The mammalian spinal cord contains the neuronal circuitry necessary to generate rhythmic locomotor activity in the absence of inputs from the higher brain centre or sensory system. This circuitry is regulated by local neuromodulatory inputs, which can adjust the strength and timing of locomotor output. The free radical gas nitric oxide has been shown to act as an important neuromodulator of spinal circuits, which control locomotion in other vertebrate models such as the tadpole and lamprey. Despite this, the involvement of the NO-mediated soluble guanylate cyclase/cyclic guanosine monophosphate secondary messenger-signalling pathway (NO/sGC/cGMP) in mammalian locomotion has largely been under-investigated. The NADPH diaphorase histochemical reaction was used to identify sources of NO in the lumbar spinal cord. The largest population NADPH diaphorase reactive neurons were located in the dorsal horn, followed by the laminae of the ventral horn, particularly around the central canal (lamina X) and lamina VII. NADPH diaphorase reactive neurons were found along a rostrocaudal gradient between lumbar segments L1 to L5. These results show that that discrete neuronal sources of NO are present in the developing mouse spinal cord, and that these cells increase in number during the developmental period postnatal day P1 – P12. NADPH diaphorase was subsequently used to identify NADPH diaphorase reactive neurons at P12 in the mouse model of ALS using the SODG93A transgenic mouse. Physiological recordings of ventral root output were made to assess the contribution of NO to the regulation induced rhythmic fictive locomotion in the in vitro isolated spinal cord preparation. Exogenous NO inhibits central pattern generator (CPG) output while facilitating and inhibiting motor neuron output at low and high concentrations respectively. Removal of endogenous NO increases CPG output while decreasing motor neuron output and these effects are mediated by cGMP. These data suggest that an endogenous tone of NO is involved in the regulation of fictive locomotion and that this involves the NO/sGC/cGMP pathway. Intracellular recordings from presumed motor neurons and a heterogeneous, unidentified sample of interneurons shows that NO modulates the intrinsic properties of spinal neurons. These data suggest that the net effect of NO appears to be a reduction in motor neuron excitability.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references (leaves 102-128).

Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 132 pages. Includes vita. Includes bibliographical references.

All cells are equipped to sense changes in their environment and make adaptive responses according to the stimuli. Signal recognition usually occurs at the cell membrane (with the exception of steroid signalling) where the ligand, which can be a small molecule, a peptide or a protein, binds its cognate receptor. This results in a change in the conformation of the receptor which in turn can regulate the production of second messengers. Second messengers can now modulate specific pathways which control gene expression and modify various aspects of cell behaviour. The signalling cascade is terminated by the removal of second messenger and/or by desensitisation of the receptor to the extracellular signal. Cyclic guanosine monophosphate (cGMP) was first identified in the rat urine and since then has emerged as an important second messenger regulating diverse cell processes. Subsequent to its discovery in mammalian cells, enzymes responsible for its synthesis (guanylyl cyclases), hydrolysis (phosphodiesterases) and its most common effectors (cGMP-dependent protein kinases) were identified. Guanylyl cyclases exist in two forms, cytosolic and membrane bound. Both have a conserved guanylyl cyclase domain, but differ in their choice of ligands, overall structure and tissue localization. It is now known that cytosolic and the membrane-bound forms are involved in eliciting distinct cellular responses. Receptor guanylyl cyclase C (GC-C) was identified as the target for a family of heat-stable enterotoxin toxins (ST) produced by enterotoxigenic E.coli. Stable toxin-mediated diarrhoeas are observed frequently in infants and contribute significantly to the incidence of Travellers’ Diarrhea. Early studies demonstrated that the effects of ST were mediated by an increase in intracellular cGMP levels in intestinal cells, and the receptor for ST was almost exclusively expressed in the apical microvilli of the intestinal brush-border epithelia. Effectors of cGMP in intestinal cells include protein kinase G (PKG), cyclic nucleotide gated ion channel 3 (CNG), and the cystic fibrosis transmembrane conductance regulator (CFTR). ST is an exogenous ligand which serves as a hyperagonist for GC-C, in comparison with the endogenous ligands guanylin and uroguanylin, which maintain fluid-ion homeostasis in the intestinal epithelia. The GC-C/cGMP signal transduction pathway also modulates intestinal cell proliferation along the crypt-villus axis by exerting a cytostatic effect on the epithelial cells, thereby regulating their turnover and neoplastic transformation. The current study describes in molecular detail two signalling pathways, one impinging on and one emerging from GC-C, which regulate colonic cell proliferation. The first part identifies the cross-talk and cross-regulation of GC-C and c-src. The second part delves into the molecular basis of GC-C/cGMP-mediated cytostasis and its effect on colonic tumorigenesis. Cross-talk between signalling pathways is believed to play a key role in regulating cell physiology. Phosphorylation of signalling molecules by protein kinases is frequently used as a means of achieving this cross-regulation. Aberrant hyperactivation of the c-src tyrosine kinase is an early event in the progression of colorectal cancer, and activated c-src specifically phosphorylates a number of proteins in the cell. It was found that c-src can phosphorylate GC-C in T84 colorectal carcinoma cells, as well as in the rat intestinal epithelia. Tyrosine phosphorylation of GC-C resulted in attenuation of ligand-mediated cGMP production; an effect which was reversed by chemical or transcriptional knockdown of c-src. These effects were found to be cell line-independent and relied only on the extent of c-src expression and activation in the cell. Mutational analysis revealed GC-C to be phosphorylated on a conserved tyrosine residue (Y820) in the guanylyl cyclase domain. The sequence of GC-C around Y820 allowed for efficient phosphorylation by c-src, and indeed, kinase assays indicated that the affinity of c-src for the GC-C Y820 peptide was one of the highest reported till date. A phospho-mimetic mutation at this site, which mimics a constitutively phosphorylated receptor, resulted in a sharp reduction of guanylyl cyclase activity of the receptor, reiterating the inhibitory role of Y820 phosphorylation on GC-C activity. Phosphorylation of GC-C at Y820 generated a docking site for the SH2 domain of c-src which could interact and thereby co-localize with GC-C on the cell membrane. Intriguingly, this interaction resulted in activation of c-src, setting-up a feed-forward loop of inhibitory GC-C phosphorylation and c-src activation. Treatment of colorectal carcinoma cells with ligands for GC-C reduces cell proliferation and inhibits tumorigenesis. It was observed that this cytostatic effect can be modulated by the status of c-src activation, and consequently, the fraction of tyrosine phosphorylated GC-C in these cells. Since activation of c-src is a frequent event in intestinal neoplasia, phosphorylation of GC-C by active c-src may be one of the means by which the cytostatic effects of GC-C agonists (guanylin and uroguanylin) in the intestine are bypassed, thereby leading to cancer progression. Colonisation of the gut with enteropathogenic microorganisms induces secretion of IFNγ from the host mucosal immune system, which subsequently activates c-src in intestinal epithelial cells. Ligand-stimulated activity of GC-C was found to be reduced in IFNγ treated cells. This could be one of the host defence mechanisms initiated in response to enterotoxigenic E. coli infection. These results provide the first evidence of cross-talk between a receptor guanylyl cyclase and a tyrosine kinase that results in heterologous desensitisation of the receptor. Populations with a higher incidence of enterotoxigenic E.coli infections appear to be protected from intestinal neoplasia. It was found that mice lacking GC-C, and therefore unable to respond to ST, displayed an increased cell proliferation in colonic crypts and enhanced carcinogen-induced aberrant crypt foci formation, which is a surrogate marker for colorectal carcinogenesis. However, pharmacological elevation of cGMP was able to efficiently induce cytostasis even in GC-C knockout mice, indicating a key role for cGMP in regulating colonic cell proliferation. Through microarray analyses, genes regulated by ST-induced GC-C activation in T84 colorectal carcinoma cells were identified. Genes involved in a number of cellular pathways were differentially expressed, including those involved in signal transduction, protein and solute secretion, transcriptional regulation and extracellular matrix formation. One of the genes found to be significantly up-regulated was the cell-cycle inhibitor, p21. The increase in p21 expression was validated at both the transcript and protein level. This p53-independent up-regulation of p21 was coupled to the activation of the cGMP-responsive kinase, PKGII, since knockdown of PKGII using specific siRNAs abolished ST-induced p21 induction. Activation of PKGII led to phosphorylation and activation of the stress responsive p38 MAPK. Similar to what was seen following knockdown of PKGII, inhibition of p38 MAPK activity attenuated the up-regulation of p21 in response to cGMP, indicating that PKGII and p38 MAPK could be a part of a pathway regulating p21 expression. It was found that active p38 MAPK phosphorylated the ubiquitous transcription factor SP1, enhancing its occupancy at the proximal p21 promoter. Therefore, SP1 could be one of the factors linking cGMP to transcription of the p21 mRNA. Chronic activation of GC-C led to nuclear accumulation of p21 in colonic cells, which entered a quiescent state. These cells arrested in the G1 phase of the cell cycle, consequent to p21-dependent inhibition of the G1 cyclin-CDK complexes. A fraction of these quiescent cells stochastically initiated a cGMP-dependent senescence programme and displayed all the hallmarks of senescent cells, including flattened cell morphology, expression of SA- galactosidase and formation of senescence-associated heterochromatic foci. Activation of senescence and loss of tumorigenicity in these cells was crucially dependent on the up-regulation of p21. This irreversible exit from the cell cycle due to cGMP-mediated activation of the PKGII/p38/p21 axis was well correlated with reduced colonic polyp formation in mice exposed to ST. In summary, these observations may provide a possible explanation for the low incidence of colorectal carcinoma seen in countries with a high incidence of ST-mediated diarrhoea. Interestingly, c-src mediated tyrosine phosphorylation of GC-C prevented p21 accumulation following ligand application. The findings described in this thesis may have important implications in understanding the molecular mechanisms involved in the progression and treatment of colorectal cancer.

Wong Cheuk Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 101-124). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vi / List of Abbreviations --- p.vii / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Methods --- p.4 / Chapter 2.1 --- Measurement of cAMP and cGMP in VSMCs --- p.4 / Chapter 2.1.1 --- Cell culture --- p.4 / Chapter 2.1.2 --- Enzyme-immunoassay colorimetric measurement for cAMP and cGMP --- p.5 / Chapter 2.1.3 --- Statistical analysis --- p.6 / Chapter 2.2 --- Measurement of apoptosis in VSMCs and bone marrow-derived stem cells --- p.6 / Chapter 2.2.1 --- Cell culture --- p.6 / Chapter 2.2.2 --- Hoechst33258 --- p.7 / Chapter 2.2.3 --- Cell Death ELISA plus --- p.7 / Chapter 2.2.4 --- Protein extraction and Western blot analysis of PKG expression --- p.8 / Chapter 2.2.5 --- Statistical analysis --- p.9 / Chapter 2.3 --- Measurement of cell proliferation in VSMCs and bone marrow-derived stem cells --- p.9 / Chapter 2.3.1 --- Cell culture --- p.9 / Chapter 2.3.2 --- Cell count --- p.10 / Chapter 2.3.3 --- MTT assay --- p.11 / Chapter 2.3.4 --- BrdU-(5`Bromo-2-deoxyuridine) ELISA colorimetric assay --- p.11 / Chapter 2.3.5 --- Statistical analysis --- p.12 / Chapter Chapter 3. --- Effects of hyperglycemia on CGRP-induced cAMP response in VSMCs / Chapter 3.1 --- Introduction --- p.13 / Chapter 3.2 --- Results --- p.18 / Chapter 3.3 --- Discussion --- p.22 / Chapter Chapter 4. --- Role of cGMP and protein kinase G in regulation of apoptosis in VSMCs / Chapter 4.1 --- Introduction --- p.26 / Chapter 4.2 --- Results --- p.30 / Chapter 4.3 --- Discussion --- p.44 / Chapter Chapter 5. --- Role of protein kinase G in regulation of proliferation in VSMCs / Chapter 5.1 --- Introduction --- p.55 / Chapter 5.2 --- Results --- p.58 / Chapter 5.3 --- Discussion --- p.67 / Chapter Chapter 6. --- Effects of aging and eNOS- and iNOS-gene deletion (using eNOS- and iNOS-knockout mice) on apoptosis of VSMCs / Chapter 6.1 --- Introduction --- p.73 / Chapter 6.2 --- Results --- p.76 / Chapter 6.3 --- Discussion --- p.79 / Chapter Chapter 7. --- Role of protein kinase G in regulation of apoptosis and proliferation of bone marrow stromal stem cells / Chapter 7.1 --- Introduction --- p.81 / Chapter 7.2 --- Results --- p.84 / Chapter 7.3 --- Discussion --- p.92 / Chapter Chapter 8. --- Overall discussion --- p.95 / Chapter Chapter 9. --- References --- p.101

Chan Siu Lan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 149-181). / Abstracts in English and Chinese. / Abstract --- p.ii / Chinese Abstract (摘要) --- p.v / Acknowledgements --- p.viii / Publications --- p.x / Table of contents --- p.xii / List of Figures --- p.xvi / List of Table and Diagram --- p.xx / Abbreviations --- p.xxi / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Major objectives and long-term significance --- p.1 / Chapter 1.2 --- Biological significance of apoptosis --- p.1 / Chapter 1.3 --- Importance of apoptosis in the study of the female reproductive system --- p.3 / Chapter 1.4 --- Specific aims of the present project --- p.3 / Chapter 1.5 --- Experimental approaches --- p.8 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- General experimental methods --- p.11 / Chapter 2.1.1 --- Culture of cells --- p.11 / Chapter 2.1.1.1 --- Culture of rabbit immortalized uterine epithelial cells --- p.11 / Chapter 2.1.1.2 --- Culture of primary mouse uterine epithelial cells --- p.12 / Chapter 2.1.1.3 --- Culture of human ovarian epithelial cancer cells --- p.13 / Chapter 2.1.2 --- Assessment of apoptotic DNA fragmentation --- p.13 / Chapter 2.1.2.1 --- DNA extraction --- p.14 / Chapter 2.1.2.2 --- Assessment of apoptotic DNA --- p.14 / Chapter 2.1.2.3 --- Assessment of apoptotic DNA by CE-LIF --- p.15 / Chapter 2.1.2.4 --- Assessment of apoptosis by Nuclear Hoechst 33248 Staining / Chapter 2.1.3 --- Assessement of protein content --- p.16 / Chapter 2.1.3.1 --- Protein extraction and western blot analysis --- p.16 / Chapter 2.1.4 --- Adenoviral infection of A2780s cells --- p.18 / Chapter 2.2 --- Preparation of solutions --- p.18 / Chapter 2.3 --- Animals and cell lines --- p.25 / Chapter 2.4 --- Statistical analysis --- p.25 / Chapter Chapter 3: --- Literature Review / Chapter 3.1 --- Morphological analysis of physiological cell death --- p.26 / Chapter 3.1.1 --- Characteristics of apoptosis --- p.27 / Chapter 3.2 --- Methods of detecting apoptosis --- p.31 / Chapter 3.3 --- Molecules controlling apoptosis --- p.33 / Chapter 3.3.1 --- Caspases --- p.33 / Chapter 3.3.2 --- The Bcl-2 family proteins --- p.34 / Chapter 3.4 --- Apoptosis signalling --- p.36 / Chapter 3.4.1 --- The death receptor-dependent pathway --- p.36 / Chapter 3.4.2 --- The mitochondria-dependent pathway --- p.38 / Chapter 3.4.3 --- The endoplasmic-reticulum-dependent pathway --- p.39 / Chapter 3.5 --- Importance of apoptosis in the female reproductive system --- p.40 / Chapter 3.5.1 --- Apoptosis in uterus epithelial cells --- p.40 / Chapter 3.5.2 --- Apoptosis in ovarian cancer cells --- p.42 / Chapter 3.6 --- Regulation of apoptosis by nitric oxide/cGMP/protein kinase G --- p.44 / Chapter 3.6.1 --- Regulation of apoptosis by nitric oxide --- p.44 / Chapter 3.6.2 --- Regulation of apoptosis by cGMP --- p.48 / Chapter 3.6.3 --- Regulation of apoptosis by soluble guanyly cyclase activator --- p.50 / Chapter Chapter 4: --- "Apoptotic DNA fragmentation caused by sodium nitroprusside, a nitric oxide donor, in uterine epithelial cells: ultrasensitive quantitation using the new capillary electrophoresis/laser-induced fluorescence (CE-LIF) technology" / Chapter 4.1 --- Abstract --- p.52 / Chapter 4.2 --- Introduction --- p.53 / Chapter 4.3 --- Results --- p.57 / Chapter 4.4 --- Discussion --- p.61 / Chapter 4.5 --- Figures of Chapter 4 --- p.66 / Chapter Chapter 5: --- Guanylyl-cyclase inhibitors NS2028 and ODQ and protein-kinase-G inhibitor KT5823 trigger apoptotic DNA fragmentation in an immortalized uterine epithelial cell line: anti-apoptotic effects of basal cGMP/PKG / Chapter 5.1 --- Abstract --- p.74 / Chapter 5.2 --- Introduction --- p.75 / Chapter 5.3 --- Results --- p.80 / Chapter 5.4 --- Discussion --- p.83 / Chapter 5.5 --- Figures of Chapter 5 --- p.89 / Chapter Chapter 6: --- "Direct, prolonged activation of soluble guanylyl cyclase by YC-1 or protein kinase G by cGMP analogs enhances the level of apoptosis in an immortalized uterine epithelial cell line, HRE-H9 cells" / Chapter 6.1 --- Abstract --- p.100 / Chapter 6.2 --- Introduction --- p.101 / Chapter 6.3 --- Results --- p.105 / Chapter 6.4 --- Discussion --- p.107 / Chapter 6.5 --- Figures of Chapter 6 --- p.114 / Chapter Chapter 7: --- "ODQ，an inhibitor of soluble guanylyl cyclase, down-regulates XIAP expression and induces apoptosis in human ovarian cancer cells" / Chapter 7.1 --- Abstract --- p.124 / Chapter 7.2 --- Introduction --- p.125 / Chapter 7.3 --- Results --- p.129 / Chapter 7.4 --- Discussion --- p.132 / Chapter 7.5 --- Figures of Chapter 7 --- p.138 / Chapter Chapter 8: --- Overall Conclusion --- p.145 / Chapter Chapter 9: --- References --- p.149

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / We evaluated the effect of sildenafil citrate (ViagraÂ) a vasodilator largely used for the treatment of male erectile dysfunction, on the gastrointestinal motility in rats. Experiments were performed on 175 male, Wistar rats, weighing 200-350g. Four groups of study were done: the sildenafil effects on the: i) Gastric emptying (GE) and gastrointestinal (GI) transit and ii) Intestinal transit (IT) of liquid in awake rats; iii) Gastric compliance in anesthetized rats and iv) Contractility of rat duodenal isolated strips. i) In 64 rats fasted for 24h with previous vascular access (right jugular vein and left carotid artery), we studied the effect of an i.v. injection (0.2mL) of sildenafil (4mg/Kg) or vehicle (0.01N HCl) on GE and GI transit of a liquid meal, as well as on arterial pressure (AP) in a separated group of rats. Animals were gavage-fed with 1.5mL of a test meal (0.5mg/mL of phenol red in 5% glucose). After 10, 20 or 30min, animals were sacrificed and submitted to a laparotomy to obstruct the pylorus, cardia and terminal ileus. The gut was removed and then divided into: stomach and consecutive three small intestine segments (40% proximal; 30% medial and 30% terminal). After processing these segments, the dye retention was determined at 560nm. The percentage of dye retention in each segment permitted to evaluate GE and GI transit. Arterial pressure was continuously monitored by a digital acquisition system during 20min before and 30min after sildenafil injection. We observed a significant increase of gastric retention in sildenafil treated rats at 10, 20, or 30min after the test meal (44,2Â2,0 vs 53,2Â2,1; 25,4Â1,3 vs 37,3Â1,6; 20,9Â2,5 vs 32,5Â2,9%, respectively), as well as a significant GI transit delay. Despite of sildenafil inducing hypotension, AP returned to basal levels 10min afterwards. Acid gastic secretion blocking pre-treatment with omeprazol did not modify the sildenafil effect on gastric retention, GI transit or AP. ii) In another group we evaluated the sildenafil (4mg/Kg) or diluente (0.01N HCl, 0.2mL) effects on the IT in awake rats, fasted for 24h. Animals were studied 3d after the insertion of a silastic cannula (0.6cm ID) into the duodenal bulb. We evaluated the progression of a radioactive liquid test meal fed (10MBq of 99mTc â 1mL of saline 0,9%) administered through the inserted cannula into the small intestine. After 20, 30 or 40min, animals were sacrificed by anesthetic overdose. After laparotomy, we removed and divided the gut in: stomach, five congruent and consecutive segments of the small intestine and the large intestine. Radioactivity counting was obtained in a gamma-chamber collimator. Sildenafil promoted an IT delay (p<0.05), indicated by shifting the center of mass to the proximal portions of the TGI (2.8Â0.2 vs 3.3Â0.1; 3.0Â0.2 vs 3.7Â0.1 and 3.4Â0.1 vs 4.2Â0.2) in relation to control group. iii) Gastric compliance study was performed on 39 anesthetized rats after 24h of fasting. Gastric volume (GV) variations were measured by plethysmography while AP was continuously monitored. We have also observed that GV increased (p<0.05) after sildenafil treatment (3mg/Kg - e.v) (3.08Â0.18; 3.10Â0.17 and 3.09Â0.17mL vs 2.91Â0.19mL) at 10, 20 and 30min after drug administation, respectively. Basal AP (105.8Â2.28mmHg) dropped by the sildenafil injection (59.8Â3.2; 64.8Â3.7 and 59.3Â4.6mmHg-p<0.05) while vehicule (0.01N HCl) did not change either GV or AP. After splanchnotomy or pre-treatments (e.v.) with methylene blue (3mg/Kg-guanilate cyclase blocker), L-NNA (3mg/Kg - NO synthase blocker) or propranolol (2mg/Kg - Ã-blocker) prevented GV increase due to sildenafil; while post-treatment with sodium nitroprusside (1mg/Kg - NO donor) raised it. iv) The in vitro contractility studies were performed on isolated duodenal strips obtained from rats (n=28) killed by cervical dislocation. Duodenal strips were suspended longitudinally in a glass chamber (10mL), filled with Tyrode solution (37oC and pH 7.4). After 1h of stabilization under 1g of initial tension, the spontaneous or induced contractility were continuously recorded by a digital acquisition system. Increasing and cummulative doses of sildenafil (0.1 to 300Âmol/L) relaxed (9.6Âmol/L of EC50) the duodenal strips. This effect was more intense than those displayed by zaprinast or papaverine (PDEs blockers) (91.6 and 78.5Âmol/L of EC50, in this order). Sildenafil showed significant antispasmodic and myorelaxant effects on the duodenal contractions induced by acetylcholine or carbamylcholine (IC50 26.7 and 16.2Âmol/L, respectively). Pre-treatment with methylene blue, ODQ (guanilato cyclase blocker) or L-NAME (NO synthase blocker) also prevented these sildenafil effects, but D-NAME (an inactive substrate for NO synthase) did not. Myorelaxant sildenafil effect was reverted by L-arginine (substrate for NO synthase) and contrarily it was largely increased by sodium nitroprusside. Forskolin adenylate cyclase activation pre-treatment also increased the myorelaxant effect of sildenafil. In summary, we have observed that sildenafil slowed down the gastrointestinal motility, delaying GE, GI and intestinal transits of a liquid meal in awake rats; Gastric compliance was also increased in anesthetized rats treated with sildenafil. Sildenafil also exhibited both antispasmodic and myorelaxant effects on isolated strips of duodenum of ex vivo rats. Besides central or peripheral sympathetic nervous system activation, sildenafil possibly acts at the gastrointestinal myocite level by activating the NO/GMPc system. / Estudamos o efeito do citrato de sildenafil (ViagraÂ), vasodilatador largamente utilizado na terapÃutica da disfunÃÃo erÃtil, sobre o comportamento motor do trato gastrintestinal (TGI) de ratos Wistar. Para tanto, utilizamos 175 animais machos, pesando entre 200 a 350g, distribuÃdos nos quatro seguintes grupos de estudo: efeitos do citrato de sildenafil sobre o i) esvaziamento gÃstrico (EG) e os trÃnsitos gastrintestinal (GI) e ii) intestinal de lÃquido em ratos acordados; iii) a complacÃncia gÃstrica de ratos anestesiados e iv) a contratilidade de tiras isoladas do duodeno de ratos ex vivo. i) Avaliamos, em 64 ratos acordados sob jejum e livre acesso à Ãgua por 24h, o efeito da injeÃÃo (0,2mL; e.v.) de sildenafil (4mg/Kg) ou veÃculo (HCl 0,01N) sobre o EG e o trÃnsito GI de lÃquido, bem como sobre a pressÃo arterial (PA). Mediante gavagem, 1,5mL da refeiÃÃo-teste (vermelho de fenol - 0,5mg/mL em glicose a 5%) foi injetada no estÃmago. Depois de 10, 20 ou 30min, sacrificamos os animais e, apÃs laparotomia, obstruÃmos o piloro, o cÃrdia e o Ãleo terminal. Removemos e dividimos o TGI em: estÃmago e segmentos consecutivos do intestino delgado (40% iniciais; 30% mediais e 30% terminais). ApÃs o processamento destas porÃÃes viscerais, determinamos as absorbÃncias das amostras a 560nm. A retenÃÃo fracional de vermelho fenol em cada segmento permitiu o cÃlculo do EG e trÃnsito GI. Em um grupo separado de animais, a PA foi monitorada continuamente por meio de um sistema digital de aquisiÃÃo de dados durante 20min antes e 30min apÃs o tratamento com sildenafil ou diluente. Comparado ao grupo controle, houve aumento significativo da retenÃÃo gÃstrica (44,2Â2,0 vs 53,2Â2,1; 25,4Â1,3 vs 37,3Â1,6; 20,9Â2,5 vs 32,5Â2,9%) nos animais tratados com sildenafil e sacrificados aos 10, 20, ou 30min, respectivamente, bem como retarde significativo no trÃnsito GI. Embora o sildenafil tenha provocado hipotensÃo, a PA retoma nÃveis basais logo apÃs 10min. O prÃ-tratamento com omeprazol (bloqueador da secreÃÃo Ãcida estomacal) nÃo modificou o efeito do sildenafil sobre os valores de retenÃÃo gÃstrica e intestinal nem nos nÃveis de PA. ii) Noutros animais (n=44), sob jejum de 24h e dotados previamente (3d) de uma cÃnula crÃnica no bulbo duodenal, estudamos o efeito do sildenafil sobre a progressÃo ao longo do intestino delgado de uma refeiÃÃo teste (10MBq de TecnÃcio ligado a fitato e diluÃdo em 1mL de salina 0,9%). Decorridos 20, 30 ou 40min da injeÃÃo (0,2mL e.v.) de sildenafil (4mg/Kg) ou diluente (HCL 0,01N), sacrificamos os animais e, apÃs laparotomia e remoÃÃo do TGI, dividimo-o em: estÃmago, cinco segmentos congruentes e consecutivos de intestino delgado e o intestino grosso. A contagem da radiatividade foi determinada num colimador de gama-cÃmara. O sildenafil promoveu retarde (p<0,05) do TI, indicado pelos retardes dos centros geomÃtricos da refeiÃÃo de 2,8 0,2 vs 3,3 0,1; 3,0 0,2 vs 3,7 0,1 e 3,4 0,1 vs 4,2 0,2 em relaÃÃo ao grupo controle, aos 20, 30 ou 40min. iii) Os estudos de complacÃncia gÃstrica foram conduzidos em 39 ratos anestesiados, sob jejum de 24h. As variaÃÃes do volume gÃstrico (VG), foram medidas por pletismografia, enquanto a PA foi monitorada continuamente por um sistema digital de aquisiÃÃo de dados. Em relaÃÃo aos valores basais (2,91Â0,19mL) o sildenafil (3mg/Kg â e.v.) aumentou (p<0,05) o VG apÃs 10, 20 e 30min (3,08Â0,18; 3,10Â0,17 e 3,09Â0,17mL). A PA basal (105,8Â2,28mmHg) caiu significativamente com o sildenafil (59,8Â3,2; 64,8Â3,7 e 59,3Â4,6mmHg) enquanto o diluente (HCl 0,01N) nÃo modificou seja o VG ou a PA. O prÃ-tratamento mediante esplancnotomia ou injeÃÃo e.v. com azul de metileno (3mg/Kg-bloqueador da guanilato ciclase), L-NNA (3mg/Kg-bloqueador da NO sintetase) ou propranolol (2mg/Kg-Ã-bloqueador) preveniram o aumento do VG pelo sildenafil; jà o pÃs-tratamento com nitroprussiato de sÃdio (1mg/Kg - e.v.) o ampliou significativamente. iv) Avaliamos ainda o efeito do sildenafil sobre a contratilidade de tiras isoladas do duodeno de ratos ex vivo (n=28), sacrificados por deslocamento cervical. Tiras dissecadas do duodeno foram suspensas longitudinalmente em cuba de vidro (10mL), plena de soluÃÃo de Tyrode (37oC e pH 7,4), e submetidas a uma tensÃo inicial de 1g. ApÃs 1h de estabilizaÃÃo, a contratilidade espontÃnea ou induzida das tiras foi registrada continuamente por um sistema digital de aquisiÃÃo de dados. O sildenafil em doses crescentes e cumulativas (0,1 a 300Âmol/L) relaxou (EC50 de 9,6Âmol/L) o duodeno, mais atà que o zaprinaste ou a papaverina (bloqueadores de FDEs) (EC50 91,6 e 78,5Âmol/L, nesta ordem). Observamos ademais que o sildenafil inibiu as contraÃÃes induzidas por acetilcolina ou carbacol (IC50 26,7 e 16,2Âmol/L, respectivamente). Jà o prÃ-tratamento com azul de metileno, ODQ (bloqueador da guanilato ciclase) ou L-NAME (bloquedor da NO sintetase), mas nÃo o D-NAME (isÃmero inativo da NO sintetase) preveniram o efeito do sildenafil. O efeito mio-relaxante do sildenafil foi ampliado pela L-arginina (substrato do NO sintetase) ou nitroprussiato de sÃdio (doador de NO). O prÃ-tratamento com forskolina (estimulador da adenilato ciclase) tambÃm aumentou o efeito mio-relaxante do sildenafil. Em resumo, observamos que o sildenafil diminui a motilidade gastrintestinal, retardando o EG, os trÃnsitos GI e intestinal de lÃquido em ratos acordados; aumenta a complacÃncia gÃstrica em ratos anestesiados alÃm de apresentar efeitos antiespasmÃdico e mio-relaxante sobre tiras isoladas de duodeno de ratos ex vivo; por estimulaÃÃo do sistema nervoso simpÃtico e tendo como provÃvel mecanismo de aÃÃo ao nÃvel do miÃcito gastrintestinal a via do NO/GMP cÃclico.

Evaluating aerodynamic noise from aircraft engines is a design stage process, so that it conform to regulations at airports. Aerodynamic noise is also a principal source of structural vibration and internal noise in short/vertical take off and landing and rocket launches. Acoustic loads may be critical for the proper functioning of electronic and mechanical components. It is imperative to have tools with capability to predict noise generation from turbulent flows. Understanding the mechanism of noise generation is essential in identifying methods for noise reduction. Lighthill (1952) and Lighthill (1954) provided the first explanation for the mechanism of aerodynamic noise generation and a procedure to estimate the radiated sound field. Many such procedures, known as acoustic analogies are used for estimating the radiated sound field in terms of the turbulent fluid flow properties. In these methods, the governing equations of the fluid flow are rearranged into two parts, the acoustic sources and the propagation terms. The noise source terms and propagation terms are different in different approaches. A good description of the turbulent flow field and the noise sources is required to understand the mechanism of noise generation. Computational aeroacoustics (CAA) tools are used to calculate the radiated far field noise. The inputs to the CAA tools are results from CFD simulations which provide details of the turbulent flow field and noise sources. Reynolds-Averaged Navier Stokes (RANS) solutions can be used as inputs to CAA tools which require only time-averaged mean quantities. The output of such tools will also be mean quantities. While complete unsteady turbulent flow details can be obtained from Direct Numerical Simulation (DNS), the computation is limited to low or moderate Reynolds number flows. Large eddy simulations (LES) provide accurate description for the dynamics of a range of large scales. Most of the kinetic energy in a turbulent flow is accounted by the large-scale structures. It is also the large-scale structures which accounts for the maximum contribution towards the radiated sound field. The results from LES can be used as an input to a suitable CAA tool to calculate the sound field. Numerical prediction of turbulent flow field, the acoustic sources and the radiated sound field is at the focus of this study. LES based on explicit filtering method is used for the simulations. The method uses a low-pass compact filter to account for the sub-grid scale effects. A one-parameter fourth-order compact filter scheme from Lele (1992) is used for this purpose. LES has been carried out for four different flow situations: (i) round jet (ii) plane jet (iii) impinging round jet and (iv) impinging plane jet. LES has been used to calculate the unsteady flow evolution of these cases and the Lighthill’s acoustic sources. A compact difference scheme proposed by Hixon & Turkel (1998) which involves only bi-diagonal matrices are used for evaluating spatial derivatives. The scheme provides similar spectral resolution as standard tridiagonal compact schemes for the first spatial derivatives. The scheme is computationally less intensive as it involves only bi-diagonal matrices. Also, the scheme employs only a two-point stencil. To calculate the radiated sound field, the Helmholtz equation is solved using the Green’s function approach, in the form of the Kirchhoff-Helmholtz integral. The integral is performed over a surface which is present entirely in the linear region and covers the volume where acoustic sources are present. The time series data of pressure and the normal component of the pressure gradient on the surface are obtained from the CFD results. The Fourier transforms of the time series of pressure and pressure gradient are then calculated and are used as input for the Kirchhoff-Helmholtz integral. The flow evolution for free jets is characterised by the growth of the instability waves in the shear layer which then rolls up into large vortices. These large vortical structures then break down into smaller ones in a cascade which are convected downstream with the flow. The rms values of the Lighthill’s acoustic sources showed that the sources are located mainly at regions immediately downstream of jet break down. This corresponds to the large scale structures at break down. The radiated sound field from free jets contains two components of noise from the large scales and from the small scales. The large structures are the dominant source for the radiated sound field. The contribution from the large structures is directional, mainly at small angles to the downstream direction. To account for the difference in jet core length, the far field SPL are calculated at points suitably shifted based on the jet core length. The peak value for the radiated sound field occurs between 30°and 35°as reported in literature. Convection of acoustic sources causes the radiated sound field to be altered due to Doppler effect. Lighthills sources along the shear layer were examined in the form of (x, t) plots and phase velocity pattern in (ω, k) plots to analyse for their convective speeds. These revealed that there is no unique convective speeds for the acoustic sources. The median convective velocity Uc of the acoustic sources in the shear layer is proportional to the jet velocity Uj at the center of the nozzle as Uc ≈ 0.6Uj. Simulations of the round jet at Mach number 0.9 were used for validating the LES approach. Five different cases of the round jet were used to understand the effect of Reynolds number and inflow perturbation on the flow, acoustic sources and the radiated sound field. Simulations were carried out for an Euler and LES at Reynolds number 3600 and 88000 at two different inflow perturbations. The LES results for the mean flow field, turbulence profiles and SPL directivity were compared with DNS of Freund (2001) and experimental data available in literature. The LES results showed that an increase in inflow forcing and higher Reynolds number caused the jet core length to reduce. The turbulent energy spectra showed that the energy content in smaller scale is higher for higher Reynolds number. LES of plane jets were carried out for two different cases, one with a co-flow and one without co-flow. LES of plane jets were carried out to understand the effect of co-flow on the sound field. The plane jets were of Mach number 0.5 and Reynolds number of 3000 based on center-line velocity excess at the nozzle. This is similar to the DNS by Stanley et al. (2002). It was identified that the co-flow leads to a reduction in turbulence levels. This was also corroborated by the turbulent energy spectrum plots. The far field radiation for the case without co-flow is higher over all angles. The contribution from the low frequencies is directional, mainly towards the downstream direction. The range of dominant convective velocities of the acoustic sources were different along shear layers and center-line. The plane jet results were also used to bring out a qualitative comparison of flow and the radiation characteristics with round jets. For the round jet, the center-line velocity decays linearly with the stream-wise distance. In the plane jet case, it is the square of the center-line velocity excess which decays linearly with the stream-wise distance. The turbulence levels at any section scales with the center-line stream-wise velocity. The decay of turbulence level is slower for the plane jet and hence the acoustic sources are present for longer distance along the downstream direction. Subsonic impinging jets are composed of four regions, the jet core, the fully developed jet, the impingement zone and the wall jet. The presence of the second region (fully developed free jet) depends on the distance of the wall from the nozzle and the length of the jet core. In impinging jets, reflection from the wall and the wall jet are additional sources of noise compared to the free jets. The results are analysed for the contribution of the different regions of the flow towards the radiated sound field. LES simulations of impinging round jets and impinging plane jet were carried out for this purpose. In addition, the results have been compared with equivalent free jets. The directivity plots showed that the SPL levels are significantly higher for the impinging jets at all angles. For free jets, a typical time scale for the acoustic sources is the ratio of the nozzle size to the jet velocity. This is ro/Uj for round jets and h/Uj for plane jets. For impinging jets, the non-dimensionlised rms of Lighthill’s source indicates that the time scale for acoustic sources is the ratio of the height of the nozzle from the wall to the jet velocity be L/Uj. LES of impinging round jets was carried out for two cases with different inflow perturbations. The jets were at Reynolds number of 88000 and Mach number of 0.9, same as the free jet cases. The impingement wall was at a distance L = 24ro from the nozzle exit. For impinging round jets, the SPL levels are found to be higher than the equivalent free jets. From the SPL levels and radiated noise spectra it was shown that the contribution from the large scale structures and its reflection from the wall is directional and at small angles to the wall normal. The difference in the range of angles where the radiation from the large scale structures were observed shows the significance of refraction of sound waves inside the flow. The rms values of the Lighthill’s sources indicate two dominant regions for the sources, just downstream of jet breakdown and in the impingement zone. The LES of impinging plane jet was done for a jet of Mach number 0.5 and Reynolds number of 6000. The impingement wall was at a distance L = 10h from the nozzle exit. The radiated sound field appears to emanate from this impingement zone. The directivity and the spectrum plots of the far field SPL indicate that there is no preferred direction of radiation from the impingement zone. The Lighthill’s sources are concentrated mainly in the impingement zone. The rms values of the sources indicate that the peak values occur in the impingement zone. The results from the different flow situations demonstrates the capability of LES with explicit filtering method in predicting the turbulent flow and radiated noise field. The method is robust and has been successfully used for moderate Reynolds number and an Euler simulation. An important feature is that LES can be used to identify acoustic sources and its convective speeds. It has been shown that the Lighthill source calculations, the calculated sound field and the observed radiation patterns agree well. An explanation for these based on the different turbulent flow structures has also been provided.