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Background: Platelet activating factor (PAF) is a phospholipid synthesized by the action of phospholipase A2 and acetyl transferase. PAF possesses a wide range of biological activities. In the lung of the fetus and newborn, PAF binds to its G protein couple receptor to evoke its biological activities via a well-defined signaling pathway. High levels of PAF receptor (PAFr) activity in fetal ovine lung vascular smooth muscle cells (PVSMC) at baseline has previously been demonstrated, a finding that is further perpetuated by conditions of hypoxia similar to fetal lung environment. Additionally in fetal ovine PVSMC, a cross-talk between PAFr-mediated cell signaling and activity of the vasodilator cyclic nucleotides cGMP and cAMP acting via their respective receptors protein kinase (PK) G and PKA has been shown. The interaction of PAF with its receptor has been implicated in the pathogenesis of persistent pulmonary hypertension in the newborn (PPHN) which has a high incidence of hospitalization and death of newborn infants. Successful transition of fetus to newborn life entails a mechanism whereby vasoconstrictors necessary for fetal existence are abrogated in the immediate newborn. Hypothesis: We hypothesize that PPHN results from the failure to down regulate PAFr- mediated activity and /or failure to up-regulate activity of the vasodilators cGMP and cAMP. PPHN is triggered by chronic intrauterine or postnatal hypoxia. Then newborn PVSMC undergo hyperplasia and hypertrophy, which over time, results in irreversible vascular remodeling. Methods: My study aims to employ in vitro models to delineate the consequences of PAF-PAFr mediated pathway in the pharmacological effects of the cAMP-PKA and cGMP-PKG signaling and the involvement of this cross-talk in the pathogenesis of PPHN. I modeled my cell culture studies to mimic the low oxygen environment of fetal lungs (hypoxia), the normal oxygen environment of newborn lungs (normoxia) and high oxygen environment (hyperoxia) to which the newborn lung may be exposed in incidental clinical condition of PPHN. I studied the effect of PAF, a vasoconstrictor, cAMP/cGMP, vasodilators, and other inhibitors of the PAFr pathway on growth of newborn PVSMC, by DNA synthesis, and measured their effects on expression of mitogenic and non-mitogenic proteins. Results: We found that both hypoxia and hyperoxia decreased cell growth even in the presence of PAF which up-regulates cell growth in fetal PVSMC. Also PAF treatment of cells resulted in down regulation of the vasodilator proteins, PKA and PKG. Conclusion: Our data suggests that in the lung of the newborn a high activity of PAF-PAFr mediated activities will worsen the condition of PPHN imposed on the newborn lung by environmental or therapeutic conditions. We can speculate that, in the long run, these findings may translate into the establishment of less toxic protein-based management of PPHN.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

Background: Asthma Link is a program that aims to increase adherence to inhaled corticosteroids among children with persistent asthma by delivering evidence-based school-supervised therapy. This program, which leverages existing infrastructure, improves asthma outcomes in children from low-income, minority families. Our aim was to elicit the perspectives of school nurses who supervise preventive medication administration. Methods: Semi-structured qualitative interviews were conducted with 12 school nurses participating in Asthma Link. Thematic analysis was used to identify themes related to barriers and facilitators to preventive medication delivery. Results: Barriers described by school nurses included communication challenges with families and providers, inconsistent supplies of the preventive medicine at school, and the perception by some families and nurses that preventive therapy should be provided at home. Facilitators included the ease of incorporating preventive medication delivery into morning routines, recognizing the positive impacts on children from families with limited resources, feeling part of the preventive health care team, and being well-positioned to engage families in preventive asthma care. Conclusions: To facilitate Asthma Link adoption, it is critical to incorporate school nurse feedback in the program’s protocol refinement. School-supervised asthma therapy programs are advised to engage school nurses in the opportunity to provide preventive care, streamline communication, and address social and logistical challenges which may impede families from bringing medication to school.

This study uses a descriptive survey design to describe and examine the relationship among school nurses’ level of education, years of experience, knowledge of asthma and identification of the school nurse’s level of proficiency based on Benner’s (1984) model of Novice to Expert. A convenience sample of school nurses employed in public schools within the state of Massachusetts with an RN degree (registered nurse) were sampled. The demographic data revealed that of the 325 participants who participated in the study, the majority of school nurses were female ranging in age from 40 to 50 (M=47.0). The majority of nurses had a bachelor’s degree in nursing and were employed in the nursing profession on an average of twenty-two years and in school nursing for ten years. Since the majority of the school nurses did not have a master’s degree, they were not certified by a national certifying body. The majority of participants indicated that they had received certification through the Board of Education in Massachusetts. Most school nurses worked full time in a public school and were responsible for between six hundred and a thousand students. The majority of nurses indicated that they did not have a school-based clinic on site, nor did they have a school-based health center or clinic to refer students. There was little variability among sample characteristics with school nurses employed in Massachusetts being a fairly homogenous group. Those surveyed were sent a packet containing four questionnaires including one on demographics, as well as an asthma questionnaire, a questionnaire assessing chronic health problems in the schools, and a self-reporting questionnaire based on Benner’s (1984) model. Further results of this study revealed that the majority of the school nurses had an average to above average knowledge of asthma. The three most common interventions performed by school nurses as well as non medical personnel for those students with chronic illness are nebulizations, inhalers, and peak flow meters. Based on the self-report model of Benner’s (1984), these same nurses viewed themselves as expert in their level of practice. Mezirow’s Adult Learning Theory as well as Benner’s (1984) model of Novice to Expert were used to support the nurses level of practice based on experience, intuition and a constellation of meaning schemes developed from previous exemplars. Results of the study indicated that although the nurses surveyed were expert in their knowledge of basic nursing concepts, none had advanced practice level courses in advanced health assessment or clinical decision making in order to effectively manage the complexities of chronic illness such as ADHD, diabetes, and epilepsy, as well as asthma, the most common chronic illness in schools today.

Mycobacterium tuberculosis, the causative agent of tuberculosis, can manipulate host cell death pathways as virulent strains inhibit apoptosis to protect its replication niche and induce necrosis as a mechanism of escape. In vitro studies revealed that similar to lytic viruses, M. tuberculosis has the ability to induce cytolysis in macrophages when it reaches an intracellular burden of ~25 bacilli. Base on this finding, we proposed the burst size hypothesis that states when M. tuberculosis invades a macrophage at a low multiplicity of infection it replicates to a burst size triggering necrosis to escape the cell and infect naïve nearby phagocytes, propagating the spread of infection. The first part of this study investigated if the in vitro observations of M. tuberculosis cytolysis were relevant to cell death of infected phagocytes during pulmonary tuberculosis in vivo. Mice infected with a low dose of M. tuberculosis revealed during TB disease, the major host cell shifted from one type of phagocyte to another. Enumeration of intracellular bacilli from infected lung cells revealed the predictions of the hypothesis were confirmed by the distribution of bacillary loads across the population of infected phagocytes. Heavily burdened cells appeared nonviable sharing distinctive features similar to infected macrophages from in vitro studies. Collectively, the data indicates that M. tuberculosis triggers necrosis in mononuclear cells when its number reaches the threshold burst size. The previous study showed during the period of logarithmic bacterial expansion, neutrophils were the primary host cell for M. tuberculosis coinciding with the timeframe of the highest rate of burst size necrosis. The second part of this study examined this link by infecting mice with one of four different M. tuberculosis strains ranging in virulence. Mice infected with the most virulent strain had the highest bacterial burden and elicited the greatest number of infected neutrophils with the most extensive lung inflammation and greater accounts of cell death. Treating these mice with a bacteriostatic agent decreased the bacterial load and infected neutrophils in a dose-dependent manner indicating necrosis induced by virulent M. tuberculosis recruited neutrophils to the lungs. Infected neutrophils can serve as a biomarker in tuberculosis as evidenced by poorly controlled infection and increased severity of lung immune pathology.

The development of methods and techniques is the driving force of scientific research. In this work, we described two large-scale screens in studying transcriptional activation and tumor suppression. In Part I, we studied transcriptional activation mechanisms by deriving and characterizing activation defective mutants. Promoter-specific transcriptional activators stimulate transcription through direct interactions with one or more components of the transcription machinery, termed the “target.” The identification of direct in vivo targets of activators has been a major challenge. We perform a large-scale genetic screen to derive and characterize tra1 alleles that are selectively defective for interaction with Gal4 in vivo. Utilizing these mutants, we demonstrated that Tra is an essential target for Gal4 activation, Gal4 and Tra1 bind cooperatively at the promoter and the Gal4–Tra1 interaction occurs predominantly on the promoter. In addition, we demonstrated that the Gal4-interaction site on Tra1 is highly selective. In Part II, we described a functional genomics approach to discover new tumor suppressor genes. A goal of contemporary cancer research is to identify the genes responsible for neoplastic transformation. Cells that are immortalized but non-tumorigenic were stably transduced with pools of short hairpin RNAs (shRNAs) and tested for their ability to form tumors in mice. ShRNAs in any resulting tumors were identified by sequencing to reveal candidate TSGs, which were then validated both experimentally and clinically by analysis of human tumor samples. Using this approach, we identified and validated 33 candidate TSGs. We found that most candidate TSGs were down-regulated in >70% of human lung squamous cell carcinoma (hLSCC) samples, and 17 candidate TSGs negatively regulate FGFR signalling pathway, and their ectopic expression inhibited growth of hLSCC xenografts. Furthermore, we suggest that by examining at the expression level of TSGs in lung cancer patients, we can predict their drug responsiveness to FGFR inhibitors. In conclusion, we have identified many new lung squamous cell cancer TSGs, using an experimental strategy that can be broadly applied to find TSGs in other tumor types.

Preclinical models of lupus indicate that T cell-B cell collaboration drives antinuclear antibody (ANA) production and sustains T cell activation. Autoreactive B lymphocytes are present in the normal repertoire but persist as ignorant or anergic cells. Mechanisms that normally limit T cell activation of autoreactive B cells remain incompletely resolved, but potentially include the absence of autoreactive effector T cell subsets and/or the presence of autoAgspecific regulatory T cells (Tregs). Several studies have addressed this issue by using experimental systems dependent on transgenic autoreactive B cells, but much less is known about the activation of autoreactive B cells present in a polyclonal repertoire. In the second chapter of this thesis, I have explored the role of effector T cells and Tregs using mice that express an inducible pseudoautoAg expressed on B cells and other antigen presenting cells (APCs). In this system, activated Th2 cells, but not naïve T cells, elicit the production of ANAs, but ANA production is severely limited by autoAg-specific Tregs. Bone marrow chimera experiments further demonstrated that this B cell activation is constrained by radioresistant autoantigen-expressing APCs (rAPC) present in the thymus as well as by non-hematopoietic stromal cells located in peripheral lymphoid tissue. Importantly, peripheral rAPC expression of autoAg induced the expansion of a highly effective subset of CD62L+CD69+ Tregs. The third chapter of this thesis focuses on the contribution of CD8+ T cells to fibrosis resulting from sterile lung injury. Type 2 effector production of IL-13 is v a demonstrated requirement in several models of fibrosis, and is routinely ascribed to CD4+ Th2 cells. However, we now demonstrate a major role for pulmonary CD8+ T cells, which mediate an exaggerated wound healing response and fibrosis through robust differentiation into IL-13-producing pro-fibrotic type 2 effectors (Tc2). Remarkably, differentiation of these Tc2 cells in the lung requires IL-21. We further show that the combination of IL-4 and IL-21 skews naïve CD8+ T cells to produce IL-21, which in turn acts in an autocrine manner to support robust IL-13 production. TGF-β negatively regulates production of IL-13 by suppressing CD8+ T cell responsiveness to IL-21. Our data illuminate a novel pathway involved in the onset and regulation of pulmonary fibrosis, and identify Tc2 cells as key mediators of fibrogenesis.

This qualitative descriptive (QD) study examined the experience of the woman newly diagnosed with obstructive sleep apnea (OSA). The study employed Leventhal’s Self- Regulatory Theory to understand women’s illness representation of OSA, cognitive and emotional coping, and situational appraisal skills in coming to terms with OSA. The specific aims were to: 1) Describe the illness representation of women with a recent diagnosis (within one year) of OSA; 2) Describe the cognitive perceptions and emotional response to diagnosis and treatment of OSA in this sample of women; and, 3) Describe the meaning of OSA and the coping strategies used by this sample of women. The overarching theme of this study of a life-altering diagnosis required participants to process the health threatening information in both a conceptual and concrete process for dealing with both the physical and emotional aspects. The first two subthemes that emerged were Making sense of it, and Making it work as the women came to terms with their symptoms, diagnosis, and adapted to their treatment. For this sample of women, both acceptance (acknowledging the diagnosis of OSA and embracing treatment), and denial (not convinced of diagnosis or need for treatment, seeking alternatives) were factors in how they made sense of the situation. The making it work subtheme dealt with the women’s experiences adapting to treatment both physically and emotionally, including the appraisal, reconsideration and adjustments when they encountered difficulties and delays. A fluid iterative process included women participants describing how they appraised their situation often moving back and forth between acceptance, denial, seeking alternatives, struggling with treatment and moving forward. In both of these subthemes, family support and the stigma of OSA and CPAP were involved in how the women accepted and adapted to treatment. The third subtheme that emerged was Paying it forward as many women felt the obligation to help themselves by adapting a healthier lifestyle for themselves, their families and to assist others impacted by OSA. Women spoke of paying it forward by offering information and support to others not yet diagnosed, or are struggling with diagnosis and treatment. Many of these women were staunch advocates for other women to be tested, for HCPs to be more aware, to be more attuned to women’s sleep history, and to refer women for treatment. Implications of these findings include enhancing recognition and awareness by women of OSA symptoms, the need for diagnostic evaluation, and partner awareness as an important component of diagnosis and successful treatment for women. Study findings support recognition of women’s presentation of OSA including unusual symptoms for earlier diagnosis and treatment. Sleep partner awareness and support appear to be relevant to women in acceptance of a life altering diagnosis. Further exploration of modifiable factors such as prompt diagnosis and individualized treatment of women with OSA could also impact potential co-morbidities. Provision of further education and awareness by HCPs and insurance companies that women may not present with classic symptoms of OSA is also needed. Targeted interventions specific to women’s experiences with OSA include development of screening tools, care guidelines and treatments that enhance applicability, acceptability, and patient satisfaction. Future advocacy work will also require supporting women in “paying it forward” to help other women diagnosed with OSA.

Purpose: The purpose of this study was to explore the way(s) in which family management of childhood asthma is affected by contextual influences as described in the Family Management Style Framework (FMSF) and to explore additional factors that affect family asthma management. Specific Aims: The specific aims of this study were 1) to describe the everyday experiences of childhood asthma management within families, 2) to explore the way(s) in which family management of childhood asthma is affected by contextual influences (social network, care providers & systems and resources) as described in the FMSF, and 3) to explore additional sociocultural factors (supported by the literature but not currently described in the FMSF) that affect asthma management in families. Framework: The Family Management Style Framework guided this study. Design: A qualitative descriptive design was used to gather data from a purposive sample of female primary caregivers. Demographic data were collected, and individual interviews were conducted using a flexible interview guide. Results: The findings support the contextual influences as described in the FMSF. An additional three contextual themes were identified: environment, emerging threats to health and work-life conditions. The themes are interrelated demonstrating the complexity of asthma management. Conclusion: Family management of asthma is challenging and complex. The findings move towards understanding the connection between family asthma management and the social determinants of health. Nurses can support families managing childhood asthma by considering each of the contextual influences when planning interventions and working on policy initiatives that support the health of children with asthma.