The Clamp-Rod Internal Fixator (CRIF) is a fracture fixation implant with growing popularity among veterinarian’s for its versatility and ease of use. Although the CRIF is currently in clinical use, relatively few reports exist describing the biomechanical properties and clinical results of this system. The objective of this study was to determine the in vitro biomechanical properties of a 5mm CRIF/rod construct to a 3.5mm Limited Contact-Dynamic Compression Plate (LC-DCP/rod) construct using a canine femoral gap model. Paired canine femora were treated with 40mm mid-diaphyseal ostectomies and randomly assigned to CRIF/rod or LC-DCP/rod. Five pairs of constructs were tested in bending and five pairs were evaluated in torsion. Single ramp to failure tests were conducted to evaluate construct stiffness, yield load, and failure mode. While CRIF/rod and LC-DCP/rod were not significantly different when evaluated in bending, LC-DCP/rod constructs are significantly more rigid than CRIF/rod constructs at higher torsional loads. Below 10degrees of twist, or 4.92Nm torque, the LC-DCP/rod and CRIF/rod were not statistically different in torsion. Catastrophic injuries of the metacarpophalangeal joint resulting in the disruption of the suspensory apparatus are the most common fatal injuries in thoroughbred racehorses. Fetlock arthrodesis is a procedure designed to mitigate suffering from injury as well as degenerative diseases affecting articulation. The objective of this study is to assess the in vitro biomechanical behavior of techniques for fetlock arthrodesis. Twelve forelimb pairs were collected from adult horses euthanized for reasons unrelated to disease of the metacarpophalangeal joint (MCP). A 14-16-hole broad 4.5mm Locking Compression Plate (LCP) was compared to a 14-16 hole broad Dynamic Compression Plate (DCP). Both constructs used a two “figure-eight” 1.25mm stainless steel wire tension band. Fatigue tests and to failure tests were conducted. There were no significant differences in stiffness between groups for fatigue tests. Stiffness increased after the first fatigue cycle for the LCP/wire (80.56+/-52.22%) and DCP/wire (56.58+/-14.85%). Above 3.5mm of axial deformation there was a statistical difference between the stiffness of the LCP/wire (3824.12+/-751.84 N/mm) and the DCP/wire (3009.65+/-718.25 N/mm) (P=0.038). The LCP/wire showed increased stiffness above 3.5mm compression compared to the DCP/wire. Under fatigue testing conditions the constructs are not statistically different.

There is a significant amount of computational literature on networks of neurons and their resulting behavior. This dissertation combines electrophysiology experiments with computational modeling to validate the assumptions and results found in this literature. First, we investigate the weak coupling assumption, which states that the phase response of a neuron to weak stimuli is separable from the stimulus waveform. For weak stimuli, there is an intrinsic neuronal property described by the infinitesimal phase response curve (IPRC) that will predict the phase response when convolved with the stimulus waveform. Here, we show that there is a linear relationship between the stimulus and phase response of the neuron, and that we are able to obtain IPRCs that successfully predict the neuronal phase response. Next, we use hybrid networks of neurons to study the phase locking behavior of networks as the synaptic time constant is changed. We verify that networks show anti-phase synchrony for fast time constants, and in-phase synchrony for slow time constants. We also show that phase models and phase response curves (PRCs) qualitatively predict phase locking observed in electrophysiology experiments. Finally, we investigate the stability of the dynamic clamp system. We determined that the maximal conductance of the current being simulated, the dynamic clamp sampling rate, the amount of electrode resistance compensation, and the amount of capacitance compensation all affect when the instability is present. There is a dramatic increase in stability when the electrode resistance and system capacitance are well compensated.

Diss. Stockholm : Karol. inst., 2004.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2016-10-18T14:55:15Z No. of bitstreams: 1 Milena Ramos Reis.pdf: 2152638 bytes, checksum: 876fac844a4f4a8b22cb82e9cdeb5f3b (MD5) / Made available in DSpace on 2016-10-18T14:55:15Z (GMT). No. of bitstreams: 1 Milena Ramos Reis.pdf: 2152638 bytes, checksum: 876fac844a4f4a8b22cb82e9cdeb5f3b (MD5) / O carvacrol, um monoterpeno fenólico encontrado nos óleos essenciais de diversas plantas do gênero Origanum, já demonstrou causar hipotensão e vasodilatação em diferentes leitos vasculares de ratos normotensos, porém, seu efeito em ratos hipertensos ainda não foi elucidado. O objetivo deste estudo foi investigar os efeitos cardiovasculares do carvacrol em ratos espontaneamente hipertensos (SHR) e comparar com normotensos Wistar, utilizando ensaios farmacológicos in vitro (estudos funcionais e celulares) e in vivo. Nos ensaios funcionais in vitro, anéis de artéria mesentérica superior isolada de animais hipertensos e normotensos foram précontraídos com FEN (1μM) e o efeito de carvacrol (10-8-10-3M) foi observado. Em SHR, este monoterpeno induziu vasodilatação dependente de concentração (pD2=5,13 ± 0,05; Emáx=115,14 ± 5,46%; N=8) e, após a remoção do endotélio funcional, a potência da droga foi alterada significantemente (pD2=4,91 ± 0,05 N=9; p<0,01), sugerindo que a resposta vasodilatadora induzida por carvacrol, provavelmente, envolve uma via dependente e outra independente do endotélio vascular, porém, esta última parece ser a majoritária e, por isso, os ensaios seguintes foram realizados na ausência do endotélio vascular. Interessantemente, quando comparada com animais normotensos, a potência farmacológica de carvacrol foi reduzida significantemente (pD2=4,91 ± 0,05; N=9; p<0,05). Em anéis de ratos hipertensos, carvacrol reduziu o influxo de Ca2+ por canais Cav tipo-L, SOC e ROC, estes resultados foram semelhantes aos obtidos em ratos normotensos. Em ratos hipertensos, mas não em normotensos, a potência farmacológica do carvacrol em anéis pré-contraídos com FEN e na presença de diferentes inibidores de canais TRP (íon Gd3+, 10-5M; 2-APB, 10-6M ou 10-5M; BCTC, 2μM; 9-fenantrol, 10-5M; ou HC03003-1, 10-5M), foi reduzida em relação ao controle na ausência destes bloqueadores, sugerindo que os canais sensíveis à estes bloqueadores (TRPC1-7, TRPM2, M4 e TRPM8, TRPV1 e TRPA1), provavelmente, estão participando dos efeitos vasculares mediados por carvacrol e podem estar envolvidos no processo hipertensivo. Em estudos de patch-clamp em células de artéria mesentérica dispersas de ratos hipertensos, carvacrol (300μM) reduziu as correntes de entrada de Ba2+ por Cav tipo-L e este efeito foi semelhante em ratos normotensos. Além disso, em células de ratos hipertensos, o Mg2+ (2,5mM), bloqueador do TRPM6 e TRPM7, reduziu as densidades de ITRPM de entrada e saída, assim como carvacrol (100μM e 300μM), na ausência ou presença do 2-APB (100μM), bloqueador de TRPM7. A presença do 2-APB provocou inibição adicional nas densidades de ITRPM pelo carvacrol (100μM, mas não 300μM). Altas concentrações intracelulares de Mg2+ reduziram the magnitude of ITRPM7. Foi evidenciado que a ITRPM no controle é menor em ratos hipertensos que em normotensos. Estes dados obtidos e os relatados na literatura são sugestivos para provável inibição de ITRPM7 por carvacrol em células mesentéricas nativas. O efeito anti-hipertensivo do carvacrol foi avaliado por administração via orogástrica (50mg/kg/dia) durante 20 dias foi capaz de reduzir a pressão arterial média dos animais SHR tratados, no 20º dia do tratamento. O tratamento subcrônico com carvacrol não alterou os pesos cardíaco e corpóreo, nem a reatividade vascular. Em conclusão, esses dados sugerem que carvacrol possui atividade anti-hipertensiva em animais SHR, que pode ser devido ao seu efeito vasodilatador em anéis de artéria mesentérica superior isolada, provavelmente, por inibição do influxo de Ca2+ por Cav tipo-L, ROC, SOC e/ou canais TRPC1, 3 ou 6, além da inibição de correntes tipo-TRPM7 em miócitos mesentéricos.

The interaural level difference (ILD) is a sound localization cue first computed in the lateral superior olive (LSO) by comparing the loudness of sounds between the two ears. In the auditory cortex, one class of neurons is excited by contralateral but not ipsilateral monaural sounds. These "EO" neurons prefer ILDs where contralateral sounds are louder than ipsilateral sounds. Another class, the "PB" neurons, are unresponsive to monaural sounds but respond predominantly to binaural ILDs, when both ears receive simultaneous sounds of roughly equal loudness (0 ILD). Behavioral studies show that ILD sensitivity is invariant to increasing sound levels. However, in the LSO, ILD response functions shift towards the excitatory ear as sound level increases, indicating level-dependence. Thus, changes in firing rate can indicate either a change in sound location or sound level, or both. This suggests a transformation in level-sensitivity between the LSO and the perception of sound sources, yet the location of this transformation remains unknown. I performed recordings in the auditory cortex of the rat to test whether neurons were invariant to overall sound level. I found that with increasing sound levels, ILD responses were level-dependent, suggesting that level invariance of ILD sensitivity is not present in the rat auditory cortex. In general, neurons follow one of two processing strategies. The tuning of cortical cells typically follows the "inheritance strategy", such that the spiking output of the cell matches that of the excitatory synaptic input. However, cortical tuning can be modified by inhibition in the "local processing strategy". In this case, neurons are prevented from spiking at non-preferred stimuli by inhibition that overwhelms excitation. The tuning strategy of cortical neurons to ILD remains unknown. I performed whole-cell recordings in the anesthetized rat and compared the spiking output with synaptic inputs to ILDs within the same neurons. I found that the PB neurons showed evidence of the local processing strategy, which is a novel role for cortical inhibition, whereas the EO neurons utilized the inheritance strategy. This result suggests that an auditory cortical circuit computes sensitivity for midline ILDs. This dissertation includes previously published/unpublished co-authored material.

abstract: Biophysical techniques have been increasingly applied toward answering biological questions with more precision. Here, three different biological systems were studied with the goal of understanding their dynamic differences, either conformational dynamics within the system or oligomerization dynamics between monomers. With Cy3 on the 5' end of DNA, the effects of changing the terminal base pair were explored using temperature-dependent quantum yields. It was discovered, in combination with simulations, that a terminal thymine base has the weakest stacking interactions with the Cy3 dye compared to the other three bases. With ME1 heterodimers, the goal was to see if engineering a salt bridge at the dimerization interface could allow for control over dimerization in a pH-dependent manner. This was performed experimentally by measuring FRET between monomers containing either a Dap or an Asp mutation and comparing FRET efficiency at different pHs. It was demonstrated that the heterodimeric salt bridge would only form in a pH range near neutrality. Finally, with DNA processivity clamps, one aim was to compare the equilibrium dissociation constants, kinetic rate constants, and lifetimes of the closed rings for beta clamp and PCNA. This was done using a variety of biophysical techniques but with three as the main focus: fluorescence correlation spectroscopy, single-molecule experiments, and time-correlated single photon counting measurements. The stability of beta clamp was found to be three orders of magnitude higher when measuring solution stability but only one order of magnitude higher when measuring intrinsic stability, which is a result of salt bridge interactions in the interface of beta clamp. Ongoing work built upon the findings from this project by attempting to disrupt interface stability of different beta clamp mutants by adding salt or changing the pH of the solution. Lingering questions about the dynamics of different areas of the clamps has led to another project for which we have developed a control to demystify some unexpected similarities between beta clamp mutants. With that project, we show that single-labeled and double-labeled samples have similar autocorrelation decays in florescence correlation spectroscopy, allowing us to rule out the dyes themselves as causing fluctuations in the 10-100 microsecond timescale. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-01-13T10:59:16Z No. of bitstreams: 1 AnnaraYveMouraSoares_DISSERT.pdf: 884763 bytes, checksum: df8a008c0603a2358c6331d336c3bd21 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-01-20T14:53:34Z (GMT) No. of bitstreams: 1 AnnaraYveMouraSoares_DISSERT.pdf: 884763 bytes, checksum: df8a008c0603a2358c6331d336c3bd21 (MD5) / Made available in DSpace on 2017-01-20T14:53:34Z (GMT). No. of bitstreams: 1 AnnaraYveMouraSoares_DISSERT.pdf: 884763 bytes, checksum: df8a008c0603a2358c6331d336c3bd21 (MD5) Previous issue date: 2016-04-05 / Esse estudo objetiva avaliar se a express?o da enzima de reparo de DNA Neil3 ? importante para o desenvolvimento funcional dos neur?nios. Estudos previos tem demonstrado que Neil3 interfere tanto na neurog?nese adulta como na fazer embrionaria. Eu utilizei whole cell patch clamp para estudar propriedades sinapticas e de membrana das c?lulas granulares do giro denteado. O giro denteado ? uma das regi?es com maior express?o de Neil3 no c?rebro e estudos previos tem demonstrado que a neurog?nese reativa em camundongos adultos ? afetada pela ausencia da enzima de reparo Neil3. Eu encontrei que a maioria das propriedades de membrana nas c?lulas granulares de camundongos knockout para Neil3 s?o normais com exce??o ? resposta de membrana ?s correntes de hiperpolariza??o e p?s-hiperpolariza??o. Diferentemente de neur?nios imaturos, as c?lulas granulares do giro denteado de camundongos com aus?ncia de Neil3, na qual as correntes de hiperpolariza??o ativadas s?o geralmente as ultimas a aparecerem durante o desenvolvimento. Al?m disso, correntes sinapticas excitatorias foram similar em amplitude mas apresentaram um decaimento ligeiramente mais rapido em c?lulas de camundongos knockout de Neil3. Esses resultados podem indicar um balan?o diferente entre os receptores AMPA e NMDA em camundongos knockout. Analises morfologicas de neur?nios preenchidos com biotina e reconstru??o post hoc n?o apresentaram grandes diferen?as na morfologia dendritica entre animais controle e knockout. Esse estudo mostra que, em rela??o diferen?as entre animais controle, neur?nios do giro denteado de animais knockout de Neil3 n?o podem ser classificados como imaturos. Eu encontrei diferen?as pontuais na corrente de hiperpolariza??o e pequenas diferen?as em propriedades sinapticas. Ainda devem ser avaliadas se essas diferen?as podem ser respons?veis por altera??es comportamentais encontradas em camundongos Neil3-knockout. Al?m disso, estudos futuros utilizando marcadores de neur?nios rec?m-nascidos s?o necess?rios para analisar o efeito da elimina??o da enzima Neil3 no desenvolvimento de neur?nios. / This study aims to assess whether the expression of the DNA repair enzyme Neil3 is important for the functional development of neurons. Previous studies have demonstrated that Neil3 interferes with both adult and embryonic neurogenesis. I have used whole cell patch clamp to study membrane and synaptic properties of granule cells of the dentate gyrus. The dentate gyrus is one of the regions with the highest expression of Neil3 in the brain, and previous studies have shown that reactive neurogenesis in adult mice is affected by Neil3 deletion. I found that most membrane properties of granule cells in Neil3-knockout mice are normal except from the membrane response to hyperpolarization currents and afterhyperpolarization currents. Different from immature neurons, granule cells of the dentate gyrus from Neil3-knockout mice, in which hyperpolarizing activated currents, are generally the last to appear during development. In addition, excitatory synaptic currents were similar in amplitude but showed a slightly faster decay in cells from Neil3-knockout mice. These results could indicate a different balance between AMPA and NMDA receptors in Neil3-knockout mice cells. Morphological analysis of neurons filled with biocytin and reconstructed post hoc showed no gross difference in dendritic morphology between dentate gyrus neurons of control and Neil3-knockout mice. This study shows that, while different from those of control littermates, dentate gyrus neurons of Neil3-knockout mice cannot be classified as ?immature?. I found specific differences in hyperpolarizing activated currents and small differences in synaptic properties. Whether these differences may account for behavioral changes found in Neil3-knockout mice is yet to be assessed. In addition, future studies using markers of newly born neurons are necessary for analyzing the effect of Neil3 deletion in developing neurons.

Ce travail de thèse s’intéresse à l’introduction de méthodologies électrochimiques dans la problématique de la caractérisation du transport de peptides pénétrants (CPPs) à travers des membranes phospholipidiques. Malgré leur charge électrique globalement positive, ces peptides sont en effet capables de traverser les bicouches lipidiques de cellules réelles ou artificielles (liposomes) et il n’existe pas à ce jour de mécanisme d’internalisation universellement admis. Dans ce contexte, nous avons dans un premier temps développé des méthodologies visant à détecter des peptides pénétrants marqués par des sondes rédox en optimisant les conditions de volume et de confinement à l’électrode. Parallèlement, nous avons tenté d’observer le passage transmembranaire de ces peptides en utilisant un dispositif dérivé du patch-clamp, dans lequel un morceau (patch)de membrane lipidique est excisé d’une vésicule géante et le suivi du passage assuré par une détection ampérométrique sur ultramicro-électrode à proximité de la membrane. La sensibilité de la technique ampérométrique et le flux de CPP s’avérant faibles, nous nous sommes tournés vers une stratégie associant fluorescence (pour la sensibilité) et commande électrochimique (pour l’extinction de la fluorescence). Dans la mesure où les phospholipides constituent la barrière dynamique à travers laquelle doivent passer les CPPs, nous avons d’abord étudié l’extinction électrochimique de phospholipides marqués par une sonde à la fois rédox et fluorescente, le NBD. Observée sur des vésicules géantes au microscope confocale, la réduction électrochimique a permis l’extinction sélective des phospholipides situés sur le feuillet externe de la vésicule, un résultat que ne permettent ni la réduction par des agents chimiques (dithionite), ni les techniques de « photobleaching ». Cette propriété a été confirmée cette fois-ci pour des CPPs marqués par le NBD qui ont été mis à incuber en présence de vésicules géantes et pour lesquels un essai préliminaire semble confirmer une extinction de fluorescence essentiellement pour les peptides associés au feuillet externe de la vésicule. / This PhD work was aimed at introducing electrochemical strategies in the general topic devoted to the characterization of the passage of cell penetrating peptides (CPPs) across phospholipidic membranes. Although positively charged, CPPs are prone to cross lipidic bilayers of real and artificial cells (liposomes) and there is no commonly admitted internalization mechanism so far. Therefore, we first developed electrochemical setups aimed at improving the amperometric detection of redox-taggedCPPs, through optimization of volume and confinement. Additionally, we have made attempts to use patch-clamp inspired setups to monitor the passage of CPPs across a membrane patched from a giant vesicle using ultramicro-electrodes in the close vicinity of the patched membrane. Since the amperometric technique displayed poor sensibility and the flux of CPP was too narrow, we changed our strategy for a methodology coupling fluorescence (for the sensitivity) and an electrochemical command (to achieve fluorescence extinction). Considering that phospholipids are forefront actors of CPP internalization, we first focused on the electrochemical quenching of phospholipids tagged with a probe displaying both redox and fluorescent properties (NBD). Observed on giant unilamellar vesicles (GUVs) with confocal microscopy, the electrochemical reduction of the NBD probe led to the selective extinction of the phospholipids located on the outer leaflet of the vesicle, a selectivity which is not observed using chemical quenchers such as dithionite or photobleaching methods. That property was extended to NBD-tagged CPPs, previously incubated with unlabeled Guvs and that preliminary experiment confirmed that the electrochemical extinction mostly concerned peptides associated to the outer leaflet of the liposome.

No description available.