Painted upon a Grecian urn for wind ensemble

Title from PDF of title page (University of Missouri--Columbia, viewed on September 9, 2013).The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Thesis advisor: Dr. W. Thomas McKenneyInstrumentation: piccolo, flute, oboe, clarinet in E♭, clarinet in B♭, bass clarinet in B♭, contrabass clarinet in B♭, bassoon, contrabassoon, soprano saxophone, alto saxophone, tenor saxophone, baritone saxophone, trumpet in B♭, horn in F1, horn in F2, trombone, bass trombone, euphonium, tuba, piano, harp, marimba, vibraphone, chimes/glockenspiel/tambourine, timpani, tom-toms, cymbals, snare drum, field drum, concert bass drum, tam-tam, brake drumMaster of Music University of Missouri--Columbia 2013.Dissertations, Academic -- University of Missouri--Columbia -- Music."May 2013"Painted Upon A Grecian Urn is a three-movement suite for modified wind ensemble of about fifteen minutes in length in which each movement represents a different legend from Greek mythology. The movements embody Athena, Orpheus, and Dionysus respectively and are titled as follows: I: Wisdom, Purity, and Reason. [4'30"] II: Noble Lyre. [6'30"] III: Ecstatic Joy and Savage Brutality. [4'0"] Each movement is united melodically, harmonically, and rhythmically by motives derived from the Hillsic scale, a variant of an all-interval pitch collection found in Nicolas Slonimsky's Thesaurus of Scales and Melodic Patterns. The compositional techniques utilized throughout include but are not limited to transpositions of scale patterns as well as inversion and symmetry

A role for Collagen I in regulating Connexin-43 mediated hemichannel activity in the proximal region of the diabetic kidney.

Background and Aims: Tubulointerstitial fibrosis represents the key underlying pathology in diabetic kidney disease and is characterised by tubular injury and extracellular matrix remodelling. In diabetic nephropathy increased Collagen I deposition is linked to inflammation and fibrosis, yet, we know little as to how collagen facilitates these effects. Pivotal to early tubular injury is impaired epithelial (E)-cadherin mediated cell adhesion; loss of which inhibits docking of connexin-mediated hemichannels and prevents gap junction intercellular communication. Uncoupled hemi-channels respond via increased release of adenosine triphosphate (ATP), an effect we have previously confirmed is linked to increased expression of inflammatory and fibrotic markers. The current study investigates if Collagen I mediates its effects through altered connexin-43 (Cx43) mediated hemichannel activity in human proximal tubule cells. Materials and Methods: Human kidney (HK-2) proximal tubule cells were cultured on Collagen I (50µg/mL) and treated for 48 hours ± TGF-β1 (2-10ng/mL). Uncoated plastic served as the control. Expression of candidate proteins was determined by immunoblotting. Cell-substrate interactions were measured using the Cytoselect cell adhesion assay. Carboxyfluorescein (200µM) dye uptake and ATP biosensing were used to measure hemi-channel activity and ATP release respectively in TGF-β1 treated HK-2 at 48 hours. Results: Immunoblot analysis confirmed that TGF-β1 increased Collagen I expression to 167±19%, 181±8% and 192±19% at 2, 4 and 10ng/mL respectively as compared to control, whilst adhesion assays confirmed that HK-2 cells exhibit increased affinity for Collagen I in the presence of TGF-β1 (10ng/mL) 0.36±0.06OD as compared to control (0.15±0.02D) (n=4, P<0.05). Interestingly, when cultured on Collagen I, cells exhibit increased expression of Cx43 to 107±16%, with co-incubation of TGF-1 exacerbating this effect to 136±7% as compared to control (n=5, P<0.001). This increase in expression was paralleled by increased hemichannel activity and ATP release. Carboxyfluorescein dye uptake increased in cells cultured on Collagen I ± TGF-1 to 149±6% (Collagen I) and 251±5% (Collagen I + TGF-1) respectively, as compared to control (n=5, P<0.001), whilst biosensing confirmed a significant increase in ATP, increased from 3.6±0.4µM (Collagen I) to 4.5±0.2µM (Collagen I + TGF-1) as compared to control (n=3, P<0.001). Conclusion: The current study confirms a role for Collagen I in regulating Connexin43 expression, changes which are exacerbated in the presence of pro-fibrotic TGF-1, the principal mediator of damage in the tubular region of the diabetic kidney. Increased expression of this protein, central to cell-cell communication, was paralleled by increased hemichannel mediated ATP release, most likely a consequence of diminished cell-cell adhesion and Gap Junction mediated intercellular communication as previously reported by the group. With studies linking increased Collagen I deposition to altered cell phenotype in tubulointerstitial fibrosis, and elevated ATP release to increased fibrosis and inflammation; this study highlights a potential role for Collagen I in exacerbating diabetic tubular injury by regulating connexin-mediated hemi-channel activity

A role for ATP in renal fibrosis as a downstream mediator of TGF-β1-evoked changes in hemichannel activity

Aims: Data from our group confirms that increased expression of Connexin-26 and Connexin-43 in biopsy material from patients with proven nephropathy is paralleled by TGF-1-evoked changes in hemichannel-mediated ATP release. With recent studies linking increased ATP to fibrosis in multiple tissue types, we have previously confirmed that incubation of proximal tubule cells with ATPS (1-100M) evokes increased expression of ECM markers. In the current study, we provide evidence of a direct role for ATP and associated downstream purinergic signalling as a mediator of TGF-1 induced altered hemichannel activity in ECM expression. Methods: Human kidney (HK2) proximal tubule cells were treated for 48hrs with either TGF-β1(10ng/mL) ± nucleotidase; apyrase (100µM) or ATPγS (10µM) ± purinergic receptor antagonist suramin (100µM). Expression of Collagen I, Collagen IV, Fibronectin and Laminin were determined by immunoblotting. Results: Immunoblotting confirmed that apyrase negated TGF-β1 upregulation of Collagen I, from 366.0±13.0% of control to 119.5% (n=3 P<0.001) and reversed loss of Collagen IV expression from 30.1±12.1% of control to 123.3±9.8% (n=3 P<0.01). The nucleotidase also negated an upregulation of Fibronectin, from 201.5±3.7% to 156.7±27.6% (n=3 P<0.01) and Laminin, from 339.5±43.1% to 173.4±42.8% (n=3 P<0.05). Suramin inhibited ATPγS-induced changes in expression of ECM, reducing expression of Collagen I from 452.9±20.6% of control to 192.1±16.0%; Collagen IV from 157.2±17.4% to 93.6±12.8%; Fibronectin from 222.6±9.5% to 103.7±5.2% and Laminin from 177.8±25.0% to 86.4±11.7% (n=3 P<0.05). Conclusions: The current study confirms that TGF-1 induced changes in hemichannel mediated ATP release may in part, contribute to tubular fibrosis in the diabetic kidney. Acknowledgement: This work is supported by Diabetes UK (BDA:16/0005427 and BDA:16/0005509)

Purinergic receptor (P2X7) activation reduces cell–cell adhesion between tubular epithelial cells of the proximal kidney

Loss of epithelial (E)-cadherin mediated cell-cell adhesion impairs gap junction formation and facilitates hemichannel-mediated ATP release in the diabetic kidney. Linked to inflammation and fibrosis, we hypothesized that local increases in inter-cellular ATP activate P2X7 receptors on neighbouring epithelial cells of the proximal tubule, to further impair cell-cell adhesion and ultimately exacerbate tubular injury. Immunoblotting confirmed changes in E-cadherin expression in human kidney cells treated with non-hydrolysable ATPγS ± the P2X7 antagonist, A438079. Atomic force microscopy based single-cell force spectroscopy quantified maximum unbinding force, tether rupture events, and work of detachment. Confocal microscopy assessed cytoskeletal reorganisation. Our studies confirmed that ATPγS downregulated E-cadherin expression in proximal kidney cells, loss of which was paralleled by a reduction in intercellular ligation forces, decreased tether rupture events and cytoskeletal remodelling. Co-incubation with A438079 restored loss of adhesion, suggesting that elevated extracellular ATP mediates tubular injury through P2X7 induced loss of E-cadherin mediated adhesion

Purinergic receptor (P2X7) activation contributes to disassembly of adherens & tight junctions in tubular epithelial cells of the diabetic kidney.

Background and aims: Preceded by a loss of cell-cell adhesion, glucose-evoked changes in connexin (Cx) expression/function are linked to increased hemichannel mediated release of adenosine triphosphate (ATP) in tubular epithelial cells of the diabetic kidney. Elevated ATP is associated with inflammation and fibrosis, and this study investigates a role for Cx43 hemichannel-mediated ATP release in regulating adherens- and tight-junction proteins, e!ects that initiate the morphological and phenotypic changes of tubular damage. Materials and methods: Human primary proximal tubule epithelial cells (HPTECs) and clonal tubular epithelial cells (HK2) were cultured in TGFβ1 (10ng/mL) ± apyrase (5U/ml), or non-hydrolysable ATPγS (100μM) at 48h. Immunoblotting assessed protein expression. Trans-epithelial electrical resistance assessed paracellular tight junction formation and atomic force microscopy force spectroscopy measured cell-cell adhesion. Carboxyfluorescein uptake and ATP biosensing measured hemichannel activity and nucleotide release. Co-incubation of cells with TGFβ1 ± Peptide5 (25μM) or A438079 (50μM) assessed e!ect of Cx43 hemichannel and purinergic receptor (P2X7) blockade respectively. Results: Immunoblotting confirmed that TGFβ1 downregulated E-cadherin (ECAD), claudin-2 and ZO-1 to 38.5±4.1%, 60.5±4.4% and 64.8±4.4% respectively, whilst N-cadherin (NCAD) expression increased to 213.3±28.0% compared to control (P<0.01; n=4). The e!ect was replicated by ATPγS, which decreased expression of ECAD, claudin-2 and ZO-1 to 43.4±6.1%, 42.0±2.6% and 45.9±1.4% respectively. NCAD increased to 181.3±6.3% (P<0.01; n=3). In a separate series of experiments, co-incubation with the ectonucleotidase apyrase partially restored ECAD expression to 51.2±3.2%, and NCAD to 133.3±9.1%, compared to control (P<0.001; n=3). Trans-epithelial resistance decreased in TGFβ1 and ATPγS treated cells from 67.7±5.5Ω.cm2to 27.6±2.0Ω.cm2 and 42.6±3.0Ω.cm2 respectively (P<0.05; n=3). Mean unbinding forces between ATPγS treated cells also decreased from 2.17±0.64nN in control cells to 1.60±0.48nN (P<0.001; n=3) confirming a loss of cell-cell adhesion. Increased carboxyfluorescein uptake (609.4±46.0%) and ATP release (6.10±0.36μM from 0.43±0.03μM) confirmed increased hemichannel mediated ATP release in TGFβ1 treated cells, an effect blocked by Cx43 mimetic, Peptide5 (163.0±10.2% and 0.60±0.20μM) (P<0.001; n=3). Co-incubation of HPTECs with TGFβ1 and Peptide5 restored expression of ECAD (108.9±17.1% from 31.5±9.2%), NCAD (154.7±10.6% from 280.5±16.7%), claudin-2 (100.9±10% from 65.3±5.4%) and ZO-1 (91.6±12.8% from 59.6±3.1%) compared to control (P<0.01; n=3). Blocking P2X7 with A438079 restored ECAD expression from 22.2±5.5% to 52.8±5.4% in TGFβ1 treated cells (P<0.001; n=3), with unbinding forces restored from 2.17±0.64nN to 2.45±0.89nN (P<0.001; n=3). Conclusion: Hemichannel mediated ATP release is downstream of TGFβ1-evoked changes to adherens- and tight-junction proteins, e!ects blocked by inhibiting P2X7 receptors or Cx43 hemichannel activity. Disassembly of these junctions is a pivotal event in progression of tubular injury in diabetic nephropathy and data suggests a potential role for Cx-mediated hemichannel activity as a future therapeutic target in diabetic kidney disease

ATP reduces functional cell-to-cell tethering between renal tubular epithelial cells

Background: Loss of epithelial (E)-cadherin mediated cell-cell adhesion impairs gap junction formation and facilitates hemichannel-mediated ATP release in the diabetic kidney. Linked to inflammation and fibrosis, we hypothesize that local increases in inter-cellular ATP activate P2X7 receptors on neighbouring epithelial cells of the proximal tubule, to further impair cell-cell adhesion and exacerbate tubular injury. Methods: Atomic force microscopy (AFM)-single cell force spectroscopy (SCFS) was used to quantify the unbinding forces needed to separate clonal tubular epithelial cells of the proximal kidney (HK2) following treatment with non-hydrolysable ATPγS (100μM) ± the P2X7 purinergic receptor antagonist A438079 (50μM) over 48hr. Densitometry was used to semi-quantify Western blot protein expression. Biophysical measurements quantified maximum unbinding force (Fmax), tether (or ligation cluster) rupture events (TREs) and work of detachment (Wd). Three force-displacement curves were acquired per cell. Data from SCFS are expressed as mean ± SEM. Sample numbers refer to separate cell passages (n) from multiple individual cells. Data was evaluated using univariate anova followed by Tukey’s multiple comparisons post-test. P<0.05 was taken to indicate statistical significance. Results: The P2-purinergic receptor agonist ATPγS (100μM), decreased E-cadherin expression by 54.4±4% compared to control (P<0.01, n=3), an effect negated by the P2X7 antagonist A438079 (50M; 113.6±9.7% of control). The agonist (100M) weakened intercellular ligation forces (Fmax) by 26.8% (P<0.001; n=3) and affected the formation of ligation clusters by reducing tether rupture events (TREs) by 22.3% (P<0.001; n=3), effects largely reversed by A438079 (50M). Total energy consumed during the pulling process prior to complete cell separation, was calculated by the integration of the retraction Force-Displacement curve and is referred as the work of adhesion Wd. Our results showed that control cells exhibited a Wd of 21.85fJ±1.1, whilst the total energy consumed in separating ATPγS (50M) treated cells was reduced to 13.32fJ±1.13, indicating a reduction of 39% (P<0.001; n=3). Co-incubation with A438079 (50M) increased Wd back to control (23.7fJ±1.22, n=3) and negated the response to ATP. Conclusion: Determining strength of adhesion in disease is challenging due to the underlying molecular assembly that regulates formation of the adherens junction. Force-displacement measurements during cell-cell separation can provide valuable information about the response to ligands and their antagonists. Nanoscale force-displacement measurements capture the molecular activity underlying ATPγS evoked changes in cell adhesion, loss of which appears to be mediated by downstream P2X7 receptor activation and supports a role for P2X7 as a potential therapeutic target in managing progression of diabetic nephropathy

Danegaptide Prevents TGFβ1-Induced Damage in Human Proximal Tubule Epithelial Cells of the Kidney

Chronic kidney disease (CKD) is a global health problem associated with a number of comorbidities. Recent evidence implicates increased hemichannel-mediated release of adenosine triphosphate (ATP) in the progression of tubulointerstitial fibrosis, the main underlying pathology of CKD. Here, we evaluate the effect of danegaptide on blocking hemichannel-mediated changes in the expression and function of proteins associated with disease progression in tubular epithelial kidney cells. Primary human proximal tubule epithelial cells (hPTECs) were treated with the beta1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± danegaptide. qRT-PCR and immunoblotting confirmed mRNA and protein expression, whilst a cytokine antibody array assessed the expression/secretion of proinflammatory and profibrotic cytokines. Carboxyfluorescein dye uptake and ATP biosensing measured hemichannel activity and ATP release, whilst transepithelial electrical resistance was used to assess paracellular permeability. Danegaptide negated carboxyfluorescein dye uptake and ATP release and protected against protein changes associated with tubular injury. Blocking Cx43-mediated ATP release was paralleled by partial restoration of the expression of cell cycle inhibitors, adherens and tight junction proteins and decreased paracellular permeability. Furthermore, danegaptide inhibited TGFβ1-induced changes in the expression and secretion of key adipokines, cytokines, chemokines, growth factors and interleukins. The data suggest that as a gap junction modulator and hemichannel blocker, danegaptide has potential in the future treatment of CKD

Collagen I Modifies Connexin‐43 Hemichannel Activity via Integrin α2β1 Binding in TGFβ1‐Evoked Renal Tubular Epithelial Cells

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel‐mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up‐regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin‐43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro‐fibrotic cytokine transforming growth factor (TGFβ1) ± the Cx43 mimetic Peptide 5 and/or an anti‐integrin α2β1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell‐substrate interactions. Collagen I and TGFβ1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2β1/integrin‐like kinase signaling. The co‐incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFβ1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin‐mediated hemichannel activity through integrin α2β1 signaling, ahead of establishing Peptide 5 as a potential intervention

Connexin mediated cell communication in the kidney, a potential therapeutic target for future intervention of diabetic kidney disease?

The ability of cells to communicate and synchronise their activity is essential for the maintenance of tissue structure, integrity and function. A family of membrane-bound proteins called connexins are largely responsible for mediating the local transfer of information between cells. Assembled in the cell membrane as a hexameric connexon, they function either as a conduit for paracrine signalling, forming a trans-membrane hemi-channel or, if aligned with connexons on neighbouring cells, form a continuous aqueous pore, or gap junction, which allows for the direct transmission of metabolic and electrical signals. Regulation of connexin synthesis and activity is critical to cellular function and a number of diseases are attributed to changes in the expression and/or function of these important proteins. A link between hyperglycaemia, connexin expression, altered nucleotide concentrations and impaired function, highlights a potential role for connexin-mediated cell communication in complications of diabetes. In the diabetic kidney, glycaemic injury is the leading cause of end stage renal failure, reflecting multiple aetiologies including glomerular hyperfiltration, albuminuria, increased deposition of extracellular matrix, and tubulointerstitial fibrosis. Loss of connexin-mediated cell-to-cell communication in diabetic nephropathy may represent an early sign of disease progression, however, our understanding of the process remains severely limited. This review focusses on recent evidence demonstrating that glucose-evoked changes in connexin mediated cell communication and associated purinergic signalling, may contribute to the pathogenesis of kidney disease in diabetes, highlighting the tantalising potential of targeting these proteins as a novel therapeutic intervention

Factors predicting drop out from, and retention in, specialist drug treatment services: A case control study in the North West of England

<p>Abstract</p> <p>Background:</p> <p>In the United Kingdom (UK), the National Treatment Agency for Substance Misuse (NTA) considers retention to be the best available measure of drug treatment effectiveness. Accordingly, the NTA has set local treatment systems the annual target of retaining 75% of clients for 12 weeks or more, yet little assessment of this target or factors that improve retention has occurred. This study aims to quantify the proportion of people retained in treatment for 12 weeks in the North West of England and to identify factors associated with premature drop out.</p> <p>Methods:</p> <p>The North West National Drug Treatment Monitoring System (NDTMS) was used to identify treatment durations for everyone beginning a treatment episode between 1^stApril 2005 and 31^stMarch 2006 (N = 16626). Odds ratios, chi-square and logistic regression analyses compared clients retained for 12 weeks to clients whose discharge record showed they had prematurely dropped out before 12 weeks. Individuals with other outcomes were excluded from analyses.</p> <p>Results:</p> <p>75% of clients (N = 12230) were retained for 12 weeks and 10% (N = 1649) dropped out prematurely. Multivariate analysis showed drop out was more likely among Asian drug users (adjusted odds ratio 1.52, 95% CI 1.12 to 2.08) than their white equivalents. Drop out was more likely among residents of Cumbria and Lancashire (adjusted odds ratio 1.80, 95% CI 1.51 to 2.15) and Greater Manchester (adjusted odds ratio 2.00, 95% CI 1.74 to 2.29) than Cheshire and Merseyside and less likely among alcohol users (adjusted odds ratio 0.73, 95% CI 0.59 to 0.91). A significant interaction between age and deprivation was observed. For those aged 18 to 24 years and 25 to 34 years, drop out was significantly more likely among those living in affluent areas. For those in the older age groups the converse effect was observed.</p> <p>Conclusion:</p> <p>In combination, the drug treatment systems of the North West achieved the Government's retention target in 2005/06. A number of factors associated with drop out were identified; these should be considered in strategies that aim to improve retention. Drop out and retention are measures that capture the joint effect of many factors. Further work is required to evaluate the effect of deprivation.</p