Gastrointestinal (GI) smooth muscle possesses distinct regional and functional properties that distinguish it from other types of visceral and vascular smooth muscle. On the basis of electrical properties and contractile phenotype, GI smooth muscles have been classified into phasic (non-sphinteric) and tonic (sphinteric) smooth muscles. The biochemical basis of phasic and tonic phenotypes of smooth muscle is not clear and is the major question of inquiry of the present study. Phosphorylation of Ser19 on the 20 kDa myosin light chain (MLC) is essential for acto-myosin interaction and contraction in both phasic and tonic muscles. The levels of MLC20 phosphorylation are regulated by Ca2+/calmodulin-dependent MLC kinase (MLCK) and MLC phosphatase (MLCP), and the activity of these enzymes are in turn regulated by various signaling molecules whose expression and activity are important in determining the strength and duration of their activity. The signaling proteins are AMP kinase (MLCK activity), Rho kinase, zipper-interacting protein kinase (ZIPK), CPI-17 and telokin (MLCP activity), phosphodiesterase 5 (PDE5) and multi-drug resistance protein 5 (MRP5). The overarching goal of the dissertation is to identify the differences in the signaling pathways that regulate MLCK and MLCP activities, and thus MLC20 phosphorylation and muscle function. Using biochemical, molecular and functional approaches, and antrum (distal stomach) and fundus (proximal stomach) of rabbit stomach as models of phasic and tonic smooth muscles, respectively, the present study characterized important differences in the signaling pathways that highly correlate with the contractile phenotype. These include: 1) tissue-specific expression of contractile proteins such as myosin heavy chain isoforms, actin, caldesmon, calponin, - and β-tropomyosin, smoothelin-A and -B; 2) higher expression of AMPK, selective feedback inhibition of MLCK activity via AMPK-mediated phosphorylation, and higher expression of telokin and activation of MLCP correlate with the rapid cyclical contractile function in phasic muscle; 3) higher expression and activation of Rho kinase/ZIPK/MYPT1 and PKC/CPI-17 pathways, preferential inhibition of MLCP activity, and sustained phosphorylation of MLC20 correlate with the sustained contraction in tonic muscle; and 4) rapid termination of cGMP signal and muscle relaxation by preferential degradation and efflux of cGMP via higher expression of PDE5 and MRP5, respectively, correlate with the brief relaxation and rapid restoration of contraction in tonic muscle. It is anticipated that these findings could be important in providing the underlying mechanisms involved in the pathophysiology of smooth muscle function and new insights for the development of therapeutic agents that should act on smooth muscle in the gut to treat motility disorders as well as in other regions such as airways and vascular smooth muscle where similar intracellular mechanisms may prevail.

Development of novel ill vitro and ill vivo models for determining primary events in HLRCC tumourigenesis Linda O'Flaherty, Mansfield College Thesis submitted for degree of Doctor of Philosophy Nuffield Department of Clinical Medicine, University of Oxford Hilary Term 2012 Germline mutations of fumarate hydratase (FR), encoding an enzyme of the tricarboxylic acid (TCA) cycle, predispose affected individuals to hereditary leiomyomatosis and renal cell cancer (HLRCC). FH-deficient cells and tissues have been shown to accumulate fumarate, exhibit S-(2-succinyl) cysteine (2SC) protein modifications and to constitutively express hypoxia-inducible factor alpha (HIF -1 a and -20.), under nonnoxic conditions. This thesis presents a phenotypic characterisation of FhI-I- mouse embryonic fibroblasts (MEFs), generated from previously reported conditional Fhl knockout mice, as a new in vitro system for investigating and identifying biochemical and metabolic pathways that are dysregulated as a result of FhI inactivation. These cell lines reproduced the aforementioned phenotypes, in addition to an observed shift from oxidative phosphorylation (OXPHOS) to glycolytic metabolism. Re-expression of either full length, mitochondrial-targeted FH (FhI-I- +FH) or cytoplasmic FH (Fhrl- +FHl'1MTS) in FhI-deficient MEFs was sufficient to reduce intracellular fumarate and to correct for the dysregulation of the Hif pathway. These results were of particular interest as they demonstrated that nonnoxic stabilisation of Hif-Ia occurs independently of the persistent mitochondrial defect observed in Fhrl- +FHl'1MTS MEFs. These findings were corroborated in vivo following the development of transgenic mouse models, ubiquitously expressing either FH or FHl'1MTS in mice with targeted inactivation of FhI in renal tubular cells. Surprisingly, the cytoplasmic-restricted FH (FHl'1MTS) transgene was just as efficient as the transgenic mice expressing mitochondrial- targeted FH at rescuing the cystic phenotype associated with Fh I-deficiency in the kidneys. As the function of cytoplasmic FH has remained poorly understood, these results go some way to extricating a role for this isofonn of FH. The results of this thesis demonstrate that these novel in vitro and in vivo models, used either alone or in combination, are a versatile and robust paradigm for studying altered cell metabolism in not only HLRCC but other diseases associated with metabolic dysregulation.

Tissue engineered blood vessels (TEBVs) have great potential as tools for disease modeling and drug screening. However, existing methods for fabricating TEBVs create homogenous tissue tubes, which may not be conducive to modeling focal vascular diseases such as intimal hyperplasia or aneurysm. In contrast, our lab has a unique modular system for fabricating TEBVs. Smooth muscle cells (SMCs) are seeded into an annular agarose mold, where they aggregate into vascular tissue rings, which can be stacked and fused into small diameter TEBVs. Our goal is to create a platform technology that may be used for fabricating focal vascular disease models, such as intimal hyperplasia. Because tubes are fabricated from individual ring units, each ring can potentially be customized, enabling the creation of focal changes or regions of disease along the tube length. In these studies, we first demonstrated our ability to modulate cell phenotype within individual SMC ring units using incorporated growth factor-loaded degradable gelatin microspheres. Next, we evaluated fusion of ring subunits to form composite tissue tubes, and demonstrated that cells retain their spatial positioning within individual rings during fusion. By incorporating electrospun polycaprolactone cannulation cuffs at each end, tubes were mounted on bioreactors after only 7 days of fusion to impart luminal medium flow for 7 days at a physiological shear stress of 12 dyne/cm2. We then created focal heterogeneities along the tube length by fusing microsphere-containing rings in the central region of the tube between rings without microspheres. In the future, microspheres may be used to deliver growth factors to this localized region of microsphere incorporation and induce disease phenotypes. Due to the challenges of working with primary human SMCs, we next evaluated human mesenchymal stem cells (hMSCs) as an alternative cell source to generate vascular SMCs. We evaluated the effects of microsphere-mediated platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and transforming growth factor beta-1 (TGF-β1) delivery on ring thickness, proliferation, and contractile protein expression over a 14 day period. Finally, we created a structurally distinct region of smooth muscle within tissue tubes by fusing human aortic SMCs in a central region between hMSC rings. In summary, we developed a platform technology for creating modular tubular tissues that may be further developed into an in vitro intimal hyperplasia model. It may also be modified to model other focal vascular diseases, such as aneurysm, or to create other types of multi-tissue tubular structures, such as trachea.

Piecewise-smooth stochastic systems are widely used in engineering science. However, the theory of these systems is only in its infancy. In this thesis, we take as an example the Brownian motion with dry friction to illustrate dynamical properties of these systems with respect to three interesting topics: (i) weak-noise approximations, (ii) first-passage time (FPT) problems and (iii) functionals of stochastic processes. Firstly, we investigate the validity and accuracy of weak-noise approximations for piecewise-smooth stochastic differential equations (SDEs), taking as an illustrative example the Brownian motion with pure dry friction. For this model, we show that the weak-noise approximation of the path integral correctly reproduces the known propagator of the SDE at lowest order in the noise power, as well as the main features of the exact propagator with higher-order corrections, provided that the singularity of the path integral is treated with some heuristics. We also consider a smooth regularisation of this piecewise-constant SDE and study to what extent this regularisation can rectify some of the problems encountered in the non-smooth case. Secondly, we provide analytic solutions to the FPT problem of the Brownian motion with dry friction. For the pure dry friction case, we find a phase transition phenomenon in the spectrum which relates to the position of the exit point and affects the tail of the FPT distribution. For the model with dry and viscous friction, we evaluate quantitatively the impact of the corresponding stick-slip transition and of the transition to ballistic exit. We also derive analytically the distributions of the maximum velocity till the FPT for the dry friction model. Thirdly, we generalise the so-called backward Fokker-Planck technique and obtain a recursive ordinary differential equation for the moments of functionals in the Laplace space. We then apply the developed results to analyse the local time, the occupation time and the displacement of the dry friction model. Finally, we conclude this thesis and state some related unsolved problems.

MicroRNAs play an important role in biological regulation. Recently miR-34a has been reported to regulate tumour cell cycle progression and apoptosis. However, the functional role of miR-34a in smooth muscle cell (SMC) differentiation from stem cells is yet unclear. Main objectives of this PhD project are to determine the functional role of miR-34a and its target genes in SMC differentiation and underlying mechanisms. Mouse embryonic stem (ES) cells were seeded on collagen coated flasks in differentiation medium to allow SMC differentiation. Upon analysis, miR-34a was significantly up-regulated during SMC differentiation. Results demonstrated that overexpression of miR-34a significantly promoted SMC-specific gene expression, while knockdown of miR-34a inhibited expression of SMC specific gene. Enforced expression and knockdown of miR-34a in differentiating ES cells up-regulated and down-regulated, respectively, several SMC transcription factors in a similar manner. It was also found that miR-34a overexpression in stem cells promoted SMC differentiation in vivo. Furthermore, deacetylase sirtuin 1 (Sirt1) was identified as one of the top targets of miR-34a. Surprisingly, Sirt1 was demonstrated to be positively regulated by miR-34a during SMC differentiation in a cellular context and RNA sequence dependent manner. VIII Mechanistically, the data suggested that miR-34a promoted differentiating stem cells arrest at G0/G1 phase, and a significant decreased incorporation of miR-34a and SirT1 RNA into Ago2-RISC complex was observed upon SMC differentiation. The results demonstrated that Sirt1 acted as a transcriptional activator in the regulation of SMC gene during ES cell differentiation. Finally, H3K9 tri-methylation around the promoter regions of the SMαA and SM22α genes was also found to be significantly inhibited by SirT1 overexpression. These findings suggest that miR-34a plays an important role in SMC differentiation from ES cells. Meanwhile, Sirt1 can be regulated by miR-34a through an unexpected pathway and it was identified as a functional modulating target in miR-34a mediated SMC differentiation.

Orientador: José Ricardo Paciência Rodrigues / Banca: Afonso Mazario / Banca: Henrique Benedito Brenelli / Banca: Cleverson Teixeira / Banca: Gilberto Uemura / Resumo: Estudos de expressão gênica têm identificado vários grupos moleculares de carcinoma de mama, com diferentes comportamentos clínico e biológico. A correlação entre "cDNA microarray" e imunoistoquímica(IQ) com marcadores para citoceratinas, Her2/neu, receptor de estrógeno(RE) e de células basais mioepiteliais (1A4, S-100 e p63), identificaram cinco grupos: (1) luminal A (RE+; Her2/neu-), (2) luminal B (RE+; Her2/neu+), (3) superexpressão de Her2/neu (RE-; Her2/neu+), (4) tipo basal (RE-; Her2/neu-; Ck 5/6 +) e (5) nenhum destes ("null"). Os de tipo luminal expressam citoceratinas de padrão luminal (Ck8/18) e os de tipo basal expressam citoceratinas 5/6 e 14 ou marcadores de células basais mioepiteliais. Avaliamos a expressão de Ck5/6, Ck8/18 e 1A4 em material de citoinclusão, comparando-a ao espécime cirúrgico. Material e Métodos: Foram selecionados 62 casos, seqüenciais, de carcinoma de mama diagnosticados por PAAF, com citoinclusão e espécime cirúrgico. Cortes de citoinclusão e do espécime cirúrgico foram imunocorados para Ck 5/6, Ck 8/18 e 1A4. Resultados e Conclusão: Os valores, em porcentagem, de sensibilidade, especificidade, valor preditivo positivo(VPP), valor preditivo negativo(VPN) e acurácia foram, respectivamente: Ck5/6 (77, 100, 100, 92 e 94); Ck8/18 ( 98, 66, 96, 80 e 95) e 1A4 ( 92, 96, 85, 98 e 95). Portanto, a identificação de Ck5/6, Ck8/18 e 1A4 por IQ em material de citoinclusão é método confiável, com resultados muito próximos aos obtidos no espécime cirúrgico e pode contribuir para a classificação dos carcinomas mamários de expressão luminal e basal, fornecendo informações importantes que possam orientar na escolha do tratamento, bem como na avaliação de fatores prognósticos e preditivos. A importância da obtenção de dados morfológicos e imunoistoquímicos sobre os carcinomas mamários através do material... (Rewsumo completo, clicar acesso eletrônico abaixo) / Abstract: Genetic expression studies have identified many molecular groups of breast carcinoma, with different clinical and biological behavior. The correlation between cDNA microarray and immunohistochemistry (IHC) with markers for cytokeratin, Her2/neu, estrogen receptor (ER) and of basal myoepithelial cells (1A4, S-100 e p63), identified five groups: (1) luminal A (ER+; Her2/neu-), (2) luminal B (ER+; Her2/neu+), (3) overexpression of Her2/neu (ER- ; Her2/neu+), (4) basal-like (ER- ; Her2/neu-; Ck 5/6 +) and (5) none of them (null). The luminal-like express cytokeratines of luminal pattern (Ck8/18) and the basal-like express cytokeratines 5/6 and 14 or markers of myoepithelial basal cells. We have evaluated the expression of Ck5/6, Ck8/18 and 1A4 in cell block comparing it to the surgical specimen. Material and Methods: 43 62 cases have been selected, sequencial, of breast carcinoma diagnosed through fine needle aspiration (FNA), with cell block and surgical specimen. Cuts of cell block and from the surgical specimen were immunostained for Ck 5/6, Ck 8/18 and 1A4. The value, in percentage, of sensibility, specificity, positive predictive value, negative predictive value, and accuracy were respectively: Ck5/6 (77, 100, 100, 92 e 94); Ck8/18 (98, 66, 96, 80 e 95) e 1A4 ( 92, 96, 85, 98 e 95). Therefore, the identification of CK5/6, 8/18 and 1A4 for IHC in cell block is a reliable method, with results very close to the ones obtained in the surgical specimen, and it can contribute to the sub classification of the breast carcinomas of luminal and basal expression, providing important information, which can orientate the treatment... (Complete abstract click electronic access below) / Doutor

The survival of Elasmobranch fishes (sharks, skates and rays) depends critically on their ability to sense odor cues. The outstanding question of detection thresholds to food odors in the shark is investigated in this study. The tracking behavior of Mustelus canis (the smooth dogfish) was analyzed using a binary choice flume designed specifically for testing odor preferences of aquatic animals. To determine threshold, odor was serially diluted until no tracking responses were observed. Sharks spent significantly more time in the odor side of the flume, regardless of their individual side bias, until the "squid juice" was diluted several orders of magnitude. For the whole flume the two greatest dilutions (10-4-10-5 at the odor source) did not cause significant choice and for the upstream flume half, all but the greatest dilution (10-5 at the odor source) caused significant odor side preference. To interpret these results fully we need to consider the structure of odor plumes and the function of the sharks' olfactory responses. Nonetheless, assuming that M. canis represent sharks in general, these findings demonstrate that their extraordinary sensitivity to food attractants may indicate aspects still unknown about life history of elasmobranch fishes including the ranges that benthic elasmobranchs are capable of traveling to feed.

Pulmonary arterial hypertension (PAH) is a disease in which pulmonary vascular resistance increases. The cell membrane of pulmonary artery smooth muscle cells (PASMC) in PAH patients is depolarised, resulting in disrupted Ca2+ signalling leading to smooth muscle constriction and PASMC proliferation and migration. In rat pulmonary artery (PA) smooth muscle the KV7 K+ channels, encoded by the KCNQ genes, have been proposed to contribute to the resting K+ current, promoting low resting tone by maintaining a negative membrane potential and low intracellular Ca2+. KV7 channel activating drugs have the potential to counteract the dysfunctional signalling during PAH by causing hyperpolarisation. This study set out to determine if the KV7 channels are expressed in human PA and if so whether they can alter vascular tone, PASMC proliferation and/or migration due to their ability to reduce intracellular Ca2+ indirectly. The effects of KV7 K+ channel modulators on human PA tone were measured using myography, while KCNQ gene expression was examined with quantitative PCR. Markers of proliferation (5-bromo-2'-deoxy-uridine (BrdU) and Ki67 antigen), were used to measure PASMC proliferation, while migration was assessed using the scratch-wound assay. Human PASMCs express all KCNQ genes, except KCNQ2. The KV7 channel blockers XE991, linopirdine and (-)chromanol 293B, constricted PAs. The KV7 channel activators retigabine and zinc pyrithione (ZnPy) relaxed PAs pre-constricted with agonists. The retigabine response was enhanced in PAs constricted with Bay K 8644, abolished in ionomycin constricted PAs and reduced in the presence of 90 mM K+, suggesting inhibition of voltage-gated Ca2+ influx. Similar experiments on rat PAs suggest that only part of the ZnPy-induced relaxation can be attributed to KV7 channel activation. The KCNQ5 gene remained in cultured PASMCs while no KV7 channel modulator altered proliferation or migration. Thus KV7.5 channels could possibly be a marker of differentiated PASMCs and/or be involved in the regulation of cell phenotype. The results imply that KV7 channels play a role in regulating PA tone and Ca2+ signalling in PA smooth. It is concluded that although KCNQ5 transcripts are preserved in proliferating PASMC, it is unlikely they play a role in PASMC proliferation or migration. In summary, KV7 channel activators may be useful in the treatment of PAH since they can prevent vasoconstriction.

Impact of drops on a dry smooth surface was studied at elevated chamber pressures and low Reynold's numbers to characterize the effect of chamber pressure on drop splashing and spreading. Two drop sizes of methanol, ethanol, propanol, hexadecane and diesel were tested for impact speeds between 1.5 - 3.3 m/s and pressure of upto 12 bars. Splash ratio, unlike the results of Xu et al, increased sharply with decreasing impact speed suggesting that drop speed is a more critical parameter for splash. Drop splashing was also found to be affected by drop shape, with drop distortion having a significant impact on splash promotion or suppression. In accordance with existing theory, drop spreading and maximum spread factor were found to be independent of pressure in the regime tested. These observations provide new insights and comparison data for evaluating and modeling the behavior of alternate fuels like ethanol.

This thesis is mainly concerned with the estimation of parameters of a first-order Smooth Threshold Autoregressive (STAR) model with delay parameter one. The estimation procedures include classical and Bayesian methods from a parametric and a semiparametric point of view.As the theoretical importance of stationarity is a primary concern in estimation of time series models, we begin the thesis with a thorough investigation of necessary or sufficient conditions for ergodicity of a first-order STAR process followed by the necessary and sufficient conditions for recurrence and classification for null-recurrence and transience.The estimation procedure is started by using Bayesian analysis which derives posterior distributions of parameters with a noninformative prior for the STAR models of order p. The predictive performance of the STAR models using the exact one-step-ahead predictions along with an approximation to multi-step-ahead predictive density are considered. The theoretical results are then illustrated by simulated data sets and the well- known Canadian lynx data set.The parameter estimation obtained by conditional least squares, maximum likelihood, M-estimator and estimating functions are reviewed together with their asymptotic properties and presented under the classical and parametric approaches. These estimators are then used as preliminary estimators for obtaining adaptive estimates in a semiparametric setting. The adaptive estimates for a first-order STAR model with delay parameter one exist only for the class of symmetric error densities. At the end, the numerical results are presented to compare the parametric and semiparametric estimates of this model.