Echo Christiana : Zu Lob vnd Ehren dem frölichen Geburtstag, vnsers Einigen Erlösers vnnd Seligmachers Jesu Christi, Gottes vnd Marien Sohn

Bibliograph. Nachweis: Harms, Dt. Illustr. Flugbl., III, 10 (Ausg. 1619); Straus, German single-leaf woodcut 1600-1700, p. 202 (Ausg. 1607)

Birgit Engell als Echo, Rollenbild, in: Richard Strauss: Ariadne auf Naxos

Signatur des Originals: S 36/F03557

Birgit Engell als Echo, Rollenbild, in: Richard Strauss: Ariadne auf Naxos

Signatur des Originals: S 36/F03558

Birgit Engell als Echo, Rollenbild, in: Richard Strauss: Ariadne auf Naxos

Signatur des Originals: S 36/F03559

Multiple gradient echo propeller (MGREP) MRI: Technical development and potential applications

The PROPELLER (Periodically Rotated Overlapping Parallel Lines with Enhanced Reconstruction) magnetic resonance imaging (MRI) technique has inherent advantages over other fast imaging methods, including robust motion correction, reduced image distortion, and resistance to off-resonance effects. These features make PROPELLER highly desirable for T2*-sensitive imaging, high-resolution diffusion imaging, and many other applications. However, PROPELLER has been predominantly implemented as a fast spin-echo (FSE) technique, which is insensitive to T2* contrast, and requires time-inefficient signal averaging to achieve adequate signal-to-noise ratio (SNR) for many applications. These issues presently constrain the potential clinical utility of FSE-based PROPELLER. ^ In this research, our aim was to extend and enhance the potential applications of PROPELLER MRI by developing a novel multiple gradient echo PROPELLER (MGREP) technique that can overcome the aforementioned limitations. The MGREP pulse sequence was designed to acquire multiple gradient-echo images simultaneously, without any increase in total scan time or RF energy deposition relative to FSE-based PROPELLER. A new parameter was also introduced for direct user-control over gradient echo spacing, to allow variable sensitivity to T2* contrast. In parallel to pulse sequence development, an improved algorithm for motion correction was also developed and evaluated against the established method through extensive simulations. The potential advantages of MGREP over FSE-based PROPELLER were illustrated via three specific applications: (1) quantitative T2* measurement, (2) time-efficient signal averaging, and (3) high-resolution diffusion imaging. Relative to the FSE-PROPELLER method, the MGREP sequence was found to yield quantitative T2* values, increase SNR by ∼40% without any increase in acquisition time or RF energy deposition, and noticeably improve image quality in high-resolution diffusion maps. In addition, the new motion algorithm was found to improve the performance considerably in motion-artifact reduction. ^ Overall, this work demonstrated a number of enhancements and extensions to existing PROPELLER techniques. The new technical capabilities of PROPELLER imaging, developed in this thesis research, are expected to serve as the foundation for further expanding the scope of PROPELLER applications. ^

The roles of costimulatory molecules in the cooperative effects of combination epinephrine and dexamethasone on type-1/type-2 cytokine production in tetanus-toxoid specific stimulated human peripheral blood mononuclear cells

A growing number of studies show strong associations between stress and altered immune function. In vivo studies of chronic and acute stress have demonstrated that cognitive stressors are strongly correlated with high circulating levels of catecholamines (CT) and corticosteroids (CS) that are associated with changes in type-1/type-2 cytokine expression. Although individual pharmacologic doses of CS and CT can inhibit the expression of T-helper 1 (Th1, type-1 like) and promote the production of T-helper 2 (Th2, type-2 like) cytokines in antigen-specific and mitogen stimulated human leukocyte cultures in vitro, little attention has been focused on the effects of combination physiologic-stress doses of CT and CS that may be more physiologically relevant. In addition, both in-vivo and in-vitro studies suggest that the differential expression of the B7 family of costimulatory molecules CD80 and CD86 may promote the expression of type-1 or type-2 cytokines, respectively. Furthermore, corticosteroids can influence the expression of β2-adrenergic receptors in various human tissues. We therefore investigated the combined effects of physiologic-stress doses of in vitro CT and CS upon the type-1/type-2 cytokine balance and expression of B7 costimulatory molecules of human peripheral blood mononuclear cells (PBMC) as a model to study the immunomodulatory effects of physiologic stress. Results demonstrated a significant decrease in type-1 cytokine expression and a significant increase in type-2 cytokine production in our CS+CT incubated cultures when compared to either CT or CS agents alone. In addition, we demonstrated the differential expression of CD80/CD86 in favor of CD86 at the cellular and population level as determined by flow cytometry in lipopolysaccharide stimulated human Monocytes. Furthermore, we developed flow cytometry based assays to detect total β2AR in human CD4+ T-lymphocytes that demonstrated decreased expression of β2AR in mitogen stimulated CD4+ T-lymphocytes in the presence of physiologic stress levels of CS and CT as single in vitro agents, however, when both CS and CT were combined, significantly higher expression of β2AR was observed. In summary, our in vitro data suggest that both CS and CT work cooperatively to shift immunity towards type-2 responses. ^

Ligand-stimulated internalization of a Dictyostelium discoideum G protein-coupled cAMP receptor

The social amoeba, Dictyostelium discoideum, undergoes a remarkable starvation-induced program of development that transforms a population of unicellular amoebae into a fruiting body composed of resistant spores suspended on a stalk. During this development, secreted cAMP drives chemotaxis of the amoebae, leading to their aggregation, and subsequent differentiation and morphogenesis. Four sequentially expressed G protein-coupled receptors (GPCRs) for cAMP play critical roles in this process. The first of these, cAR1, is essential for aggregation as it mediates chemotaxis as well as the propagation of secreted cAMP waves throughout aggregating populations. Ligand-induced internalization has been shown to regulate a variety of GPCRs. However, little was known at the outset of this study about the role of internalization in the regulation of cAR1 function or, for that matter, in developmental systems in general. For this study, cAMP-induced cAR1 internalization was assessed by measuring (1) the reduction of cell surface binding sites for [ 3H]cAMP and (2) the redistribution of YFP-tagged receptors to the cell's interior, cAMP was found to induce little or no loss of ligand binding (LLB) in vegetative cells. However, the ability to induce LLB increased progressively over the initial 6 hrs of development, reaching ∼70% in cells undergoing aggregation. Despite these reductions in surface binding, detectable cAR1-YFP redistribution could be induced by cAMP only after the cells reached the mound stage (10 hrs) and was found to occur naturally by the ensuing slug stage (18 hrs). Site-directed substitution of a cluster of 5 serines in the receptor's cytoplasmic tail that was previously shown to be the principal site of cAMP-induced cAR1 phosphorylation impaired both LLB and receptor redistribution and furthermore resulted in mound-stage developmental arrest, suggesting that phosphorylation of cAR1 is a prerequisite for its internalization and that cAR1 internalization is required for post-aggregative development. To assess the involvement of clathrin mediated endocytosis, Dictyostelium cells lacking the clathrin light chain gene (clc-) or either of two dynamin genes were examined and found to be defective in LLB and, in the case of clc- cells, also cAR1 redistribution and turnover. Furthermore, cAR1 overexpression in clc- cells (like the serine mutant in wild-type cells) promoted developmental arrest in mounds. The mound-arrest phenotype was also recapitulated in a wild-type background by the specific expression of cAR1 in prestalk cells (but not prespore cells), suggesting that development depends critically on internalization and clearance of cAR1 from these cells. Persistent cAR1 expression following aggregation was found to be associated with aberrant expression of prestalk and prespore genes, which may adversely affect development in the prestalk cell lineage. The PI3 kinase-TORC2 signal transduction pathway, known to be important for Dictyostelium chemotaxis and internalization of yeast pheromone receptors, was examined using chemical inhibitors and null cells and found to be necessary for cAR1 internalization. In conclusion, cAR1 was shown to be similar to other GPCRs in that its internalization depends on phosphorylation of cytoplasmic domain serines, utilizes clathrin and dynamin, and involves the TORC2 complex. In addition, the findings presented here that cAR1 internalization is both developmentally regulated and required for normal development represent a novel regulatory paradigm that might pertain to other GPCRs known to play important roles in the development of humans and other metazoans. ^

DEVELOPMENT AND IMPLEMENTATION OF A REMOTE AUDIT TOOL FOR HIGH DOSE RATE (HDR) 192IR BRACHYTHERAPY USING OPTICALLY STIMULATED LUMINESCENCE DOSIMETRY

This work aimed to create a mailable and OSLD-based phantom with accuracy suitable for RPC audits of HDR brachytherapy sources at institutions participating in NCI-funded cooperative clinical trials. An 8 × 8 × 10 cm3 prototype with two slots capable of holding nanoDot Al2O3:C OSL dosimeters (Landauer, Glenwood, IL) was designed and built. The phantom has a single channel capable of accepting all 192Ir HDR brachytherapy sources in current clinical use in the United States. Irradiations were performed with an 192Ir HDR source to determine correction factors for linearity with dose, dose rate, and the combined effect of irradiation energy and phantom construction. The uncertainties introduced by source positioning in the phantom and timer resolution limitations were also investigated. It was found that the linearity correction factor was where dose is in cGy, which differed from that determined by the RPC for the same batch of dosimeters under 60Co irradiation. There was no significant dose rate effect. Separate energy+block correction factors were determined for both models of 192Ir sources currently in clinical use and these vendor-specific correction factors differed by almost 2.6%. For Nucletron sources, this correction factor was 1.026±0.004 (99% Confidence Interval) and for Varian sources it was 1.000±0.007 (99% CI). Reasonable deviations in source positioning within the phantom and the limited resolution of the source timer had insignificant effects on the ability to measure dose. Overall measurement uncertainty of the system was estimated to be ±2.5% for both Nucletron and Varian source audits (95% CI). This uncertainty was sufficient to establish a ±5% acceptance criterion for source strength audits under a formal RPC audit program. Trial audits of eight participating institutions resulted in an average RPC-to-institution dose ratio of 1.000 with a standard deviation of 0.011.

Development and Implementation of the Use of Optically Stimulated Luminescent Detectors in the Radiological Physics Center Anthropomorphic Quality Assurance Phantoms

The Radiological Physics Center (RPC) uses both on-site and remote reviews to credential institutions for participation in clinical trials. Anthropomorphic quality assurance (QA) phantoms are one tool the RPC uses to remotely audit institutions, which include thermoluminescent dosimeters (TLDs) and radiochromic film. The RPC desires to switch from TLD as the absolute dosimeter in the phantoms, to optically stimulated luminescent dosimeters (OSLDs), but a problem lies in the angular dependence exhibited by the OSLD. The purpose of this study was to characterize the angular dependence of OSLD and establish a correction factor if necessary, to provide accurate dosimetric measurements as a replacement for TLD in the QA phantoms. A 10 cm diameter high-impact polystyrene spherical phantom was designed and constructed to hold an OSLD to study the angular response of the dosimeter under the simplest of circumstances for both coplanar and non-coplanar treatment deliveries. OSLD were irradiated in the spherical phantom, and the responses of the dosimeter from edge-on angles were normalized to the response when irradiated with the beam incident normally on the surface of the dosimeter. The average normalized response was used to establish an angular correction factor for 6 MV and 18 coplanar treatments, and for 6 MV non-coplanar treatments specific to CyberKnife. The RPC pelvic phantom dosimetry insert was modified to hold OSLD, in addition to the TLD, adjacent to the planes of film. Treatment plans of increasing angular beam delivery were developed, three in Pinnacle v9.0 (4-field box, IMRT, and VMAT) and one in Accuray’s MultiPlan v3.5.3 (CyberKnife). The plans were delivered to the pelvic phantom containing both TLD and OSLD in the target volume. The pelvic phantom was also sent to two institutions to be irradiated as trials, one delivering IMRT, and the other a CyberKnife treatment. For the IMRT deliveries and the two institution trials, the phantom also included film in the sagittal and coronal planes. The doses measured from the TLD and OSLD were calculated for each irradiation, and the angular correction factors established from the spherical phantom irradiations were applied to the OSLD dose. The ratio of the TLD dose to the angular corrected OSLD dose was calculated for each irradiation. The corrected OSLD dose was found to be within 1% of the TLD measured dose for all irradiations, with the exception of the in-house CyberKnife deliveries. The films were normalized to both TLD measured dose and the corrected OSLD dose. Dose profiles were obtained and gamma analysis was performed using a 7%/4 mm criteria, to compare the ability of the OSLD, when corrected for the angular dependence, to provide equivalent results to TLD. The results of this study indicate that the OSLD can effectively be used as a replacement for TLD in the RPC’s anthropomorphic QA phantoms for coplanar treatment deliveries when a correction is applied for the dosimeter’s angular dependence.

ATP- and carbachol -stimulated 1,2-diacylglycerol production during sensitization of adenylyl cyclase

Stimulation of LM5 cells with the phorbol ester 4$\beta$-phorbol 12-myristate 13-acetate (PMA), causes a 2-4 fold sensitization of hormonally-stimulated adenylyl cyclase (AC) activity. This effect is thought to be due to protein kinase C (PKC)-mediated phosphorylation of either G$\sb{\rm i}$ or the catalytic subunit of AC. PKC are components of the phosphatidylinositol-4,5-bisphosphate phospholipase C (PIP$\sb2$-PLC) pathway. The currently accepted model of this pathway is that its activation by an agonist results in the production of inositol 1,4,5-triphosphate (IP$\sb3$) which causes Ca$\sp{++}$ mobilization, and 1,2-diacylglycerols (DAG) which activate PKC. Based on this model, we predicted that stimulation of purinergic and muscarinic receptors with the agonists ATP and carbachol (CCh), respectively in the LM5 cells, should sensitize AC. Surprisingly we found that only stimulation of the purinergic receptors in these cells caused a sensitization of PGE$\sb1$-stimulated AC measured in cell-free assays.^ We hypothesized that ATP-and CCh-stimulated differential DAG production contributes to the effectiveness of these two agonists to sensitize PGE$\sb1$-stimulated AC activity. To test this hypothesis directly, we performed a combined high-performance liquid chromatography and gas-liquid chromatography analysis of the DAG produced in the LM5 cells in response to stimulation with ATP and CCh.^ We found that both ATP and CCh increased levels of 23 species of DAG. Relative to the control levels (0.261 nmol DAG/100 nmol phospholipid) the CCh-induced increase in DAG levels was 280% (0.738 $\pm$ 0.051 nmol DAG/100 nmol phospholipid) whereas the ATP-induced levels increased 180% (0.441 t 0.006 nmol DAG/100 nmol phospholipid). Neither agonist created new species or eliminated the existing ones. The major species which comprised $\approx$50% of the total cellular DAG in all of the groups were 16:0-18:1, 18:0-18:1, 18:1-18:1, and 18:0-20:4. CCh was more effective than ATP at stimulating these major DAG species.^ It is concluded that factor(s) other than DAG contribute(s) to the differences between ATP-and CCh-sensitization of PGE$\sb1$-stimulated AC activity in the LM5 cells. ^

Inhibitor of cytochrome c messenger RNA -protein interaction in stimulated rat skeletal muscle

The purpose of this work was to examine the possible mechanisms for the regulation of cytochrome c gene expression in response to increased contractile activity in rat skeletal muscle. The working hypothesis was that increased contractile activity enhances cytochrome c gene expression through a cis-element. A 110% increase in cytochrome c mRNA concentration was observed in tibialis anterior (TA) muscle after 9 days of chronic stimulation. Similar difference (120%) exists between soleus (SO) muscle of higher contractile activity and white vastus lateralis (WV) muscle of lower contractile activity. These results suggest that the endogenous cytochrome c gene expression is regulated by contractile activity. Cytochrome c-reporter genes were injected into skeletal muscles to identify the cis-element that is responsible for the regulation. Although the data was inconclusive, part of it suggested the importance of the 3$\sp\prime$-untranslated region (3$\sp\prime$-UTR) in mediating the response to increased contractile activity.^ RNA gel mobility shift (GMSA) and ultraviolet (UV) cross-linking assays revealed specific RNA-protein interaction in a 50-nucleotide region of the 3$\sp\prime$-UTR in unstimulated TA muscle. Computer analysis predicted a stem-loop structure of 17 nucleotides, which provides a structural basis for RNA-protein interaction. These 17 nucleotides are 100% conserved among rat, mouse and human cytochrome c genes and their 13 pseudogenes, suggesting a functional role for this region. The RNA-protein interaction was significantly less in highly active SO muscle than in inactive WV muscle and was dramatically decreased in stimulated TA muscle due to a protein inhibitor(s) associated with ribosome. It is possible that cytochrome c mRNAs undergoing translation are subject to a compartmentalized regulatory influence.^ The conclusion from these results is that increases in contractile activity induce or activate a protein inhibitor(s) associated with ribosome in rat skeletal muscle. The inhibitor decreases RNA-protein interaction in the 3$\sp\prime$-UTR of cytochrome c mRNA, which may result in increased mRNA stability and/or translation. ^