Apoptosis is an intricately regulated cellular process required for the health and homeostasis of living systems. The mitochondrial apoptotic pathway depends on the BCL-2 family of pro- and anti-apoptotic members whose interactions regulate cell fate. BAX and BAK are key pro-apoptotic proteins required for mitochondrial permeabilization during apoptosis. While the mitochondrial death program relies heavily on its protein components, evidences support equally crucial roles for lipids and lipid metabolism in promoting or hindering apoptosis at the mitochondria. To gain insight into the interplay between lipids and BCL-2 proteins we used a liquid chromatography (LC)-mass spectrometry (MS)-based comparative lipidomics approach to uncover lipid changes in the absence of BAX and/or BAK. Our analysis revealed novel functions for BAX and BAK in inflammation and ceramide metabolism. A targeted LC-MS workflow was also developed for characterization of a novel lipid class involved in type 2 diabetes. Targeted LC-MS revealed altered oxysterol metabolism following perturbation of the Sonic hedgehog pathway. Taken together, our findings demonstrate interesting connections among lipids, cell death and disease. / Chemistry and Chemical Biology

Les Hedgehogs (Hhs) sont des morphogènes indispensables au développement et à l'homéostasie de l'organisme, notamment dans la formation de l'intestin. La littérature y révèle l'importance d'Indian Hh dans le contrôle de la prolifération, de la différenciation des entérocytes et dans l'attraction des cellules immunitaires. Par contre, sa délétion ne récapitule pas l'inhibition de la voie Hh, soulevant ainsi l'hypothèse que Sonic Hh aurait des rôles complémentaires dans l'intestin. Nous avons donc voulu identifier les implications de Shh dans l'histologie, la prolifération et la différenciation de l'épithélium intestinal chez la souris adulte. Pour ce faire, nous avons utilisé le système de délétion conditionnelle Cre/loxP afin d'abolir l'expression de Shh au niveau de l'épithélium de l'intestin et du côlon, à l'aide du promoteur de la Villine . Des traitements au DSS sur 7 jours ont permis de simuler une colite ulcéreuse (CU) chez les animaux afin d'étudier les maladies inflammatoires intestinales (MII). Aucune anomalie histologique n'a été observée par coloration H&E, mais l'axe crypte-villosité était réduit de 16%. Une immunofluorescence dirigée contre PCNA relie ce résultat avec une réduction de 24% du nombre de cellules prolifératives dans l'iléon. Ensuite, Shh ne serait pas impliqué dans la différenciation des entérocytes et des cellules entéroendocrines, contrairement à Ihh. Par contre, les observations montrent une diminution de l'expression de Klf4 en qPCR et un défaut dans l'ultrastructure des vésicules de sécrétion des cellules caliciformes en microscopie électronique. La production des mucines acides et leur fucosylation sont aussi affectées par coloration. Il en résulte un défaut dans la sécrétion de la bicouche de mucus procurant une défense physique. Ensuite, une diminution de l'expression de Sox9 et de la taille des granules dans les cellules de Paneth est observée en absence de Shh. Une diminution de l'autophagie, associée à un réticulum endoplasmique relâché, semble être à la base de ce phénotype en immunobuvardage. Il en résulte un défaut dans la production des agents antimicrobiens, tels que le lysozyme et les a-défensines, en qPCR, suggérant un possible défaut dans la défense antimicrobienne. Bien que les altérations de la barrière intestinale laissent supposer un rôle dans l'inflammation, les souris expérimentales soumises au traitement DSS suggèrent que Shh ne module pas le développement de la CU mais pourrait avoir un rôle dans les étapes de restitution. Finalement, le niveau d'expression des effecteurs Hhs en qPCR lors des Mn et de la CU chez les souris de type sauvage révèle qu'Ihh et Glil sont fortement réduits dans ces pathologies. Les résultats obtenus démontrent que Shh et Ihh ont des rôles distincts dans l'intestin. Shh semble influencer positivement la prolifération et participe à la différenciation terminale des cellules caliciformes et de Paneth ainsi que dans le processus d'autophagie. Ensemble, les Hhs sont essentiels à l'homéostasie et sont impliqués dans la pathologie des MII.

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.

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.

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.

A lateral jet in a supersonic crossflow creates a highly complex three-dimensional flow field which is not easily predicted. The aim of this research was to assess the use of a RANS based CFD method to simulate a lateral jet in supersonic crossflow interaction by comparing the performance of available RANS turbulence models. Four turbulence models were trialled in increasingly complex configurations; a flat plate, a body of revolution and a body of revolution at incidence. The results of this numerical campaign were compared to existing experimental and numerical data. Overall the Spalart-Allmaras turbulence model provided the best fit to experimental data. The performance of the lateral jet as a reaction control system was assed by calculating the force and moment amplification factors. The predicted flowfield surrounding the interaction was analysed in detail and was shown to predict the accepted shock and vortical structures. The lateral jet interaction flowfield over a body of revolution was shown to be qualitatively the same as that over a flat plate. An experimental facility was designed and manufactured allowing the study of the lateral jet interaction in Cranfield University’s 2 ½” x 2 ½” supersonic windtunnel. The interaction was studied with a freestream Mach number of 1.8, 2.4 & 3.1 and over a range of pressure ratios (50≤PR≤200). Levels of unsteadiness in the interaction were measured using high bandwidth pressure transducers. The level of unsteadiness was quantified by calculating the OASPL of the pressure signal. OASPL was found to increase with increasing levels of PR or MPR and to decrease with increases of Mach number. The levels of unsteadiness found were low with the highest levels found downstream of the jet.

abstract: In the last few decades, the rapid development of electronic music technologies has changed the way society interacts with music, which in turn impacts the profession of music therapy. Except for a few cases, music therapy has not extensively explored the integration of new technology. However, current research trends show a willingness and excitement to explore the possibilities (Nagler, 2011; Ramsey, 2011; Magee, et al., 2011; Magee & Burland, 2008; Magee 2006). The project described in this paper intends to demonstrate one of these possibilities by combining modern technologies to create an interactive musical system with practical applications in music therapy. In addition to designing a practical tool, the project aims to question the role of technology in music therapy and to initiate dialogue between technologists and music therapists. The project, entitled MIST: A Musical Interactive Space for Therapy, uses modern gestural technology (the Microsoft® Kinect®) to capture body movements and turn them into music. It is intended for use in a clinical setting with children with mild to moderate disabilities. The system is a software/hardware package that is inexpensive, user-friendly, and portable. There are two functional modes of the system: the first sonifies specific movement tasks of reaching and balancing; the second is an interactive musical play space in which an entire room becomes responsive to presence and movement, creating a sonic playground. The therapeutic goals of the system are to motivate and train physical movement, encourage exploration of space and the body, and allow for musical expression, play, auditory perception, and social interaction. / Dissertation/Thesis / The instruction manual for MIST software / M.M. Media Arts and Sciences 2011

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-01-05T16:59:19Z No. of bitstreams: 1 GeissyLainnyDeLimaAraujo_DISSERT.pdf: 3295602 bytes, checksum: 5e99ad6700c5552b3b1ad3651c7bce8c (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-01-08T18:10:33Z (GMT) No. of bitstreams: 1 GeissyLainnyDeLimaAraujo_DISSERT.pdf: 3295602 bytes, checksum: 5e99ad6700c5552b3b1ad3651c7bce8c (MD5) / Made available in DSpace on 2016-01-08T18:10:33Z (GMT). No. of bitstreams: 1 GeissyLainnyDeLimaAraujo_DISSERT.pdf: 3295602 bytes, checksum: 5e99ad6700c5552b3b1ad3651c7bce8c (MD5) Previous issue date: 2014-10-15 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / O Sonic Hedgehog (Shh) ? um morf?geno com importantes a??es no sistema nervoso central (SNC) em desenvolvimento, assim como na vida adulta em quadros de les?o tecidual e processos tumorig?nicos. A rela??o da sua via de sinaliza??o com prolifera??o, diferencia??o e sobreviv?ncia celular ? amplamente estudada em regi?es ventrais do SNC. No entanto, o papel da sinaliza??o por Shh em egi?es dorsais, como o telenc?falo dorsal, origem do c?rtex cerebral, n?o est? bem documentada. A partir do cultivo de c?lulas de roedores retiradas do telenc?falo dorsal em desenvolvimento, observamos a influ?ncia do Shh sobre a prolifera??o e diferencia??o das c?lulas-tronco neurais. Utilizando v?deo-microscopia de tempo intervalado, podemos avaliar o tempo de ciclo celular, tamanho de c?lulas progenitoras antes da divis?o celular e tipo de divis?o sofrida pelas c?lulas na presen?a ou na aus?ncia de sinaliza??o por Shh. Verificamos um aumento do n?mero de c?lulas em estado proliferativo assim como um aumento de c?lulas reativas para o marcador astrocit?rio GFAP com o tratamento com Shh. Em contrapartida, ap?s bloqueio da sinaliza??o por Shh, observamos um menor n?mero de c?lulas em estado proliferativo, desacelera??o do ciclo celular, aumento da morte celular e redu??o da astrogliog?nese. Por fim, com intuito de avaliar a influencia do Shh in vivo, n?s injetamos f?rmacos agonista (Purmorfamina) e antagonista (Ciclopamina) da via de sinaliza??o dessa prote?na em diferentes per?odos da gesta??o de roedores. Ao avaliar os animais na vida p?s-natal, observamos um aumento no n?mero de progenitores gliais gerados com o tratamento com Purmorfamina na subst?ncia branca, enquanto na subst?ncia cinzenta n?o parece haver altera??o dessa popula??o em ambos os tratamentos. Al?m disso, a popula??o de c?lulas astrocit?rias, evidenciada por marcadores espec?ficos, parece estar alterada com a manipula??o da sinaliza??o por Shh. Em conjunto, nossos dados sugerem que a Shh est? presente no telenc?falo dorsal em per?odos precoces do desenvolvimento e influencia a gera??o, sobreviv?ncia e prolifera??o de progenitores e c?lulas gliais.

No description available.

This thesis studied the effects of ear training on motivation and the ability to identify sounds under a layer of pink noise, using two versions of a training application. The first version was a serious game that utilized common game design features with the aim of increasing the player’s motivation to keep playing. The second version, called the basic training application was used as a base-line and modeled after the training method used by Shafiro (2008) in his experiment #2. 14 participants (divided into two groups of 7) were tested on their ability to identify sounds and how motivated they felt to continue training. Results pointed to the basic training application being better at increasing the participants’ sound identification ability, and the serious game being better at retaining the participants’ motivation to keep playing. Future studies should aim to combine features from both versions of the training application.