No description available.

No description available.

No description available.

Background: Myrmecia pilosula (the jack jumper ant, JJA) is the principal cause of ant venom anaphylaxis in Australia. Whereas honeybee and wasp venom allergy can be treated by venom immunotherapy (VIT), no such treatment is available for ant sting allergy. In addition, information on the natural history of JJA sting allergy is required to identify those most likely to benefit from immunotherapy. The main objectives of this research were to establish: (i) the prevalence, natural history and determinants of reaction severity for JJA allergy, and; (ii) the efficacy and tolerability of JJA VIT. Methods: A search of the Royal Hobart Hospital (RHH) forensic register, a random telephone survey, and a review of emergency department (ED) presentations were performed. Three hundred eighty-eight JJA allergic volunteers were assessed, including serum venom-specific IgE RAST, and then followed up for accidental stings over a 4-year period. Finally, a randomised double-blind, placebo-controlled, crossover trial of JJA VIT was performed. Laboratory parameters measured during the trial were; leukocyte stimulation index (SI), IL-4 production, IgE RAST, histamine release test (HRT), leukotriene release test (LRT) and basophil activation test (BAT). Intradermal venom skin testing (VST) was also performed at trial entry. Findings: The prevalence of JJA sting allergy was 2.7% in the Tasmanian population, compared to 1.4% for honeybee. People aged 35 or older had a greater risk of both sting allergy and hypotensive reactions. Four deaths were identified, all in adults with significant comorbidities. During follow-up, 79 (70%) of 113 accidental jack jumper stings caused systemic reactions. Only prior worst reaction severity predicted the severity of follow-up reactions, with the majority of people experiencing similar or less severe reactions when stung again. Sixty-eight otherwise healthy JJA allergic adult volunteers were enrolled in the clinical trial. Systemic reactions to therapy were recorded in 34% during VIT. Objectively defined systemic reactions to sting challenges arose in 1/35 after VIT (mild self-limiting urticaria only) versus 21/29 in the placebo group. Treatment with oxygen, intravenous adrenaline infusion and volume resuscitation was effective and well tolerated. Hypotension was always accompanied by a relative bradycardia, which was severe and treated with atropine in two patients. In the placebo group, only VST and HRT were predictive of sting challenge results. Although IgE RAST, leukocyte SI and IL-4 production, LRT and BAT all correlated well with VST, they did not predict sting challenge outcome. After successful VIT, venom-induced leukocyte IL-4 production tended to fall, whereas IgE RAST increased and a natural decline in HRT reactivity was reversed. Interpretation: VIT is highly effective in prevention of JJA sting anaphylaxis and is likely to be of most benefit to people with a history of severe systemic reactions, which usually occur in people aged over 35. Neurocardiogenic mechanisms &/or direct cardiac effects may be important factors in some anaphylaxis deaths. Systemic reactions to immunotherapy are common and require immediate access to resuscitation facilities. The HRT warrants further investigation as a test for selecting those most likely to benefit from VIT. None of the tests evaluated appear to be reliable markers of successful VIT.

Desensitisation is defined as a failure of a signaling pathway to respond to chronic or repeated stimulation. The β-adrenergic receptor signaling pathway of the healthy adult heart is known to desensitise, and then regain the sensitivity to stimulation if given enough time to rest between stimulations (resensitisation). The fetal heart does not desensitise, and in animal models of heart failure, a permanent desensitisation have been observed. No isolated element of the signaling pathway have yet been proven to be the sole modulator of the desensitisation behavior. Therefore a mathematical model of the signaling pathway has been constructed, minimized against theoretical desensitisation data and tested for resensitisation. The minimal models and the original model were capable of describing the theoretical de- and resensitisation of the pathway, and only one receptor type with three states was required in the minimal models, but one feedback from the kinases either to phosphorylation of the receptor or to breakdown of cAMP. The original model was also capable of describing experimental data of contraction force from chicken cardiac tissue. The cardiac tissue displays the peak behavior of the desensitisation when stimulated with ISO for ten minutes, and resensitises in less than 5 minutes.

In light of the significant modulation of receptor activity previously shown by a peptide (designated L247), designed to mimic the third extracellular loop of the human P2Y2 receptor, the aim of this study was to use this peptide as an immunogen to generate and fully characterise polyclonal rabbit antibodies to the P2Y2 receptor. Other aims of this study were to characterise epithelial and endothelial cells for a thorough expression profile of P2Y receptor mRNA transcripts in order to provide a rapid screen for the molecular determinants of these receptors in these cells. These studies also aimed to confirm previously published pharmacology, thus, to set the basis for western blot studies using P2Y receptor antibodies. Bovine aortic endothelial cells that co-express P2Y1 and P2Y2 receptors; EAhy926, a human endothelial fusion cell line, that express P2Y2 receptors; and ECV304 human bladder cancer cell line, known to express P2Y2-like and P2Y11-like receptors were used in this study. The dose dependent accumulation of inositol phosphates and cAMP response to potent P2Y11 agonists and RT-PCR studies confirmed the functional expression of both P2Y2 and P2Y11 receptors in ECV304 cells. Likewise, the dose dependent accumulation of inositol phosphates in response to potent P2Y2 and P2Y6 agonists and the presence of mRNA transcripts confirmed the expression of functional P2Y2/4- like and P2Y6- like receptors in EAhy926 cells. Polyclonal antiserum raised against L247 peptide was affinity purified and the purified fractions showed strong immunoreactivity with immobilised immunogenic antigen in ELISA. In western blot analysis L247 rabbit polyclonal anti-P2Y2 antibody detected strong bands in ECV304 and EAhy926 cells. On pre-absorption with the immunogenic peptide these responses were abolished suggesting that this antibody is antigen specific. Agonist induced P2Y2 receptor desentisation studies in ECV304 cells showed that prolonged agonist incubation caused the receptor sequestration. The loss of bands caused by P2Y2 receptor desensitisation and sequestration in membrane enriched fractions of agonist incubated ECV304 cells confirmed the specificity of L247 antibody. This antibody also showed no immunoreactivity in 1321N1 human brain astrocytoma cells devoid of any P2Y receptor subtypes cells. Deglycosylation studies revealed that the P2Y2 receptors are glycosylated in ECV304 cells. The polyclonal rabbit anti-P2Y2 receptor antibodies obtained from commercial sources produced completely different immunoreactive profiles with multiple bands even in 1321N1 cells. Furthermore, in comparison to L247 anti-P2Y2 antibody the commercial antibody showed no difference between normal and agonist incubated cells suggesting that this antibody may not be recognising the P2Y2 receptors in ECV304 cells. Likewise polyclonal rabbit antibodies to other P2Y receptors either showed no response or showed strong immunoreactive profile with multiple bands even in 1321N1 cells suggesting that these antibodies may not have been extensively characterized. Furthermore, immunofluorescence studies with commercial anti-P2Y2 antibodies showed that they may be only recognising non-denatured receptors. These studies suggest that the L247 anti-P2Y2 antibody raised against peptide designed to mimic specific region in the third extracellular loop of human P2Y2 receptor is highly specific and sensitive and provides an important tool to study endogenously expressed P2Y2 receptors in both non-denatured and denatured state. These studies indicate that this strategy of generating antibodies may be used to generate highly specific antibodies to other P2Y receptor subtypes.

Abstract This thesis addresses the issue of the effects of (repeated) exposure to death-related news content systematically and programmatically through a four-phase research project using a Terror Management Theory (TMT) framework. The central research questions that are posed include, ‘What are the effects for individuals of exposure to death in TV news?’; ‘When will individuals personalise death-related TV news as opposed to feel desensitised to it?’; and, ‘How do individuals cope with repeated exposure to death in TV news?’ The first three chapters provide an extensive literature review that integrates current research from the media effects and mass communication literature with that of experimental findings based on TMT. This leads to an overview of the research program. Then, a series of empirical chapters present findings from six experiments, using a mixed methods approach that incorporates both quantitative and qualitative data and analyses. Finally, in Chapter 9 trends within the quantitative and qualitative data across the studies are discussed along with the theoretical and broader implications of the findings. Overall, there are three primary aims of the research. (1.) To examine a) whether death in news media can prime personal mortality salience, thus eliciting death thought accessibility and cultural worldview fluid compensation defensive outcomes as theorised by TMT (increased nationalism, endorsement of affiliation needs and self-esteem bolstering), and b) whether it is only particular portrayals of death in news media that work this way (i.e., whether there are critical factors such as viewer-victim similarity or level of exposure, as identified in the media effects literature) that play a significant moderating role. (2.) To explore whether it is necessary for the outcomes of exposure to death in news media to be defensive or whether there are alternative and more pro-social outcomes related to the extent that the viewer elaborates cognitively on the content or views more rationally (as implicated in Cozzolino, Staples, Meyers, & Sambceti, 2004). This could be as a function of individual differences (e.g., in cognitive thinking style) or as a function of the situational or contextual factors that prompt one to consider death-related news content more personally (emotionally) versus rationally. (3.) To ask about the “repeated” nature of death primes in news media, given that news media is unique in its daily emphasis on death-related content. Towards this aim we seek to answer the following: Does repeated exposure lead to accentuation of the defensive fluid compensation effects or does it lead to diminished effects because of desensitisation and depersonalisation? This third aim is potentially the most complex and is an under-researched area with important real-world implications. Specifically, Study 1 addresses reactions to death in TV news using a written stimulus task for a range of dependent variables– namely, death thought accessibility, cultural worldview endorsement, and cultural worldview defence. Examining the same dependent variables, Studies 2 and 3 explore the effects of actual TV news footage of a bus crash with multiple fatalities and the role of viewer-victim similarity. Study 4 examines what happens when explicit instructions to imagine your own death are given while watching the same TV news footage. Next, Study 5 examines whether more pro-social effects rather than the typical TMT defensive reactions are possible when a method by Cozzolino et al. (2004) that involves deeper death reflection and the role of cognitive elaboration are explored. Finally, Study 6 addresses the question of repeated exposure to death in TV news, with a focus on whether prior death exposure leads to attenuation or heightening of typical TMT defensive outcomes. Together, results from the six studies indicate that exposure to death-related TV news does not lead inevitably to defensive reactions. While there is strong evidence that death in TV news increases death thought accessibility (especially compared to a non-death TV news control), critically, whether personal mortality salience (as evidenced by self and other death thoughts) is resultant is more variable. Qualitative data shows that people have a range of defensive strategies and resources available to them and that we are honed at detecting personal relevance. Rather than viewing desensitisation as a negative by-product of TV news consumption it seems that the self-protective features of desensitisation are note-worthy. Detachment or neutrality seems to help individuals cope with the barrage of death-related images and sound bytes broadcast via TV news. Conversely, a sensitivity to detect personal relevance helps serve an important surveillance function also geared towards self-protection and meaning making. When there is maximal similarity with the victims of TV news stories portraying death, we can expect viewers to perceive high personal relevance, to personalise news content and to process the content more emotionally, as opposed to feeling desensitised. Although the buffering role of high rational thinking was weak overall, contrary to TMT-based predictions higher rational thinkers were found to be more prone to cultural worldview defence in a number of instances. The theoretical implications for TMT, social identity-based theories, Cozzolino et al.s (2004) work, and relevant media effects literature are discussed. The primary implication for TMT is evidence that death-related TV news footage has the capacity to make personal mortality salient and that higher death thought accessibility often can be evoked by death-related TV news. However, when subsequent measurement of cultural worldview defence is undertaken after a three-minute delay, higher death thought accessibility does not necessarily lead to consistent evidence of defensive fluid compensation effects. These two dependent variables have not been measured together in the literature to date, so these findings provide a significant theoretical distinction for TMT. While death in TV news more likely promotes procreation or family-related defensiveness than national bias, a range of factors (such as detecting self-relevance, viewer-victim similarity, and one’s ability to adopt a rational thinking style) moderate effects in various situations. In particular, factors such as contextual news features, rational thinking, shock value or spontaneous realisation of relevance, and reminders of one’s own family or of one’s own or others’ death are important.

Les récepteurs couplés aux protéines G (RCPG) peuvent activer des voies de signalisation indépendantes des protéines G. Cependant, la régulation de ces voies, et en particulier leur désensibilisation, est peu connue. Le récepteur de la sérotonine de type 4 (R-5-HT4) est un RCPG exprimé dans le cerveau et les organes périphériques. Il est impliqué dans des fonctions physiologiques importantes comme la mémoire, l'apprentissage, la prise de nourriture, le contrôle respiratoire et la mobilité gastro-intestinale. Le R-5-HT4 est couplé à la protéine Gs. De plus, il active la voie Src/ERK1/2, indépendamment des protéines G et des β-arrestines.Nous avons montré que GRK5, physiquement associé à la région C-terminale (C-ter) du R-5-HT4 inhibait la voie Src/ERK1/2 couplée au récepteur, mais pas la voie Gs. Ce résultat a été observé dans la lignée de cellules HEK-293 mais aussi dans des neurones de collicules en culture. Cette inhibition nécessite deux séquences d'évènements : l'association de la β-arrestine1 à une région riche en sérines et thréonines, localisée dans le domaine C-ter du récepteur et la phosphorylation par GRK5, de la β-arrestine1 (en sérine 412) liée au récepteur. La β-arrestine1 phosphorylée empêche l'activation de Src, constitutivement liée au récepteur, nécessaire à l'activation d'ERK1/2. Ceci constitue la première démonstration que la phosphorylation d'une β-arrestine par une GRK régule la signalisation indépendante des protéines G. En plus de ces résultats, nous avons démontré que l'activation d'ERK1/2 par le R-5-HT4, indépendante des β-arrestines, implique la libération d'un ligand induite par une métalloprotéase, conduisant à la transactivation d'un autre récepteur. Par une approche protéomique, nous avons également identifiés plusieurs partenaires potentiels du R-5-HT4. L'étude de ces partenaires pourrait apporter un éclairage supplémentaire sur les voies de signalisation du récepteur et leur régulation. / G protein-coupled receptors (GPCRs) have been found to trigger G protein-independent signalling. However, the regulation of G protein-independent pathways, especially their desensitization, is poorly characterized.The 5-Hydroxytryptamine 4 receptor (5-HT4R) is a GPCR widely expressed in the brain and at the periphery. It is implicated in important physiological functions such as memory, cognition, feeding, respiratory control and gastrointestinal motility. 5-HT4R couples to the Gs/cAMP/PKA pathway. Moreover, this receptor can activate a Src/ERK pathway independently of both G proteins and β-arrestins.Here, we show that the G protein-independent 5-HT4R-operated Src/ERK pathway, but not the Gs pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor C-terminus, in both HEK-293 cells and colliculi neurons. This inhibition requires two sequences of events: the association of β-arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C-terminal domain and the phosphorylation by GRK5 of β-arrestin1 (at Ser 412) bound to the receptor. Phosphorylated β-arrestin1 prevents in turn activation of Src constitutively bound to 5-HT4R, a necessary step in receptor-stimulated ERK signalling. This is the first demonstration that β-arrestin phosphorylation by a GRK regulates G protein-independent signalling.In addition to these results, we also demonstrated that the β-arrestin-independent activation of ERK1/2 by the 5-HT4R involves a metalloprotease-dependant ectodomain shedding and transactivation of another receptor. By a proteomic approach, we also identified several potential partners of the 5-HT4R. Study of these proteins may provide a better understanding of 5-HT4R signalling and his regulation.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第20294号 / 人健博第42号 / 新制||人健||4(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 二木 淑子, 教授 精山 明敏, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM