Sonic hedgehog (Shh) is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. When cells receive the Shh signal a cascade of effects begin which in turn regulate downstream target genes. The genes controlled by Sonic hedgehog provide messages instructing cells how to differentiate or when to divide. Disruption of the hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. Patched, the receptor for Shh, acts as a tumour suppressor and is mutated in naevoid basal cell carcinoma syndrome (NBCCS). NBCCS patients display a susceptibility to tumour formation, particularly for basal cell carcinoma (BCC). The discovery of Patched mutations in sporadic BCCs and other tumour types further highlights the importance of this pathway to human cancer. The identification of genes regulated by hedgehog is crucial for understanding how disruption of this pathway leads to neoplastic transformation. It is assumed that the abnormal expression of such genes plays a large role in directing cells to divide at inappropriate times. Only a small number of genes controlled by Shh have been described in vertebrate tissues. In the work presented in this thesis a Sonic hedgehog responsive embryonic mouse cell line, C3H/10T1/2, was used as a model system for hedgehog target gene discovery. Known downstream target genes were profiled to determine their induction kinetics, building up a body of knowledge on the response to Shh for this cell type. During this work, it was discovered that C3H/10T1/2 cells do not become fully competent to respond to Shh stimulation until the cells reach a critical density, a factor that had to be taken into account when determining timepoints of interest for further investigation. Several techniques were employed to identify genes that show expression changes between Shh stimulated and control cells. In one of these techniques, RNA from cell cultures activated with Shh was used to interrogate cDNA microarrays, and this provided many insights into the downstream transcriptional consequences of hedgehog stimulation. Microarrays consist of thousands of spots of DNA of known sequence gridded onto glass slides. Experiments using this technology allow the expression level of thousands of genes to be measured simultaneously. Independent stimulation methods combined with northern blotting were used to investigate individual genes of interest, allowing genuine targets to be confirmed and false positives eliminated. This resulted in the identification of eleven target genes. Seven of these are induced by Sonic hedgehog (Thrombomodulin (Thbd), Glucocorticoid induced leucine zipper (Gilz), Brain factor 2 (Bf2), Nuclear receptor subfamily 4, group A, member 1 (Nr4a1), Insulin-like growth factor 2 (Igf2), Peripheral myelin protein 22 (Pmp22), Lim and SH3 Protein 1 (Lasp1)), and four are repressed (Secreted frizzled related proteins 1 and 2 (Sfrp1 and Sfrp2), Macrophage inflammatory protein-1 gamma (Mip-1?), and Anti-mullerian hormone (Amh)). The majority of these represent novel downstream genes not previously reported as targets of Shh. The new target genes have a diverse range of functions, and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery. The identification of novel Sonic hedgehog responsive genes provides candidates whose abnormal expression may be decisive in initiating tumour formation and future studies will investigate their role in development and disease. It is expected that such findings will provide vital clues to the aetiology of various human cancers, and that an understanding of their roles may ultimately provide greater opportunities in the future design of anti-tumour therapies.

La niche des cellules souches hématopoïétiques représente un compartiment complexe et radiosensible. Sa protection est nécessaire pour la restauration de l'hématopoïèse faisant suite à la myélosuppression due à l'exposition aux rayonnements ionisants. Nous avons dans un premier temps étudié l'effet des RI sur les progéniteurs endothéliaux et mésenchymateux de la niche par une étude de radiosensiblilité et une étude d'évaluation de la mort cellulaire. Nous avons proposé par la suite une stratégie innovante de thérapie génique basée sur la sécrétion locale et à court terme du morphogène Sonic hedgehog visant à favoriser la réparation de niche vasculaire et de stimuler les cellules souches hématopoïétiques et les cellules progénitrices résiduelles. Nous avons étudié la réponse hématopoïétique des singes irradiés à 8-Gy gamma après une seule injection intra-osseuse de cellules souches mésenchymateuses xénogéniques, multipotentes et d'origine adipocytaire transfectées avec un plasmide pIRES2-eGFP codant la protéine Shh. La durée de thrombocytopénie et celle de neutropénie ont été significativement réduites chez les animaux greffés et les clonogènes sont normalisés à partir du 42e jour. Les aires sous la courbe des numération des plaquettes et des neutrophiles entre 0 et 30 jours ont été significativement plus élevée chez les animaux traités que chez les témoins. La greffe d'explants de MatrigelTM colonisés ou non avec des ASC chez des souris immunodéprimées a démontré une activité pro-angiogénique notable des ASC transfectées avec le plasmide Shh . Le suivi à long terme (180 à 300 jours) a confirmé une reconstitution durable dans les quatre singes greffés. Globalement cette étude suggère que la greffe de cellules souches multipotentes Shh-peut représenter une nouvelle stratégie pour la prise en charge des dommages radio-induits de la niche.

Audio is an effective but often overlooked component of World Wide Web delivery. Of the nearly twenty billion web pages estimated to exist, statistically few use sound. Those few using sound often use it poorly and with hardly any regard to theoretical and rhetorical issues. This thesis is an examination of the uses of audio on the World Wide Web, specifically focusing on how that use could be informed by current and historical rhetorical theory. A theoretical methodology is applied to suggest the concepts and disciplines required to make online audio more meaningful and useful. The thesis argues for the connection between the Web and the modern orator, its embodiment, its place in sound reproduction technology, and awareness of the limitations placed on it by design and convention.

This study examines the significance of sounds and silence in Joseph Conrad’s novella Heart of Darkness (1902). The importance of visual effects has repeatedly been analyzed and therefore, it is high time to explore the text from an auditory perspective. By comparing and contrasting Victorian city sounds to sounds in the wilderness, I show that the urban and rural worlds have a great deal in common. Furthermore, by deconstructing the seemingly stable binary opposition of sound and silence as well as that of civilization and wilderness, it becomes evident that they are related and depend on one another. This paper also examines noise and how it is used as a means of power. Moreover, it deals with Thomas Edison’s invention, the phonograph, as an implicit discursive device in the text. In conclusion, it is argued that the sonic environment is of high significance and should therefore not be ignored and readers must try to close their eyes in order to hear what is said.

The world is urbanizing rapidly with more than half of the global population now living in cities. Improving urban environments for the well-being of the increasing number of urban citizens is becoming one of the most important challenges of the 21st century. Even though it is common that city planners have visions of a ’good urban milieu’, those visions are concerning visual aesthetics or practical matters. The qualitative perspective of sound, such as sonic diversity and acoustic ecology are neglected aspects in architectural design. Urban planners and politicians are therefore largely unaware of the importance of sounds for the intrinsic quality of a place. Whenever environmental acoustics is on the agenda, the topic is noise abatement or noise legislation – a quantitative attenuation of sounds. Some architects may involve acoustical aspects in their work but sound design or acoustic design has yet to develop to a distinct discipline and be incorporated in urban planning.My aim was to investigate to what extent the urban soundscape is likely to improve if modern architectural techniques merge with principles of acoustics. This is an important, yet unexplored, research area. My study explores and analyses the acoustical aspects in urban development and includes interviews with practitioners in the field of urban acoustics, situated in New York City. My conclusion is that to achieve a better understanding of the human living conditions in mega-cities, there is a need to include sonic components into the holistic sense of urban development.

<p>The two major contributing populations to the outflow tract of the heart are the secondary heart field and the cardiac neural crest. These two populations are responsible for providing the myocardium that supports the outflow tract valves, the smooth muscle that surrounds these valves and the outflow vessels themselves, and the septum that divides the primitive, single outflow tract into an aorta and pulmonary trunk. Because the morphogenesis of this region is so complex, its development is regulated by many different signaling pathways. One of these pathways is the Sonic hedgehog pathway. This thesis tests the hypothesis that Sonic hedgehog induces secondary heart field proliferation, which is necessary for normal outflow tract development. To address this hypothesis, I took advantage of small chemical antagonists and agonists to determine how too little or too much hedgehog signaling would affect the secondary heart field, both in in vitro explants and in vivo. I have determined that Sonic hedgehog signaling maintains proliferation in a subset of secondary heart field cells. This proliferation is essential for generating enough myocardium and smooth muscle and also for the cardiac neural crest to septate the outflow tract into two equal-sized vessels. Up-regulating hedgehog signaling induces proliferation, which is quickly down-regulated, showing that the embryo exhibits a great deal of plasticity. Together, these studies have shown that Sonic hedgehog promotes proliferation in a subset of the secondary heart field and that the level of proliferation must be tightly regulated in order to form a normal outflow tract.</p> / Dissertation

<p>Development requires a delicate balance of proliferation and differentiation. Too little proliferation can result in dysfunctional tissues, while prolonged or heightened proliferation can result in tumor formation. This is clearly seen with the granule neuron precursors (GNPs) of the cerebellum. Too little proliferation of these cells during development results in ataxia, whereas too much proliferation results in the cerebellar tumor medulloblastoma. While these cells are known to proliferate in response to Shh, it is not clear what controls the differentiation of these cells in vivo.</p><p> Previous work from our lab has identified basic fibroblast growth factor (bFGF) as a candidate differentiation factor for these cells. In this thesis, I characterize some of the cellular and molecular mechanisms involved in FGF-mediated inhibition (FMI) of Shh-induced GNP proliferation. In addition, I employ FGFR knockouts and a bFGF gain-of-function mouse to determine whether FGF signaling is necessary and/or sufficient for differentiation of GNPs during cerebellar development. Finally, the question of whether bFGF can be effective as a therapeutic agent for in vivo tumor treatment is tested in a transplant model.</p><p> These experiments indicate that FGF signaling is neither necessary nor sufficient for GNP differentiation during cerebellar development. However, transplanted tumors are potently inhibited by bFGF treatment. Furthermore, FMI is shown to occur around the level of Gli2 processing in the Shh pathway, implying that such a treatment has promise to be widely effective in treatment of Shh-dependent medulloblastomas.</p> / Dissertation

Some fishes use sound to communicate. The majority of these soniferous fishes use superfast sonic muscles to set the vibration of the swim bladder which results in sound emission and sound amplification. Carapus, a benthic ophiidiform genus, use a slow contracting sonic muscle to pull the anteriormost part of the swim bladder, upon termination of the pulling action, the front part of the swim bladder is snap back setting the swimbladder fenestrum to vibrate. This vibration gives rise to the sound. Other ophiidiform fishes may also use a similar way to emit sounds. Among the soniferous percoids, an advanced perform suborder, glaucosomatid, pempherid, and terapontid share a fenestrum-like structure in the front part of their swim bladder. Previous molecular study suggested that the first two groups form a clade (monophyletic group). It is of great interest to compare the proteomic features of these groups with that of the ophiidiform representatives so that the effects of function and phylogeny to the proteomic characteristics of the sonic muscle can be compared. A species was selected for each of these four groups and their proteomics were analyzed. Results of this study, however, revealed the protein composition of the sonic muscles in the ophiidiform species was more similar to that of the pempherid species. The proteins contribute to the close relationship between these two groups was discussed. A total of 484 protein spots were found in these four species and only five were presented in the sonic muscles of all four species, but absent in the white muscles; and only three of them were successfully identified as: Flotillin-1 (spot 6), HBS1-like protein (spot 8), and Ras-related protein ralB-B (spot 10). Their functions which may be related to the specific role of the sonic muscle were discussed.

The Marcellus Shale is an important resource play prevalent in several states in the eastern United States. The productive zone of the Marcellus Shale has variations in rock properties such as clay content, kerogen content and pore aspect ratio, and these variations may strongly effect elastic anisotropy. The objective of this study is to characterize surface seismic sensitivity for variations in anisotropic parameters relating to kerogen content and aspect ratio of kerogen saturated pores. The recognized sensitivity may aid to characterize these reservoir from surface seismic observations for exploration and production of hydrocarbon. In this study, I performed VTI anisotropic modeling based on geophysical wireline log data from Harrison County, WV. The wireline log data includes spectral gamma, density, resistivity, neutron porosity, monopole and dipole sonic logs. Borehole log data were analyzed to characterize the Marcellus Shale interval, and quantify petrophysical properties such as clay content, kerogen content and porosity. A rock physics model was employed to build link between petrophysical properties and elastic constants. The rock physics model utilized differential effective medium (DEM) theory, bounds and mixing laws and fluid substitution equations in a model scheme to compute elastic constants for known variations in matrix composition, kerogen content and pore shape distribution. The seismic simulations were conducted applying a vertical impulse source and three component receivers. The anisotropic effect to angular amplitude variations for PP, PS and SS reflections were found to be dominantly controlled by the Thomsen Ɛ parameter, characterizing seismic velocity variations with propagation direction. These anisotropic effect to PP data can be seen at large offset (>15o incidence angle). The most sensitive portion of PS reflections was observed at mid offset (15o-30o). I also analyzed seismic sensitivity for variations in kerogen content and aspect ratio of structural kerogen. Elastic constants were computed for 5%, 10%, 20% and 30% kerogen content from rock physics model and provided to the seismic model. For both kerogen content and aspect ratio model, PP amplitudes varies significantly at zero to near offset while PS amplitude varied at mid offsets (12 to 30 degree angle of incidences). / text

The choroid plexus is an organ within each brain ventricle comprised of elaborate folds of epithelium (CPe) and vasculature. It performs numerous functions essential for brain development and health, including secretion of cerebrospinal fluid (CSF) and acting as the blood-CSF barrier. Functionality requires: (1) that CPe and vasculature develop in register and in close proximity, so that the CPe ensheaths the vasculature at a high surface area to volume ratio, which permits efficient CSF secretion; and (2) that CPe barrier integrity is sustained throughout choroid plexus expansion. Genetic experiments in mouse embryos have identified a central role for Sonic hedgehog (Shh) in coordinating these developmental challenges. Specifically, Shh is secreted by differentiated CPe and drives choroid plexus expansion. In the absence of Shh, a hypoplastic choroid plexus forms, which is deficient in CPe, vasculature, and villous folds. Two choroid plexus cell populations respond to Shh: (1) rhombic lip-resident CPe progenitor cells and (2) vascular pericytes. Here, I present evidence that canonical Shh signaling to CPe progenitors alone is insufficient to fully drive their proliferation at normal rates. Rather, Shh-triggered pericytes appear to secondarily boost CPe progenitor cell proliferation, in addition to acting in vascular development. Shh-triggered pericytes also appear necessary for formation of the characteristic folds of the choroid plexus. Thus, pericytes coordinate the expansion of choroid plexus epithelium and vasculature. Notch signaling was also explored and was found to inhibit the differentiation of CPe progenitors, maintaining them in a proliferative state. Notch activation in CPe progenitors leads to invaginated tubules from the overproliferating CPe progenitor domain, without associated vascular growth or villous folds. Folding morphogenesis may thus be regulated by vascular components such as pericytes, and require that vascular growth match CPe growth. To identify Shh-induced pericyte signaling programs that might underlie these developmental processes, expression profiling was performed on dsRed-labeled pericytes isolated from Shh-deficient versus wild-type choroid plexuses. Candidate genes, including several involved in lipid metabolism, were identified. Collectively, this work points to pericytes as central in orchestrating the coordinated elaboration of multiple choroid plexus cell types, producing the complex tissue architecture required for efficient CSF production.