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Every summer Camp Hopewell in Oxford, Mississippi hosts its annual summer camps. Over the course of a week, kids between the ages of seven and fifteen run, play, hike, and canoe. It's the pretty standard summer camp fair, but there is something that makes a week at Camp Hopewell different. Every child that comes to camp has been diagnosed with Type I Diabetes. Some have had it for years and consider camp their second home while some have just been diagnosed and still live in fear of their condition. For this one week, however, they all have something in common, and while they eat, sleep, and play, they learn to take care of their own Diabetes. Diabetes Camp is a 25 minute documentary film that is meant to show audiences the remarkable occurrences at Camp Hopewell through the eyes and voices of the campers and the staff that work there.

Die proximale spinale Muskelatrophie (SMA) ist eine autosomal rezessive Erb-krankheit, welche durch fortschreitende Muskelatrophie mit Betonung der pro-ximalen Extremitäten, sowie zunehmende motorische Lähmungen charakterisiert wird. Bedingt wird diese neurodegenerative Erkrankung durch Mutation bzw. Deletion des SMN1-Gens auf Chromosom 5q13. Dies führt zu reduzierten Mengen des ubiquitär exprimierten SMN-Proteins, da der Verlust des SMN1-Gens nicht durch das noch verbleibende SMN2-Gen kompensiert werden kann. Die SMN-Promotor-Region enthält ein CRE II bindendes Element, welches Effekte von zyklischem Adenosinmonophosphat (cAMP) vermittelt und so die SMN-Transkription in untersuchten Zellen stimuliert. Ausgehend von diesem Befund stellte sich die Frage, ob cAMP dem Mangel an volllängen SMN bei der SMA entgegen wirkt. Daher wurden für diese Dissertation neurosphärenbildende kortikale Vorläuferzellen und primär kultivierte Motoneuronen von Smn+/+; SMN2- und Smn–/–;SMN2-Mausembryonen untersucht, um zu klären, ob die cAMP-Behandlung der Zellen zu einer Hochregulierung des SMN2-Transkripts führt, und durch die resultierende Erhöhung des SMN-Proteingehalts morphologische und funktionelle Defekte kompensiert werden. Die Untersuchung zeigte eine signifikante Zunahme des SMN2-Transkriptgehalts in Anwesenheit von cAMP. Dadurch kam es zu einem Anstieg der SMN-Proteinmenge im Soma, Axon und Wachstumskegel von Smn–/–;SMN2-Motoneuronen. Die Verteilungs-störung des SMN-Interaktionspartners hnRNP R mit fehlender kontrolltypischer Anreicherung im distalen Axon und Wachstumskegel von Smn–/–;SMN2-Motoneuronen wurde ebenfalls durch cAMP kompensiert. Smn-defiziente Mo-toneurone zeigten im Vergleich zu Kontrollzellen kleinere Wachstumskegel sowie ein Defizit an β-Aktin im distalen Axon. Zudem fehlte in Smn–/–;SMN2-Motoneuronen die bei Kontrollen ausgeprägte Zusammenlagerung von N-Typ spezifischen Ca2+-Kanälen in der Präsynapse, die nach Kontakt mit der β2-Kette des endplattenspezifischen Laminin-221 spontan öffnen und so einen in-trazellulären Kalziumanstieg bewirken, wodurch es zu Erregbarkeitsstörungen und Axonelongationsdefekten bei Smn-defizienten Motoneuronen kommt. Die Behandlung der Smn-defizienten Motoneuronen mit cAMP führte zur Vergrößerung der Wachstumskegelfläche und zu einer im Verlauf des Axons zunehmenden Anfärbung mit β-Aktin. Außerdem kam es zu einer Erhöhung der Menge an Cav2.2-Kanalprotein in den Wachstumskegeln Smn-defizienter Motoneurone, was mit einer erhöhten Erregbarkeit korrelierte und zu einer Normalisierung der Axonlänge von Smn–/–;SMN2-Motoneuronen auf Laminin-221 führte. Die Ergebnisse dieser Arbeit lassen die Vermutung zu, dass Smn-defiziente Motoneurone in vivo Defekte im präsynaptischen Bereich der Motorendplatte aufweisen. In Zukunft können mit dem beschriebenen in vitro Assay weitere Substanzen, welche die SMN2-Traskription stimulieren, auf ihr kompensatorisches Potential getestet werden. / Proximal autosomal recessive spinal muscular atrophy (SMA) is caused by mutation or deletion of the SMN1-gene on chromosome 5q13. The SMN promotor region contains a CRE II binding element, which mediates effects of cyclic adenosine monophosphate and stimulates the SMN transcription in examined cells. In animal models of SMA, spinal motoneurons exhibit reduced axon elongation and growth cone size. These defects correlate with reduced ß-actin protein levels in distal axons. In this study I examined primary cultured motoneurons from Smn+/+;SMN2- and Smn-/-;SMN2-mice embryos. The examination could show a significant increase of the SMN2 transcript by treating the cells with cAMP. The Smn protein level increases in the soma, axonal department and growth cones of Smn-deficient motoneurons which were treated with cAMP in cell culture. I could also show that Smn–deficient motoneurons exhibit severe defects in clustering Cav2.2 channels in axonal growth cones and that treating with cAMP compensate these defects. Growth cone size, axonal length, hnRNP R protein levels and ß-actin protein levels in distal axons being normalized by cAMP treating of the Smn-/-;SMN2-motoneurons. Other substances, which stimulate the SMN2 transcription, can be tested in the future with the in this study established in vitro assay.

Thesis advisor: Charles Hoffman / The fission yeast Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway. Elevated cAMP levels activate protein kinase A (PKA) to inhibit transcription of genes involved in sexual development and gluconeogenesis, including the fbp1⁺ gene, which encodes fructose-1,6-bisphosphatase. Glucose-mediated activation of PKA requires the function of nine git genes (git=glucose insensitive transcription), encoding adenylate cyclase, the PKA catalytic subunit and seven “upstream” proteins required for glucose-triggered adenylate cyclase activation. This thesis describes the cloning and characterization of the git10⁺ gene, which is identical to swo1⁺ and encodes the S. pombe Hsp90 chaperone protein. This discovery is consistent with the previous identification of the Git7 protein as a member of the Sgt1 Hsp90 co-chaperone family. Glucose repression of fbp1⁺ transcription is impaired by both hsp90⁻ and git7⁻ mutant alleles, as well as by chemical inhibition of Hsp90 activity and temperature stress. Unlike the swo1⁻ and git7⁻ ts mutant alleles, the git10-201 allele and git7-93 allele support cell growth at 37º and show no cytokinesis defect, while severely reducing glucose repression of an fbp1-lacZ reporter, suggesting a separation-of-function defect. A physical interaction between Git7 and Hsp90 in S. pombe was also detected and findings in this thesis suggest their involvement in the initial assembly of the cAMP complex. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Charles Hoffman / Cyclic adenosine monophosphate (cAMP) and the second messengers that modulate several biological processes are regulated by adenylyl cyclase (AC) and cyclic nucleotide phosphodiesterases (PDEs). ACs and PDEs are comprised of superfamilies of enzymes that are viewed as druggable targets due to their many distinct biological roles and tissue-specific distribution. As such, small molecule regulators of ACs and PDEs are important as chemical probes to study the roles of individual ACs or PDEs and as potential therapeutics. In the past, our lab has expressed 15 mammalian PDE genes in S. pombe, replacing the endogenous Cgs2 PDE. High throughput screens for PDE inhibitors identified novel compounds that show relevant biological activity in mammalian cell culture assays. The aim of this thesis is to develop tools to understand the mechanism of interaction between key regulators of the cAMP pathway and small molecules. The current study is comprised of two parts. In the first part of this thesis, I developed a genetic screen that detected alleles whose proteins remain active in the presence of BC54 and was to confirm the effect of the PDE4BT407A mutation using cell-based assays and in vitro enzyme assays. In the second part of this thesis, I developed and carried out HTSs using a PKA-repressed GFP reporter that can identify compounds that reduce PKA activity, which would include PDE activators and AC or GNAS1 inhibitors. To date, I have identified three AC inhibitors that appear to act on several of the ten different mammalian ACs. To our knowledge, this is the first time a large HTS has identified AC inhibitors, where inhibition was assessed inside the cells. The findings in this thesis will be useful in the design of more effective PDE inhibitors and in the development of novel chemical probes for studying cAMP signaling in mammalian cells. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Charles Hoffman / Cyclic adenosine monophosphate (cAMP) and the second messengers that modulate several biological processes are regulated by adenylyl cyclase (AC) and cyclic nucleotide phosphodiesterases (PDEs). ACs and PDEs are comprised of superfamilies of enzymes that are viewed as druggable targets due to their many distinct biological roles and tissue-specific distribution. As such, small molecule regulators of ACs and PDEs are important as chemical probes to study the roles of individual ACs or PDEs and as potential therapeutics. In the past, our lab has expressed 15 mammalian PDE genes in S. pombe, replacing the endogenous Cgs2 PDE. High throughput screens for PDE inhibitors identified novel compounds that show relevant biological activity in mammalian cell culture assays. The aim of this thesis is to develop tools to understand the mechanism of interaction between key regulators of the cAMP pathway and small molecules. The current study is comprised of two parts. In the first part of this thesis, I developed a genetic screen that detected alleles whose proteins remain active in the presence of BC54 and was to confirm the effect of the PDE4BT407A mutation using cell-based assays and in vitro enzyme assays. In the second part of this thesis, I developed and carried out HTSs using a PKA-repressed GFP reporter that can identify compounds that reduce PKA activity, which would include PDE activators and AC or GNAS1 inhibitors. To date, I have identified three AC inhibitors that appear to act on several of the ten different mammalian ACs. To our knowledge, this is the first time a large HTS has identified AC inhibitors, where inhibition was assessed inside the cells. The findings in this thesis will be useful in the design of more effective PDE inhibitors and in the development of novel chemical probes for studying cAMP signaling in mammalian cells. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis (M.S.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / 2031-01-01

This dissertation is an historical anthropology investigating the late 19th century liturgical landscapes of the National Camp‐Meeting Association for the Promotion of Holiness, an organization of Methodist clergy who sought ecclesiastical and social reform primarily through camp‐meeting revivals promoting the experience of entire sanctification. National camp meetings drew from the liturgical and architectural traditions of early 19th century frontier revivalism, yet, as this dissertation argues, these meetings were not simply an appropriation of the structure of Second Great Awakening revivals for the purpose of promoting holiness theology in decidedly more urban areas of the Northeast and Mid‐Atlantic. Rather, these meetings were a (re)imagining of the cultural practice of the camp‐meeting through a Victorian system of symbolic meanings, a middle‐class, (ex)urban geographic context, and a distinctive set of liturgical performances, social interactions, and cognitive‐environmental and architectural cues designed to elicit a changed subjectivity among attendees. Each of these transformations shaped the social space, architectural configuration, and site selection of the liturgical landscapes of the National Camp‐Meeting Association, and it is these spatial and material traces that offer a substantial body of data for the interpretation of past religious and ritual landscapes in North America. Such interpretation of revival landscapes is possible through a process of cross‐mending archival sources (diaries, autobiographies, biographies, historic correspondence, newspaper reports, sermon texts, organizational documents, maps, photographs), material culture, archaeological reports, geo‐spatial and environmental data to reconstruct and thickly interpret the ritual landscapes of three early meetings of the National Camp‐Meeting Association for the Promotion of Holiness – Vineland, New Jersey, Manheim, Pennsylvania, and Round Lake, New York. In its results, this dissertation argues for a significant connection between Methodism, geographic regions, and 19th century holiness practices, and an interpretation of holiness revivalism as a means of renegotiating moral orders amidst industrialization, urbanization, vacationing, and changing social fault lines in the church including race and gender.

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.