Streptococcus agalactiae CAMP factor is a protein exotoxin that contains 226 amino acid residues and forms oligomeric pores on susceptible cell membranes and liposomes. In this study, fragments of CAMP factor were created and recombinantly expressed to identify functional domains that are involved in membrane binding, oligomerization, and membrane insertion. Altogether, six truncated forms of CAMP factor were created and assayed. CAMP1-113, CAMP1-170, CAMP57-226, and CAMP171-226 showed different levels of hemolytic activity. CAMP1-56 and CAMP114-226 did not show hemolytic activity or oligomerization ability, but showed binding ability. CAMP114-226 inhibited the hemolytic activity of wild-type CAMP factor, most likely through ‘one-sided’ oligomerization. From the comparison of these fragments, it emerges that the region between residues 57 and 113 plays a crucial role in oligomerization and membrane insertion. The high binding efficiency of CAMP114-226 suggests this region has great responsibility on membrane binding. The hemolytically inactive fragments showed higher binding efficiency than some of the active fragments. For the hemolytic fragments, higher binding efficiency gave stronger hemolysis. These findings support that CAMP factor has different functional regions for pore-formation.

ABSTRACT CAMP factor is a 25kDa extracellular protein from Streptococcus agalactiae (Group B streptococci) that contains 226 amino acid residues. CAMP factor has been characterized as a pore-forming toxin (PFT). The typical mechanism of pore formation of PFTs involves three main stages, namely binding of toxin monomers to the membrane surface, oligomerization of the monomers on the cell membrane, and finally the insertion of oligomers into the membrane. This study focused on second stage, and investigates the oligomerisation of CAMP factor on sheep red blood cell membranes. It is known that the hemolytic activity of CAMP factor is greatly enhanced by interaction with sphingomyelinase from Staphylococcus aureus. We here focused on understanding the role of sphingomyelinase in the oligomerisation step. Experimental data were obtained using Förster resonance energy transfer (FRET) studies. The fluorescence dyes IAEDANS and Fluorescein-5-maleimide were used as donor/acceptor fluorophores and attached to mutant single cysteine residues in CAMP factor. Samples of donor- and acceptor-labelled protein were mixed and incubated with red cell membranes that had or had not been pre-treated with sphingomyelinase. Energy transfer was monitored with time-resolved and steady-state fluorescence measurements. In the time-resolved experiments, the fluorescence lifetime of the donor was measured in the presence and the absence of the acceptor, on membrane samples that were or were not treated with sphingomyelinase. We observed a decrease in the fluorescence lifetime of the donor with the presence of the acceptor. The decrease in lifetime due to acceptor interaction signifies the occurrence of energy transfer between the donor and acceptor fluorophores, which indicates proximity due to oligomerisation of the CAMP factor protein on the cell membrane. This was only observed when the membranes had been treated with sphingomyelinase. When membranes were used that had not been treated with sphingomyelinase, the donor lifetimes are very low, suggesting the inability of the CAMP factor to undergo membrane insertion and oligomerisation.

Childhood cancer has obvious impacts on the children themselves, but also has impacts on their families and siblings. While studies have been conducted on the impacts of cancer on children and their parents, there has been limited consideration of the impacts of cancer on healthy siblings. Some studies have suggested that siblings of children with cancer are the most emotionally disregarded and distressed of all family members as a result of their sibling having cancer. Exposure to stress has a negative impact on the immune system and places siblings at risk for health problems. Thus, finding mechanisms to ameliorate stress are critical. Early intervention and treatment may serve as protective factors against risky behavior and lead to more normative child development and well-being. One method of improving the psychosocial adjustment of siblings of children with cancer has been through interventions such as camp experiences that include opportunities for campers to experience social support. There is a need to better understand the efficacy of intervention programs designed to reduce stress across a variety of settings. The focus of this study was if campers experience social support (emotional, informational, and instrumental) at a three and one-half day residential camp, and the elements of camp campers’ identified as leading to social support. Participant observations of camp and semi-structured interviews with selected campers were used to help gain insights concerning the impact of the camp experience on siblings of children with cancer. Coding of responses was undertaken, which led to the identification of themes and subthemes. Findings provided a narrative description of how campers perceived social support. In addition, social comparison was identified as a critical mechanism for meeting camper’s biological, physiological and behavioral needs. Overarching perceptions of campers were that people at camp were nice and staff was instrumental in facilitating universality and a cycle of reciprocity. Implications of this study suggest intentionally designed camps have the ability to impact a broad range of attitudes and behaviors. The study also supports theory of how structures and processes of social relationships work in relation to good health; and can be applied to a camp setting.

Childhood cancer has obvious impacts on the children themselves, but also has impacts on their families and siblings. While studies have been conducted on the impacts of cancer on children and their parents, there has been limited consideration of the impacts of cancer on healthy siblings. Some studies have suggested that siblings of children with cancer are the most emotionally disregarded and distressed of all family members as a result of their sibling having cancer. Exposure to stress has a negative impact on the immune system and places siblings at risk for health problems. Thus, finding mechanisms to ameliorate stress are critical. Early intervention and treatment may serve as protective factors against risky behavior and lead to more normative child development and well-being. One method of improving the psychosocial adjustment of siblings of children with cancer has been through interventions such as camp experiences that include opportunities for campers to experience social support. There is a need to better understand the efficacy of intervention programs designed to reduce stress across a variety of settings. The focus of this study was if campers experience social support (emotional, informational, and instrumental) at a three and one-half day residential camp, and the elements of camp campers' identified as leading to social support. Participant observations of camp and semi-structured interviews with selected campers were used to help gain insights concerning the impact of the camp experience on siblings of children with cancer. Coding of responses was undertaken, which led to the identification of themes and subthemes. Findings provided a narrative description of how campers perceived social support. In addition, social comparison was identified as a critical mechanism for meeting camper's biological, physiological and behavioral needs. Overarching perceptions of campers were that people at camp were nice and staff was instrumental in facilitating universality and a cycle of reciprocity. Implications of this study suggest intentionally designed camps have the ability to impact a broad range of attitudes and behaviors. The study also supports theory of how structures and processes of social relationships work in relation to good health; and can be applied to a camp setting.

Diss. / Bibliogr. p. 331-363.

This dissertation examines factors that are associated with college outcomes (i.e., college GPA, persistence, and academic probation) for a migrant Latina/o college student population participating in the federally funded College Assistance Migrant Program (CAMP). The study also examines the impact of CAMP services on college outcomes and socio-cognitive perceptions (i.e., college academic self-efficacy, academic resilience, and school connectedness). Further, the study compared participant's level of involvement in the CAMP program across universities and qualitatively examined students' open-ended responses on how the CAMP program is useful and how it can be improved. Participants were 245 CAMP participants from four universities including: Northeastern University, Central State University, Pacific Northwest University, and Southwestern University. High school achievement and academic resilience were significant positive predictors of college GPA. Living on-campus was a negative predictor. Financial aid in the form of loans, having family responsibilities, and working full-time off campus were negative predictors of persistence, while involvement in CAMP's personal and academic counseling services was a positive predictor. Higher levels of academic self-efficacy, academic resilience, and CAMP's academic and financial assistance predicted being less likely to obtain academic probation status. Student's open ended responses revealed CAMP services helped students to: integrate socially into the university system, become more resilient when facing academic, personal, and cultural barriers, navigate the higher education landscape, and provided students with financial assistance. However, students also reported that the program could be improved. Implications for practice are discussed.

Following starvation, Dictyostelium discoideum cells aggregate, a response that requires chemotaxis to cyclic AMP (cAMP) and the relay of the cAMP signal by the activation of adenylyl cyclase (ACA). Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG does have a role in early development. When the responses of rasG⁻ cells to cAMP were compared with the responses of rasC⁻ strain, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Characterization of a rasC⁻/rasG⁻ mutant revealed that both cAMP chemotaxis and adenylyl cyclase (ACA) activation were negligible in this strain. The ectopic expression of carA from the actin 15 promoter restored early developmental gene expression to the rasC⁻/rasG⁻ strain, rendering it suitable for an analysis of cAMP signal transduction. Since there was negligible signaling through either the cAMP chemotactic pathway or the adenylyl cyclase activation pathway in this strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The mutational analysis of Switch I and Switch II regions also defined the key residues that generate functional differences between RasC and RasG. Rap1 is also activated in response to cAMP but its position in the signal transduction cascade was clarified by the finding that its activation was totally abolished in rasC⁻/rasG⁻/[act15]:carA and in rasG⁻ cells, but only slightly reduced in rasC⁻ cells. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC/rasG⁻4act15]:carA strain identifies RasG⁻/RasC⁻ as the presumptive monomeric GTPases required for this activation. The phenotypes of the vegetative ras null mutants were also examined. The results indicate that RasG plays an important role in cytokinesis. The partial absence of chemotaxis to folate in rase cells compared to the total absence of chemotaxis to folate in rasC⁻/rasG⁻, and rasC⁻/rasG⁻/[act15]:carA cells suggests a compensatory role of RasC for RasG during this process, a similar phenomenon to that observed for cAMP chemotaxis by aggregating cells.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 水谷武史氏の博士論文として提出された

Cells of the cardiovascular system translate incoming extracellular signals from hormones and drugs through binding of cell surface receptors, and activation of intracellular signaling cascades allowing modulation of specific cellular function. cAMP and cGMP are ubiquitous second messengers that activate specific signaling machinery used to promote or inhibit cellular functions such as cell migration, cell adhesion and proliferation. Increases in intracellular cAMP or cGMP levels occurs through activation of adenylyl cyclase (cAMP) or guanylyl cyclase (cGMP) or by inhibition of the cAMP and cGMP hydrolyzing enzymes, cyclic nucleotide phosphodiesterases (PDEs). Cyclic nucleotides achieve signaling specificity through compartmentation, a mechanism allowing effective regulation of cAMP or cGMP signaling in discrete parts of the cell in a spatial and temporal manner. Cells of the cardiovascular system such as platelets, vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs) maintain cyclic nucleotide compartmentation through coordinating signaling complexes containing a cAMP or cGMP effector protein and PDEs. Studies reported in this thesis demonstrate that human platelets, VECs and VSMCs each contain distinct cyclic nucleotide signaling complexes, and that based on their composition and selective subcellular localization, regulate specific cellular functions. In platelets, subcellular localization of PDE5 results in differential regulation of PDE5 and selective regulation of Ca2+ release from endoplasmic reticulum stores, an initial step in platelet aggregation and provides a potential therapeutic target in preventing thrombosis. VECs utilize multiple signaling systems to regulate cellular function including cAMP signaling pathways and modification of phosphatidylinositols. These studies demonstrate that a PDE3B-based signaling complex allows integration of both cAMP and phosphatidylinositol-3-kinase-γ (PI3Kγ) signals resulting in increased cell adhesion and cell spreading. Finally, studies in VSMCs demonstrate that PDE5 localization in cells allows cAMP/cGMP cross talk through PDE5 and PDE3A. These results are discussed in the context of further understanding the role of PDEs in mediating cAMP and cGMP signaling and modulation of cell function in cells of the cardiovascular system. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2011-06-28 13:31:51.428

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