A two-dimensional, nonlinear dynamic analysis is conducted on a moored breakwater configuration to investigate snap loads in mooring lines. Breakwaters are structures used to attenuate or eliminate waves and protect shorelines, harbors, and other natural and man-made marine structures from wave damage. The breakwater in this investigation is modeled both as a point mass and as a rigid body. Both models are subjected to free undamped motions and forced undamped wave motion. Energy is dissipated through the use of a coefficient of restitution applied when a mooring line becomes taut (i.e., reaches its natural length). The mooring line is modeled as an inextensible cable with no axial or bending resistance when slack. Snap loading arises when a mooring line transitions suddenly from a slack condition to a taut condition. The analysis was conducted on a breakwater configured upside down and hanging by two mooring lines. The length of the mooring lines, coefficient of restitution, size and shape of the breakwater, initial position of the breakwater, amplitude of wave forcing, ratio of vertical to horizontal forcing, and frequency of forcing were all varied in the analysis. The results show that the rotations of the rigid body and the wave forcing have a significant role in the analysis, indicating that a rigid-body model for a moored breakwater under wave forcing is the more accurate model. / Master of Science

When a rope quickly transfers from a slack state to a taut state, a snapping action occurs and produces a large tensile force which is known as a snap load. Energy is dissipated during this snap load, and it is proposed to use synthetic fiber ropes as a type of passive earthquake damper in order to take advantage of this phenomenon. This thesis is the second phase of a multi-stage research project whose goal is to investigate and develop what will be known as Snapping-Cable Energy Dissipators (SCEDs). The experimental data that was collected in the Master'­s Thesis of Nicholas Pearson was organized and analyzed as a part of this research in order to evaluate the behavior of the ropes during the snapping action. Additional tests were also conducted for this project under more controlled conditions in order to better understand how the ropes change throughout a sequence of similar snap loadings and also to determine the amount of energy that is dissipated. The data from both projects was then used as input parameters for a mathematical model that was developed to characterize the behavior of the ropes during a snap load. This model will be utilized in subsequent research involving the finite element analysis of the seismic response of structural frames containing SCEDs. / Master of Science

Energy is lost when a rope transfers from a slack state to a taut state. This transfer is called a snap load and can be very violent. It is proposed to use synthetic fiber ropes as a type of passive control device in new or existing structures to mitigate seismic response. Experimental static and snap load (dynamic) tests were conducted on various synthetic fiber ropes. An eleven-foot-tall drop tower was built in the Virginia Tech Structures and Materials Laboratory in order to conduct these tests. Force and acceleration of the drop plate, which slides vertically within the drop tower, were measured with respect to time for all dynamic tests. Acceleration data was integrated using the trapezoidal or midpoint rule to obtain velocity and displacement values. Plots were made for each test in order to give a better representation of the results. These plots include representations of force and acceleration vs. time, force vs. absolute displacement, force vs. velocity, and force, acceleration, velocity, and displacement vs. time (during the initial taut phase only). Test results show that energy was dissipated in all of the dynamic drop tests, which was expected. Also, the displacement of each rope did not return to zero at the same time that the force returned to zero after the initial snap load. This proves that the ropes undergo some permanent elongation under load. The stiffness of each rope increased with continuous testing. As more tests are conducted on each rope, the strands are pulled tighter into the braided configuration, which causes the rope to become stiffer. / Master of Science

A three-dimensional, nonlinear dynamic analysis is conducted on a fully submerged, rigid, solid cylinder to be used as a breakwater. The breakwater could potentially be used as a single cylinder to protect small structures. Alternatively, multiple cylinders could be positioned in series to protect shorelines, harbors, or moored vessels from destructive incident water waves. The cylinder is positioned with its axis horizontal and is moored to the seafloor with four symmetrically placed massless mooring lines connected at the ends of the cylinder. The mooring lines are modeled as both linearly elastic ("regular") springs and compressionless springs. All six degrees of freedom of the structure are considered. The breakwater is modeled in air with a net buoyant force acting through the cylinder's center of gravity. The six "dry" natural frequencies of the structure are computed. Both linear and nonlinear free vibrations of the structure are considered. Linear damping is used to model the fluid and mooring damping effects. Normal and oblique harmonic wave forces at various frequencies and amplitudes are applied to the cylinder. The effects of the forcing amplitude and frequency, and the coefficient of damping, on the motion of the breakwater are studied. The results show that more erratic behavior occurs for the breakwater with compressionless springs, mainly due to the development of snap loads in the mooring lines. / Master of Science

The geometrically nonlinear response of a shallow, circular, cylindrical panel under a midspan line load and induced strain actuation is presented. The panel is a laminate of piezoelectric material perfectly bonded to the convex and concave surfaces of a core of passive material. Since the curved edges are free and the straight edges are pinned a fixed distance apart, the response of the panel is independent of the axial coordinate. Hence, the governing ordinary differential equations are of the same form as for a shallow circular arch. Without induced strain actuation, the panel exhibits snap-through behavior under the midspan load. Induced strain distributions are determined at a constant midspan load to displace the panel to an inverted configuration in a stable manner. This adaptive structure may find application as an electromechanical, nonlinear spring with a digital-like, load-displacement response characteristic. / Master of Science

No description available.

L’étude de l’assemblage et de l’acheminement à la membrane plasmique des complexes de signalisation des RCPGs va jouer un rôle crucial dans le développement de nouveaux médicaments ayant moins d’effets secondaires. Des outils permettant l’étude de ces phénomènes existent déjà, mais un outil polyvalent qui permettrait d’étudier plusieurs aspects pourrait grandement faciliter et accélérer ces études. L’étiquette SNAP est un candidat intéressant puisqu’avec cette étiquette il est possible de marquer une seule construction avec une variété de différents substrats. Une construction encodant pour le récepteur β2AR avec une étiquette SNAP à son extrémité C-terminale a été ingénérée. Cette construction est apte à lier son ligand, à être acheminée à la membrane plasmique et à homodimériser. La protéine exprimée a été marquée avec le fluorophore BG-430. De la fluorescence non spécifique a été détectée dans la cellule (même en absence de l’étiquette SNAP sur le récepteur). Un essai BRET a été développé, utilisant la construction HA-β2AR-SNAP en tant qu’accepteur et est fonctionnel. L’étiquette SNAP, comme utilisée ici, ne présente pas un aussi bon candidat qu’attendu, puisque le substrat n’ayant pas réagi demeure coincé dans la cellule. Le facteur activateur de plaquette (PAF) et son récepteur (PAFR) jouent un rôle critique dans plusieurs réponses inflammatoires et le récepteur FP est impliqué dans l’accouchement prématuré. Une meilleure compréhension des complexes de signalisation associés à ces récepteurs pourrait être la première étape dans la compréhension de leur signalisation dans des situations normales ou de maladies. Des expériences de BRET étudiant des interactions de bases entre les récepteurs et leurs partenaires d’interactions connus ont été réalisées. Elles ont permis de déterminer que : les récepteurs PAF et FP homodimérisent, que les récepteurs PAF et FP hétérodimérisent, que les protéines Gβγ interagissent de manière constitutive avec ces récepteurs et qu’aucun signal de BRET n’a été détecté avec la protéine Gα et ce, en présence ou en absence de stimulation par un agoniste (suggérant qu’il est nécessaire d’optimiser le système présentement utilisé). / Studies of the assembly and plasma membrane trafficking of G protein-coupled receptor (GPCR) signalling complexes will play an important role in the development of new drugs with fewer side effects. Tools for this purpose have already been developed, but a more versatile tool which would allow assessment of multiple aspects of GPCR trafficking and protein/protein interaction could greatly facilitate and expedite these studies. The SNAP tag is without doubt an interesting candidate for this task. Using this tag, it is possible to label one receptor construct, with a variety of different substrates. A construct encoding the β2AR receptor was engineered with a C-terminal SNAP tag. This construct, when expressed in HEK 293 cells, was able to bind ligand, to traffic to the plasma membrane and to form homodimers. The expressed protein was then labelled with the BG-430 fluorophore. Non-specific fluorescence was detected in the cell, even in the absence of the SNAP tag. A BRET assay was developed using the HA-β2AR-SNAP as a BRET acceptor. The SNAP tag as used in this initial study was not as good as thought previously because uneacted substrate remains trapped in cells (i.e. the background was too high). Platelet-activating factor (PAF) and its receptor (PAF-R) play a critical role in many inflammatory responses and the receptor for prostaglandin F (FP) plays a role in pre-term labour. A better understanding of signalling complexes associated with these receptors might be the first step in a better understanding of their signalling in health and disease. As an initial step in this direction, we used BRET to study basic interactions between these receptors and known signalling partners. PAF-R and FP receptors homodimerize. A heterodimer between the FP and PAF receptors was also detected. Gβγ subunits interact with these receptors in a constitutive way. In the case of the Gα, no BRET signal was detected with or without agonist stimulation suggesting more work is required to optimize the system.

O óxido nítrico (NO) é um mensageiro químico detectado em vários tipos celulares como células endoteliais, neurônios e macrófagos, desempenhando também funções variadas como vasodilatação, neurotransmissão e indução de morte celular. O NO é gerado pela atividade da enzima óxido nítrico sintase (NOS), a qual foi detectada em vários órgãos incluindo o sistema reprodutor. O sistema NOS/NO parece desempenhar papel importante na maturação oocitária entre outras funções. No entanto, apesar das evidências, há poucos estudos sobre o possível papel desse sistema em oócitos da espécie bovina. O presente estudo teve por objetivo investigar a importância do sistema NOS/NO na maturação in vitro (MIV) de oócitos bovinos. Para isso, foram avaliados os efeitos da inibição da NOS durante a MIV na presença de concentrações crescentes do inibidor de NOS (L-NAME, 0-1mM) e do aumento do NO durante a MIV na presença de concentrações crescentes do doador de NO (SNAP, 0-1mM) sobre a taxa de maturação (metáfase II) e formação de blastocistos após a fecundação in vitro. Também foi avaliado o efeito da pré-maturação com butirolactona (10µM BLI), seguida de MIV, na presença ou não de doador ou inibidor de NO em cada fase de cultivo, sobre o desenvolvimento de blastocistos. Após 22h de MIV os grupos tratados com L-NAME apresentaram uma taxa de metáfase II (MII) inferior (~80%, P<0,05) ao controle (95,5%). A taxa de blastocisto foi similar entre os grupos (34 a 41,5%, P>0,05). Apenas 7,2% (P<0,05) dos oócitos maturados com a maior concentração de SNAP (10-3M), atingiram o estádio de MII. Os demais tratamentos (71,6; 72,4 e 54,8% para controle, 10-7 e 10-5M, respectivamente) não diferiram entre si (P>0,05). Altos níveis de SNAP (10-3M) bloquearam o desenvolvimento embrionário, enquanto os demais tratamentos apresentaram cerca de 38% de blastocistos (P>0,05). As taxas de blastocistos (26,3 a 34,1%) e de eclosão (14,8 a 22,0%) adicionando ou não L-NAME em baixa concentração (10-7M) durante a pré-maturação, a maturação ou ambas as fases foram similares (P>0,05). Também não houve diferença (P>0,05) na taxa de blastocistos (25,5 a 39,7%) com a adição ou não de baixa concentração de SNAP (10-7M) durante a pré-maturação, a maturação ou ambas as fases. O grupo com SNAP (10-7M) na pré-maturação e MIV superou a taxa de eclosão (27,5%, P<0,05) dos demais grupos que tiveram SNAP adicionado somente no bloqueio, na MIV ou sem adição de SNAP (14,2 a 18,6 %, P>0,05). Esses resultados exibiram o duplo efeito do NO sobre oócitos bovinos, que tiveram a maturação reduzida com a inibição da síntese de NO e a maturação nuclear e citoplasmática bloqueadas pelo excesso de NO. / Nitric oxide (NO) is a chemical messenger detected in several cell types such as endothelial cells, neurons and macrophages, performing also varied functions as vasodilatation, neurotransmission and induction of cell death. NO is generated by the activity of the enzyme nitric oxide synthase (NOS), which has been detected in several organs including the reproductive system. The NOS/NO system seems to play an important role in oocyte maturation besides other functions. However, despite the evidence, there are few studies on the possible role of this system in bovine oocytes. The present study aimed to investigate the importance of the NOS/NO system on in vitro maturation (IVM) of bovine oocytes. The effects of NOS inhibition during IVM in the presence of increasing concentrations of NOS inhibitor (L-NAME, 0-1mM) and of the increase in NO during IVM with the NO donor SNAP (0-1mM) on maturation rates (metaphase II) and on blastocyst development after in vitro fertilization were assessed. The effect of prematuration with butyrolactone I (10µM BLI) followed by IVM, in the presence or not of NO inhibitor or donor in each culture period on blastocyst development was also investigated. After 22h IVM, L-NAME treated groups showed a lower (~80%, P<0.05) metaphase II (MII) rate when compared with controls (95.5%). Blastocyst rates were similar among all groups (34 to 41.5%, P>0.05). Only 7.2% (P<0.05) of oocytes matured with the highest SNAP concentration (10-3M) reached MII. The other treatments (71.6; 72.4 and 54.8% for control, 10-7 and 10-5M, respectively) were similar among them (P>0.05). High SNAP concentration (10-3M) blocked blastocyst development, while the other treatments presented about 38% blastocyst rates (P<0.05). Blastocyst (26.3 to 34.1%) and hatching rates (14.8 and 22.0%) were similar (P>0.05) when low L-NAME concentration (10-7M) was added or not during prematuration, maturation or both. Blastocyst rates (25.5 to 39.7%) were also similar (P>0.05) whether SNAP (10-7M) was added or not during prematuration, IVM or both. When low concentration of SNAP (10-7M) was added during both prematuration and IVM, hatching rates were increased (27.5%, P<0.05) when compared with oocytes cultured in the presence of SNAP only during prematuration or IVM or without it (14.2 to 18.6 %, P>0.05). These results shoe the dual effect of NO on bovine oocytes, which had maturation rates decreased when NO synthesis was inhibited and nuclear maturation and blastocyst development were blocked by excess NO.

Introdução: Receptores TRPV1 são canais catiônicos nociceptivos não seletivos. Eles desempenham um papel na sensação de dor na córnea. No presente trabalho, descreveremos a resposta do TRPV1 na córnea de camundongos e TRPV1 -/- a estímulos químicos e o efeito do SNAP sobre este receptor. Métodos: camundongos C57BL/6 e TRPV1 -/- foram comparados. As observações incluíram o influxo de cálcio na cultura, observação direta e o comportamento de limpeza do olho após o desafio na córnea com 1µM da capsaicina (CAP) com ou sem 1mM do colírio SNAP. Resultados: As características da superfície ocular não eram diferentes entre ambos os genótipos, exceto que a sensibilidade para CAP é inferior em TRPV1 -/- (p = 0,0019 e 0,0095, respectivamente). A imunocoloração revelou que TRPV1 é expresso na camada basal do epitélio da córnea, apenas nos camundongos C57BL/6. CAP estimula o influxo de cálcio em cultura de células epiteliais e a sensibilidade corneana em C57BL/6 in vivo foi reduzida na presença do SNAP (p = 0,0329 e 0,01, respectivamente). Conclusão: A ausência dos receptores TRPV1 no epitélio da córnea não afeta o fenótipo da superfície ocular de camundongos. A resposta para tirar colírios revelou que ela poderia funcionar como uma terapia analgésica devido ao seu antagonismo com o TRPV1. / TRPV-1 receptors are non-selective nocioceptive cation channels. They play a role in cornea sensation. In the present work we describe the response of corneas of TRPV1-/- and mice to chemical stimuli and the effect of SNAP on this receptor. TRPV1-/- and C57BL/6 mice were compared. The observations included the calcium influx in culture, direct observation and the behavior of eye wipe after corneal challenge with 1?M Capsaicin (CAP) with or not 1mM SNAP eye drops. Ocular surface characteristics were not different between both genotypes, except that the sensitivity to CAP is lower in TRPV1-/- (p=0.0019 and 0.0095, respectively). Immunostaining revealed that TRPV 1 is expressed in the basal layer of the cornea epithelia, only in the C57 mice. CAP stimulated calcium influx in epithelial cells in culture and cornea sensitivity in C57 mice in vivo was reduced in the presence of SNAP (p=0.0329 and 0.01, respectively). The absence of TRPV-1 receptors in the cornea epithelia did not affect the ocular surface phenotype in mice. The response to SNAP eye drops revealed that it could work as an analgesic therapy due to its antagonism to the TRPV1.

Introdução: Receptores TRPV1 são canais catiônicos nociceptivos não seletivos. Eles desempenham um papel na sensação de dor na córnea. No presente trabalho, descreveremos a resposta do TRPV1 na córnea de camundongos e TRPV1 -/- a estímulos químicos e o efeito do SNAP sobre este receptor. Métodos: camundongos C57BL/6 e TRPV1 -/- foram comparados. As observações incluíram o influxo de cálcio na cultura, observação direta e o comportamento de limpeza do olho após o desafio na córnea com 1µM da capsaicina (CAP) com ou sem 1mM do colírio SNAP. Resultados: As características da superfície ocular não eram diferentes entre ambos os genótipos, exceto que a sensibilidade para CAP é inferior em TRPV1 -/- (p = 0,0019 e 0,0095, respectivamente). A imunocoloração revelou que TRPV1 é expresso na camada basal do epitélio da córnea, apenas nos camundongos C57BL/6. CAP estimula o influxo de cálcio em cultura de células epiteliais e a sensibilidade corneana em C57BL/6 in vivo foi reduzida na presença do SNAP (p = 0,0329 e 0,01, respectivamente). Conclusão: A ausência dos receptores TRPV1 no epitélio da córnea não afeta o fenótipo da superfície ocular de camundongos. A resposta para tirar colírios revelou que ela poderia funcionar como uma terapia analgésica devido ao seu antagonismo com o TRPV1. / TRPV-1 receptors are non-selective nocioceptive cation channels. They play a role in cornea sensation. In the present work we describe the response of corneas of TRPV1-/- and mice to chemical stimuli and the effect of SNAP on this receptor. TRPV1-/- and C57BL/6 mice were compared. The observations included the calcium influx in culture, direct observation and the behavior of eye wipe after corneal challenge with 1?M Capsaicin (CAP) with or not 1mM SNAP eye drops. Ocular surface characteristics were not different between both genotypes, except that the sensitivity to CAP is lower in TRPV1-/- (p=0.0019 and 0.0095, respectively). Immunostaining revealed that TRPV 1 is expressed in the basal layer of the cornea epithelia, only in the C57 mice. CAP stimulated calcium influx in epithelial cells in culture and cornea sensitivity in C57 mice in vivo was reduced in the presence of SNAP (p=0.0329 and 0.01, respectively). The absence of TRPV-1 receptors in the cornea epithelia did not affect the ocular surface phenotype in mice. The response to SNAP eye drops revealed that it could work as an analgesic therapy due to its antagonism to the TRPV1.