with five different agents attenuated this effect. Fibrin and platelet clots were associated with TCE significantly more frequently in surgically stressed mice. Surgery promotes the formation of fibrin and platelet clots around TCE and this appears to be the mechanism for the increase in metastases seen following surgery.

I developed a spatially explicit individual-based model (IBM) to capture the dynamic behavioral interaction between a fierce predator (killer whale, Orcinus orca) and a clever prey (dusky dolphin, Lagenorhynchus obscurus), and to answer the ultimate question of costs vs. benefits for dusky dolphins when making anti-predator decisions. Specifically, I was interested in calculating time/distance budgets for dusky dolphins in the presence/absence of killer whales and the presence/absence of movement and behavioral rules, which presumably evolved in response to spatial and temporal variations in predation risk. Results reveal that dusky dolphins rest less, travel more and have reduced foraging time when killer whales are present. These effects are more pronounced with increased presence of killer whales. The model suggests that a strong reason favoring the adoption of short and long-term anti-predator mechanisms is increased survival resulting from decreased encounters with killer whales. Further, a mother with calf rests less and travels more when killer whales are present relative to a dolphin without calf. However, a mother with calf on average, flee shorter distances and have fewer encounters with killer whales than a dolphin without calf. Thus, despite ecological costs, it makes evolutionary sense for dusky dolphins to adopt anti-predator rules. Bioenergetic consequences for dusky dolphins with and without calf were estimated as total energetic costs and foraging calories lost due to low/high presence of killer whales. I calculated total energy costs as: Foraging costs (FC) Locomotor costs (LC) (Travel) or LC (Travel) LC (Flee) based on the absence, as well as low/high presence of killer whales. Foraging costs contributed significantly to total energetic costs estimated. Travel costs are minimal owing to proximity to deep waters. The total energy costs were not significantly higher from low or high presence of killer whales for mother with calf, but increases by about 90 kcal/day for a dusky without calf. However, I estimate foraging calories lost due to increased killer whale presence is almost 5 times more for mother with calf. Therefore, it might be important to consider indirect predation risk effects by social type in future studies on animal bioenergetics.