In cardiac myocytes, multiple receptor mediated signalling pathways converge on cyclic nucleotide production. These second messengers act to achieve changes in cellular function. Despite this, each signalling molecule and receptor can achieve distinct sub-cellular effects. This has led to the theory of cyclic nucleotide compartmentation, which has been postulated to be mediated by phosphodiesterases (PDEs). Research in this field has focused on compartmentation using β-adrenergic stimulation. As an extension of this work, we investigated the effects of two agonists, prostaglandin E2 (PGE2; 10 nM) and forskolin (FSK; 30 nM), on various cellular parameters in the presence of either cilostamide (1 µM) a selective PDE3 inhibitor, or Ro 20-1724 (10 µM) a selective PDE4 inhibitor. In myocytes treated with PGE2, unloaded cell shortening and intracellular calcium transients exhibited significantly different (p<0.05) values of 147 ± 10% and 138 ± 5% of pre-treatment (t=0) values, respectively, in the presence of PGE2 and Ro 20-1724 (all n=5). However, values were not significantly different in cells pre-treated with cilostamide. Conversely, FSK resulted in significant increases of 153 ± 9% (n=5; P>0.05) and 189 ± 20% (n=5; P>0.05) of t=0 in cells treated with cilostamide and Ro 20-1724, respectively. PGE2 enhanced ICa,L was not altered using either PDE inhibitor. However, with FSK as an agonist, a significant increase in peak ICa,L from -6.0 ± 0.8 pA/pF to -7.7 ± 0.4 pA/pF (n=5; P>0.05) was observed in cells pre-treated with Ro 20-1724. SR calcium loading was also increased, but only in cells pre-treated with Ro 20-1724, with values of 127 ± 11% and 156 ± 47% of t=0 (n=5) for FSK and PGE2, respectively. Our results demonstrate that a unique pattern of regulation exists for PGE2 and that it is different from what was found previously with isoproterenol. We have shown that this is achieved by functionally localizing PDEs to distinct compartments. Specifically, PDE4 is localized at the SR, PDE3 at the sarcomere, and a combination of both at the calcium channel. However, our ICa,L results also indicate that the location of the receptor and adenylate cyclases must be considered relevant to compartmentalizing the cAMP signal. / Thesis (Master, Physiology) -- Queen's University, 2009-07-15 11:01:49.571

Thesis (M.S.)--Florida State University, 2005. / Advisor: Dr. W. Ross Ellington, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed June 15, 2005). Document formatted into pages; contains x, 51 pages. Includes bibliographical references.

No description available.

Nesta dissertação, são apresentados os processos de caracterização, classificação e de compartimentação geomecânica dos maciços rochosos, com a utilização dos dados pertinentes às fases de estudos nos projetos de engenharia. Como área de pesquisa analisou-se os dados referentes aos estudos da fundação da barragem, de uma Usina Hidroelétrica, localizada no Rio Pelotas, entre os estados do Rio Grande do Sul (Margem esquerda) e Santa Catarina (Margem Direita) em um sítio constituído por uma seqüência de derrames basálticos da Formação Serra Geral na Bacia do Paraná. Este maciço revela-se verticalmente heterogêneo, constituído por uma seqüência de 15 (quinze) derrames (relativamente pouco espessos) que possuem distintas características tanto sobre os aspectos geomecânicos, estruturais e tectônicos; quanto da diferenciação litológica de cada um - que compreende a seqüência de basalto denso, basalto vesículo amigdaloidal e brecha basáltica. O intuito da pesquisa foi mostrar a caracterização do maciço abrangendo aspectos estratigráficos, litológicos, estruturais, tectônicos e geomecânicos, para que fosse possível a formulação da compartimentação e dos modelos geológicos e geomecânicos do maciço. Na composição da base de dados foi realizado um levantamento de campo por meio de investigações de superfície e subsuperfície, para que posteriormente fossem realizados os trabalhos de gabinete e as análises laboratoriais. Para o estabelecimento do modelo geológico orientou-se pelos dados litoestratigráficos, estruturais e tectônicos obtidos através dos mapeamentos de superfície e das investigações de subsuperfície. A compartimentação geomecânica foi composta por informações oriundas dos itens de caracterização do maciço (alteração, fraturamento, Rock qualitiy designation - RQD, e permeabilidade); e pela classificação geomecânica, quanto a esta, empregaram-se as metodologia Rock Mass Rating - RMR, Quality - Q e Geological Strength Index - GSI. O propósito dessa classificação visava estabelecer um paralelo entre os parâmetros metodológicos utilizados e os resultados obtidos - em que se notou uma grande dependência da compartimentação geomecânica ao modelo geológico. / In this dissertation, the processes of characterization, classification and geomechanical compartmentation of the rocky mass range are investigated with the utilization of data resulting from the study phases in engineering projects. As a research area, the data referring to the studies of the foundation of a hydroelectric power plant dam, located on Rio Pelotas, between the states of Rio Grande do Sul (left bank) and Santa Catarina (right bank) on a site formed by a sequence of basaltic flood of Serra Geral formation at the Paraná Basin have been analyzed. This rock mass range, vertically heterogeneous, is formed by a sequence of 15 (fifteen) floods (relatively little solid) which possess distinct characteristics on the geomechanical, structural and tectonic aspects, as well as on the lithological differentiation of each one - which includes the sequence of dense basalt, vesicles-amygdaloidal basalt and basaltic breach. The intention of the research was to show the characterization of the mass range, embracing stratigraphic, lithological, structural, tectonic and geomechanical aspects, so that it would be possible the formulation of the compartmentation and geological and geomechanical models of the mass range. In the database composítion, a field survey was carried out through surface and subsurface investigations, so that afterwards the laboratorial analysis could be accomplished. For the establishment of the geological model, the lithostratigraphic, structural and tectonic data have been examined, which have been obtained through the mapping of the surface and subsurface investigations. The geomechanical compartmentation was put together by the information resulting from the characterization items of the mountain range (alteration, fracture, Rock quality designation - RQD and permeability); and by the geomechanical classification; for that matter, the Rock Mass Rating - RMR, Quality - Q and Geological Strength Index - GSI methodologies have been employed. The purpose of this classification was to establish a parallel between the methodological parameters used and the results obtained - where a large dependence of the geomechanical compartmentation on the geological model had been observed.

Pathways and mechanisms for human memory T cell differentiation and maintenance have largely been inferred from studies of peripheral blood, though the majority of T cells are found in lymphoid and mucosal sites. We present here a novel, multidimensional, quantitative analysis of human T cell compartmentalization and maintenance over six decades of life in blood, lymphoid and mucosal tissues obtained from 56 individual organ donors. Our results reveal that the distribution and tissue residence of naïve, central and effector memory, and terminal effector subsets is contingent on both differentiation state and tissue localization. Moreover, T cell homeostasis driven by cytokine or TCR-mediated signals is dependent on CD4+ or CD8+ T cell lineage, subset differentiation and tissue localization, and cannot be inferred from blood. Our data provide an unprecedented spatial and temporal map of human T cell compartmentalization and maintenance, supporting new pathways for human T cell fate determination and homeostasis. Memory T cells can remain in tissues as non-circulating, resident memory populations, which provide optimal protection against infection at barrier surfaces. Lung-resident memory T cells (TRM) mediate in situ protection to respiratory pathogens, though mechanisms for their maintenance and retention are unknown. Through whole transcriptome profiling, we identify a cohesive network of genes enriched in lung CD4+ TRM, including Itgad (CD11d), Cd69, and IFN-associated responders. We find that upregulation of CD11d enhances CD69 expression through type I IFN signaling downstream of homotypic cell adhesion, and is required for optimal T cell differentiation and lung retention. Moreover, blockade of IFNαR1 reduces CD11d expression and retention of influenza-generated lung TRM, suggesting that CD11d-dependent type I IFN signaling promotes TRM establishment. Our results implicate CD11d and type I IFN in retaining lung CD4+ TRM cells, and identify potential targets for modulating tissue immunity.

Nesta dissertação, são apresentados os processos de caracterização, classificação e de compartimentação geomecânica dos maciços rochosos, com a utilização dos dados pertinentes às fases de estudos nos projetos de engenharia. Como área de pesquisa analisou-se os dados referentes aos estudos da fundação da barragem, de uma Usina Hidroelétrica, localizada no Rio Pelotas, entre os estados do Rio Grande do Sul (Margem esquerda) e Santa Catarina (Margem Direita) em um sítio constituído por uma seqüência de derrames basálticos da Formação Serra Geral na Bacia do Paraná. Este maciço revela-se verticalmente heterogêneo, constituído por uma seqüência de 15 (quinze) derrames (relativamente pouco espessos) que possuem distintas características tanto sobre os aspectos geomecânicos, estruturais e tectônicos; quanto da diferenciação litológica de cada um - que compreende a seqüência de basalto denso, basalto vesículo amigdaloidal e brecha basáltica. O intuito da pesquisa foi mostrar a caracterização do maciço abrangendo aspectos estratigráficos, litológicos, estruturais, tectônicos e geomecânicos, para que fosse possível a formulação da compartimentação e dos modelos geológicos e geomecânicos do maciço. Na composição da base de dados foi realizado um levantamento de campo por meio de investigações de superfície e subsuperfície, para que posteriormente fossem realizados os trabalhos de gabinete e as análises laboratoriais. Para o estabelecimento do modelo geológico orientou-se pelos dados litoestratigráficos, estruturais e tectônicos obtidos através dos mapeamentos de superfície e das investigações de subsuperfície. A compartimentação geomecânica foi composta por informações oriundas dos itens de caracterização do maciço (alteração, fraturamento, Rock qualitiy designation - RQD, e permeabilidade); e pela classificação geomecânica, quanto a esta, empregaram-se as metodologia Rock Mass Rating - RMR, Quality - Q e Geological Strength Index - GSI. O propósito dessa classificação visava estabelecer um paralelo entre os parâmetros metodológicos utilizados e os resultados obtidos - em que se notou uma grande dependência da compartimentação geomecânica ao modelo geológico. / In this dissertation, the processes of characterization, classification and geomechanical compartmentation of the rocky mass range are investigated with the utilization of data resulting from the study phases in engineering projects. As a research area, the data referring to the studies of the foundation of a hydroelectric power plant dam, located on Rio Pelotas, between the states of Rio Grande do Sul (left bank) and Santa Catarina (right bank) on a site formed by a sequence of basaltic flood of Serra Geral formation at the Paraná Basin have been analyzed. This rock mass range, vertically heterogeneous, is formed by a sequence of 15 (fifteen) floods (relatively little solid) which possess distinct characteristics on the geomechanical, structural and tectonic aspects, as well as on the lithological differentiation of each one - which includes the sequence of dense basalt, vesicles-amygdaloidal basalt and basaltic breach. The intention of the research was to show the characterization of the mass range, embracing stratigraphic, lithological, structural, tectonic and geomechanical aspects, so that it would be possible the formulation of the compartmentation and geological and geomechanical models of the mass range. In the database composítion, a field survey was carried out through surface and subsurface investigations, so that afterwards the laboratorial analysis could be accomplished. For the establishment of the geological model, the lithostratigraphic, structural and tectonic data have been examined, which have been obtained through the mapping of the surface and subsurface investigations. The geomechanical compartmentation was put together by the information resulting from the characterization items of the mountain range (alteration, fracture, Rock quality designation - RQD and permeability); and by the geomechanical classification; for that matter, the Rock Mass Rating - RMR, Quality - Q and Geological Strength Index - GSI methodologies have been employed. The purpose of this classification was to establish a parallel between the methodological parameters used and the results obtained - where a large dependence of the geomechanical compartmentation on the geological model had been observed.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 166-179). Also issued on the Internet.

La protéine kinase AMPc-dépendante (PKA) joue un rôle crucial dans la régulation neurohormonale de la fonction cardiaque. L’activation aiguë de la PKA est bénéfique car elle conduit à une augmentation de la contraction cardiaque en phosphorylant les acteurs clés du couplage excitation-contraction. En revanche, son activation chronique est délétère et ces effets semblent faire intervenir la régulation de protéines nucléaires pouvant conduire au remodelage hypertrophique et à l'insuffisance cardiaque. La localisation subcellulaire de la PKA, assurée par des protéines d’ancrage (AKAPs), est importante pour la rapidité et la spécificité d’action des hormones mettant en jeu la voie de l’AMPc. Les niveaux d’AMPc sont régulés par l’activité des adénylate cyclases et des phosphodiestérases (PDEs), et l’état de phosphorylation des protéines cibles de la PKA dépend de l’activité des Ser/Thr phosphatases (PPs). Dans le cœur, les PDEs les plus importantes dégradant l’AMPc sont les PDE3 et les PDE4. Les principales PPs cardiaques sont PP1, PP2A et PP2B. Dans une première partie de mon travail, j’ai mis au point, dans les cardiomyocytes de rats adultes, une mesure de l’activité de la PKA en temps réel dans les compartiments cytoplasmiques et nucléaires. J’ai utilisé pour cela des sondes de type AKAR (A-kinase activity reporters) basées sur le transfert d’énergie de fluorescence (FRET) et localisées spécifiquement dans le noyau ou dans le cytoplasme par des séquences d’adressage ou d’exclusion nucléaires. J’ai ainsi pu montrer qu’une stimulation maintenue des récepteurs β-adrénergiques active la PKA de façon plus importante dans le cytoplasme que dans le noyau, et que cette activation se développe lentement au niveau nucléaire que dans le cytoplasme. De ce fait, une stimulation brève des récepteurs β-adrénergiques active maximalement la PKA dans le cytoplasme, mais de façon marginale dans le noyau. Dans une seconde partie de l’étude, je me suis intéressée au rôle des PDE3 et PDE4 ainsi qu’à celui de PP1, PP2A et PP2B dans la régulation de l’activité PKA cytoplasmique et nucléaire, en réponse à une stimulation β-adrénergique. J’ai montré que la PDE4, mais pas la PDE3, régule l’activité de la PKA cytoplasmique et nucléaire. L’utilisation de souris invalidées pour les gènes Pde4b et Pde4d a révélé que l’isoforme PDE4B est prédominante pour la modulation de l’activité PKA cytoplasmique, alors que les deux isoformes PDE4B et PDE4D contribuent à la régulation de l’activité PKA nucléaire. Finalement, j’ai montré que la PP1 et la PP2A, mais pas la PP2B, participent à la terminaison des réponses β-adrénergiques dans le cytoplasme, alors qu’au niveau nucléaire, la PP1 semble jouer un rôle majeur. En conclusion, ce travail a mis en évidence le rôle des phosphodiestérases et des phosphatases dans l’intégration différentielle des réponses PKA à une stimulation β-adrénergique dans le cytoplasme et le noyau de cardiomyocytes adultes. / The cAMP-dependent protein kinase (PKA) exerts short term beneficial effects on cardiac function by phosphorylating several key excitation-contraction coupling (ECC) proteins. However, its chronic activation is deleterious on the long term, and this may involve regulation of nuclear effectors ultimately leading to hypertrophic remodelling and heart failure. The subcellular localization of PKA, mediated by anchoring proteins (AKAPs), is important for the speed and specificity of hormones that activate the cAMP pathway. The levels of cAMP are regulated by adenylyl cyclase and phosphodiesterases (PDEs), and PKA activity is counterbalanced by Ser/Thr phosphatases (PPs). In heart, the most important PDEs that degrade cAMP belong to the PDE3 and PDE4 famillies, whereas the major cardiac PPs are PP1, PP2A and PP2B. In a first part, I developed, in adult rat cardiomyocytes, a technique to measure PKA activity in real time specifically in the cytoplasm and the nucleus. For this I used genetically-encoded fluorescence resonance energy transfer (FRET) sensors called AKAR (A-kinase activity reporters) that can be targeted specifically to the nucleus or the cytoplasm by nuclear localization or exclusion sequences, respectively. Using this approach, I showed that maintained β-adrenergic stimulation activates PKA more efficiently and more potently in the cytoplasm than in the nucleus, and that the kinetics of PKA activation was much slower in the nucleus than in the cytoplasm. Accordingly, a short β-adrenergic stimulation maximally activated PKA in the cytoplasm but marginally activated PKA in the nucleus. In a second part, I characterized the respective contribution of PDE3, PDE4, and PP1, PP2A and PP2B families in the regulation of cytoplasmic and nuclear PKA activity in response to β-adrenergic stimulation. PDE4, but not PDE3, regulates PKA activity in the cytoplasm and in the nucleus. The use of knock out mice for Pde4b and Pde4d genes revealed that PDE4B plays a predominant role to modulate β-AR stimulation of cytoplasmic PKA, whereas in the nucleus both PDE4B and PDE4D isoforms contribute. Finally, I showed that both PP1 and PP2A, but not PP2B, participate to the termination of β-adrenergic PKA responses in the cytoplasm, whereas PP1 appears to play a major role in the nuclei. In conclusion, this work highlights the role of phosphodiesterases and phosphatases in the differential integration of PKA responses to β-adrenergic stimulation in the cytoplasm and the nucleus of adult cardiomyocytes.

This dissertation presents the development of a multielectrode microcompartment platform for understanding signal transduction in the nervous system. The design and fabrication of the system and the characterization of the system for pharmacological and electrophysiological measurements of cultured neurons is presented in this work. The electrophysiological activity of cultured dorsal root ganglion (DRG) neurons and cortical neurons is shown on the MEA substrate. These recordings were measured and tied to the toxicological effects of the chemotherapeutic drug vincristine on DRGs. Conventional electrophysiological recordings (via a patch micropipette) are made routinely to record action potentials and ion channel activity in neurons. Moreover, Campenot chambers (traditional compartmented culture systems) have been used for the last thirty years to study the selective application of drugs to neurons. Both of these techniques are useful and well established; however they have their limitations. For instance, Campenot chambers cannot be used very well for small processs-producing neurons, since the barriers are difficult to tranverse. Moreover, conventional patch recordings are labor-intensive, especially when more than one microelectrode needs to be positioned. The developed system is composed of a two compartment divider, each compartment capable of housing axons or cell bodies. Underneath the divider, the substrate has 60 electrodes, arranged in several lines to accommodate several different neurite tracks. Neurons can be stimulated and their activity can be recorded in both of the compartments. The neurotoxin and chemotherapeutic drug vincristine was tested in the system on the DRGs. The drug caused length-dependent axonal degeneration in the DRGs when applied locally. Moreover, electrophysiological activity in both compartments showed that only the activity in the axonal compartment was affected, leading us to believe that the mechanism behind the degeneration is localized to the distal axon.

Previous studies have suggested that phosphodiesterase (PDE) hydrolysis of cyclic adenosine monophosphate (cAMP) is important in the generation of specific and segregated cAMP signals within cells. The purpose of this study was to determine if PDE compartmentation was important in cardiac ventricular myocytes. Therefore, we investigated the effects of β-adrenergic (β-AD) stimulation with isoproterenol in the presence of cilostamide, a PDE3 inhibitor, or Ro 20-1724, a PDE4 inhibitor, on unloaded cell shortening, L-type calcium currents and intracellular calcium levels in freshly dissociated adult rat ventricular myocytes. PDE3 inhibition resulted in a 216 ± 17 % (n=8) increase in unloaded cell shortening after ten minutes of isoproterenol exposure, whereas isoproterenol produced a statistically smaller increase of 155 ± 12 % (n=8) in the presence of PDE4 inhibition. There was a non-significant trend for PDE4 inhibition to produce larger increases in calcium currents (179 ± 17 % (n=4) of controls) than PDE3 inhibition (155 ± 10 % (n=6) of controls). Both PDE3 and PDE4 inhibitors had similar effects on isoproterenol-stimulated increases of calcium transient amplitude with values of 209 ± 14 % (n=8) and 185 ± 12 % (n=8), respectively. Determination of sarcoplasmic reticulum (SR) calcium load using caffeine pulse experiments demonstrated that PDE4 inhibition and isoproterenol superfusion produced a statistically larger increase in SR-calcium loading (139 ± 9 % (n=6)) than PDE3 inhibition and isoproterenol superfusion (113 ± 9 % (n=6)). These results suggest that PDE3 may be active in proximity to the contractile apparatus of cardiac myocytes, whereas PDE4 may be localized in a domain consisting of the L-type calcium channel and junctional SR. Consequently, our study provides functional evidence for differential localization of PDE isoforms in cardiac myocytes. / Thesis (Master, Physiology) -- Queen's University, 2008-01-18 10:14:29.671 / CIHR OGS OGSST