Dissertationen zum Thema „Animal mechanics“

Decorin is required for the normal fibrillogenesis and spatial arrangement of collagen. As collagen is important in determining the elastic behaviour of the lung, we hypothesized that lung tissue mechanics would be altered in decorin deficient (Dcn-/-) mice. Complex impedance, pressure-volume curves, and length-stress curves of lung parenchyma were measured in C57BL/6 mice, 6 Dcn-/- and 6 wildtype ( Dcn+/+), both in vivo and in vitro. Immunohistochemistry and Western blotting were performed to identify decorin and biglycan in the lung tissues. In vivo, airway resistance was decreased and lung compliance was increased in Dcn-/- mice. In vitro, length-stress curves showed increased compliance in the Dcn-/- mice. Immunohistochemistry showed decorin staining in the airway and vessel walls of Dcn+/+ but not Dcn-/- mice; Western blots showed that biglycan levels were not different in the Dcn-/- mice. These data support a critical role for decorin in the formation of the lung collagen network. Lack of decorin alters lung tissue mechanical behaviour. Additionally, the data from Dcn+/+ mice were compared to those from other species, and is consistent with the evidence in the literature that mouse lungs differ structurally from other species. Finally, differences observed in vivo vs. in vitro suggest that measurements made in the strip more accurately reflect lung tissue properties.

Although rodents have been widely used in respiratory research, there are still only limited data comparing respiratory mechanics between different species of small animal. In order to provide further insight into the mechanical behavior of the respiratory systems of different sized small animals, accurate measurements of respiratory impedance (Zrs) were made in four different rodent species and in the developing rat over a broad range of frequencies at various levels of positive end-expiratory pressure (PEEP). PEEP dependencies of airway and tissue properties were interpreted in terms of physiological phenomena such as airway closure and airway-parenchymal interdependence forces. In adult animals, Zrs was fitted to a model including a Newtonian resistance (R) in series with a constant-phase tissue compartment. In general, rodent respiratory parameters obeyed the same scaling laws described in other species, but rabbits had a relatively higher elastance than one would predict from previously published allometric relationships. This is probably due to the rabbit's proportionately smaller airspace volume. R normalized to body weight was lower in smaller species suggesting that they have proportionately wider airways compared to larger animals. By using computer models of the asymmetric airway tree to estimate airway resistance (Raw), we confirmed that the larger of two isomorphic rodent species has relatively higher Raw. Moreover, we showed that both the airway dimensional scaling differences and the asymmetric arrangement of the individual airways are responsible for the relative differences in Raw between smaller and larger animals. Finally, in the developing rat, elastance and resistance normalized to lung weight decreased progressively with age, suggesting that intrinsic changes in the mechanical properties of the respiratory system occur with development. Parenchymal interdependence forces manifested themselves in animals as young as 10 days of age, with PEEP

This thesis contains four manuscripts examining the respiratory mechanics of normal and stuttering speech. The first study (J.Appl.Physiol. 75 (2):696-703, 1993) examined lung volume (V scL) during stutterers' relatively fluent speech. We showed that stutterers used the V scL extremes. This suggested that stutterers recruited their respiratory muscles in a different manner than normals to maintain subglottic pressure (Psg). We therefore investigated respiratory muscle recruitment patterns in normals and stutterers. In the second study, (submitted to J.A.P.), we modified the Campbell pressure volume diagram by the addition of abdominal pressure (Pab) and by the use of a surrogate relaxation curve. The addition of Pab allowed us to differentiate between diaphragmatic and non diaphragmatic inspiratory muscles and to quantify expiratory muscle recruitment. The surrogate curve provided a good approximation of the true relaxation curve (non significant difference between surrogate and true relaxation curves: P $>$.10). The third study (submitted to J.A.P.) used the modified diagram to examine respiratory muscle recruitment patterns, instantaneous Psg, voluntary hyperinflation and intrinsic positive end-expiratory pressures (PEEPi) during normal and stuttering speech. Stutterers used consistent muscle recruitment patterns and consistent V scL but Psg was not as well controlled as in normals. Because a lack of control of Psg could have an immediate effect on the glottis, particularly at high or low V scL, we measured instantaneous Psg and flow in the final study (to be submitted to J.A.P.) to give us instantaneous glottal resistance. In this study we were able to plot a 'family' of glottal resistances that covered normal speech. This provided a normal envelope of resistances. When stutterers were fluent they remained within both the flow and Psg limits set by the normals' envelope. When they were fluent both Psg and flow extended beyond the normals' envelope. We conclude

This study investigates dyspnea and the mechanics of breathing during progressive exercise. Three subject groups, athletes, normal sedentary subjects and chronic obstructive diseased patients were studied during progressive exercise testing to exhaustion on a cycle ergometer. Subjects rated dyspnea on a Borg Scale. Inspiratory flow, esophageal/gastric pressures and rib cage/abdominal displacements were measured.
Subjects demonstrated two patterns of dyspnea response to changes in esophageal (pleural) pressure. All athletes, two normals and five patients were termed "low dyspnea responders", (LDR), whereas the remaining subjects were termed "high dyspnea responders", (HDR).
LDR demonstrated large, rapid negative gastric pressure swings, coupled with outward abdominal displacement during early inspiration when compared to HDR, suggesting that LDR utilized abdominal muscle relaxation at the onset of inspiration. This mechanism appears to provide an extra inspiratory force, contributing to the increasing pleural pressures required. This breathing pattern appears to diminish the sensation of dyspnea at a given pleural pressure.

This thesis examines changes in the mechanical behaviour of the canine and human respiratory systems to changes in lung volume below normal functional residual capacity (FRC). In open chested dogs lung elastance (E$ sb{ rm L}$) increased and lung resistance (R$ sb{ rm L}$) changed little with decreases in positive end-expiratory pressure (PEEP) of the ventilatory circuit. The dominance of plastoelastic lung tissue properties at low lung volumes was used to interpret the lack of change in R$ sb{ rm L}$. Computed tomography demonstrated that pleural effusion (PE) created atelectasis in dependent caudal lung regions which contributed to the overall lung volume loss. PE produced a decrease in only lung vertical height while chest wall dimensions changed both vertically and horizontally. E$ sb{ rm L}$ and R$ sb{ rm L}$ increased while elastance and resistance of the chest wall were little affected by these shape and density changes. In close-chested, anesthetised, paralysed, ventilated humans a decrease in PEEP below normal FRC caused an increase in R$ sb{ rm L}$, E$ sb{ rm L}$ and both chest wall elastance and resistance. Median sternotomy caused E$ sb{ rm L}$ to increase with increasing PEEP while the negative volume dependence of R$ sb{ rm L}$ remained. Most of the difference between open-chested and closed-chested E$ sb{ rm L}$ was presumably due to lung collapse in the open-chested state.

The kinematics of wiping movements to the back were examined in spinal adult Rana Catesbeiana. The aim was to identify the elements of the back wipe and their functional role. The data show that there are three essential phases of the wiping movement: a placing phase; a flexion of the hip and knee; and a whisk/extension phase. The first phase is the only one which is dependent upon stimulus location. The spinal frog adjusts the hindlimb to account for stimulus location in the rostro-caudal direction. There is no adjustment for stimulus position along the medial-lateral axis of the body. It is proposed that the second phase serves as a preparatory movement for the extension portion of the wipe. When the wipe is partitioned into these phases, the motion was found to be planar for the first and third phases. At the end of the first phase there was a transition between the two planes.

Irregular terrain is difficult for small robots to traverse, so the research in this thesis endeavours to develop a jumping robot as a solution to this problem. Gliding is proposed as a means of reducing the landing impact forces, and potentially extending the range of each jump. The biomechanics of jumping and gliding are introduced from fundamental principles, before various examples from nature are described and contrasted. Flying squirrels glide quickly between trees by stretching out their patagia, membranous skin folds spreading between the wrists and ankles. Several hypotheses exist to explain the evolution of gliding flight in these animals. These are investigated by filming northern flying squirrels leaping across a range of short distances while measuring the corresponding take-off and landing forces. Evidence is provided that the evolution of controlled flight was most likely stimulated by the need to reduce landing impact forces. A model is proposed for the skin structure of flying squirrel patagia, which is likely to be specialised from normal mammalian skin to facilitate gliding flight and the high speed transition to other arboreal locomotion. This predicts that the skin would be thin and its stiffness highest along the length of the animal, with the behaviour more elastic perpendicularly. Uni-axial tensile test results from a single southern flying squirrel are consistent with the hypotheses. A biomimetic jumping and gliding robot, Glumper, is described which relies on a power-amplifying energy storage and catchrelease mechanism. A novel, bi-stable dog clutch device was developed that enables the robot to launch itself automatically using an on-board power supply. The robot has selfdeploying gliding membranes and allowance is made in the design for the adjustment of pitch to improve landing control. After testing the performance of the robot, consideration is given to its power requirement for full autonomy.

The ability to control balance and stability are essential to prevent falls during locomotion. Maintenance of stable locomotion is challenging especially when complicated by amputation and prosthesis use. Humans employ several motor strategies to maintain stability during walking on complex terrain: decreasing walking speed, adjusting stride length and stance width, lowering the center of mass, and prolonging the double support time. The mechanisms of selecting these motor strategies by the primary motor cortex are unknown and cannot be studied directly in humans. There is also little information about dynamic stability of prosthetic gait with bone-anchored prostheses, which are thought to provide sensory feedback to the amputee through osseoperception. Therefore, the Specific Aims of my research were to (1) evaluate dynamic stability and the activity of the primary motor cortex during walking with different constraints on the base of support and (2) develop an animal model to evaluate mechanics and stability of prosthetic gait with a bone-anchored prosthesis. To address these aims, I developed a feline model that allows for investigating (1) the role of the primary motor cortex in regulation of dynamic stability of intact locomotion, (2) skin and bone integration with a percutaneous porous titanium implant facilitating prosthetic attachment, and (3) dynamic stability of walking on a bone-anchored prosthesis. The results of Specific Aim 1 demonstrated that the area and shape of the base of support influence the margins of dynamic stability during quadrupedal walking. For example, I found that the animal is dynamically unstable in the sagittal plane and frontal plane (although to a lesser degree) during a double-support by a forelimb and the contralateral hindlimb. Elevated neuronal activity from the right forelimb representation in the primary motor cortex during these phases suggests that the motor cortex may contribute to selection of paw placement location and thus to regulation of stability. The results of Specific Aim 2 on the development of skin-integrated bone-anchored prostheses demonstrated the following. Skin ingrowth into 3 types of porous titanium pylons (pore sizes 40-100 μm and 100-160 μm and nano-tubular surface treatment) implanted under skin of rats was seen 3 and 6 weeks after implantation, and skin filled at least 30% of available implant space. The duration of implantation, but not implant pore size (in the studied range) or surface treatment statistically influenced skin ingrowth; pore size and time of implantation affected the implant extrusion length (p<0.05). The implant type with the slowest extrusion rate (pore size 40-100 μm) was used in a feline model of prosthetic gait with skin-integrated bone-anchored prosthesis. The developed implantation methods, rehabilitation procedures and feline prostheses allowed 2 animals to utilize skin- and bone-integrated prostheses for dynamically stable locomotion. Prosthetic gait analysis demonstrated that the animals loaded the prosthetic limb, but increased reliance on intact limbs for weight support and propulsion. The obtained results and developed animal model improve the understanding of locomotor stability control and integration of skin with percutaneous implants.

The potential of the crural fascia to increase the articulation of the posterior thigh muscles through the in series connection of the structures, suggests that the crural fascia may influence the endpoint force direction of the muscles by partially redirecting the muscular force output. Furthermore, not only the in series connections should be considered but also how the parallel alignment of the crural fascia and the triceps surae may influence the force direction from the muscles. A redirection in force may, in turn, affect the intra-limb coordination or contribute to the selection of a task variable muscle activation pattern. The central objective was to evaluate the role of the synergistically located, posterior, distal musculature and connective tissue during locomotion. The central hypothesis was that the crural fascia would redirect the force output from the posterior thigh muscles to the endpoint and consequently increase propulsion within the limb. We selected to perform our studies in the spontaneously locomoting decerebrate cat, which allows us to investigate acute treatments applied to the hindlimb. The overall objective was accomplished by: (1) evaluating the role of the crural fascia during level walking; (2) determine the acute effect of denervating the triceps surae muscles and disrupting the crural fascia during level walking; and (3) evaluating the change in force direction output of selective stimulation of muscles in different limb configurations before and after complete fasciotomy. Our findings demonstrated that the crural fascia not only assists in propulsion but also acts to stabilize the distal limb. Furthermore, the acute denervation of the triceps surae resulted in a decrease in leg length and an increase in ankle yield during the weight acceptance phase of stance. This suggests that the conservation of the limb length as a task level variable is an adaptation rather than an immediate response.

This dissertation proposed a novel experimental model combining a defect configuration with an active instrumented fixation device to investigate the influence of mechanics on bone healing. The proposed defect configuration aimed to minimise physiological loading within an experimental fracture gap and the instrumented fixator was used for the application of controlled displacements and in vivo stiffness monitoring of the healing process. This thesis has provided a novel approach to advance current knowledge and understanding of mechanobiology, which has been limited in previous experimental models.

The intervertebral disc (IVD) is a complex orthopaedic tissue that is located between the vertebrae in the spine. Degeneration of the IVD is thought to be a contributor to low back pain (LBP), which affects up to 80% of the population at enormous economic cost. The role of the intervertebral disc in supporting and resisting applied loading to the spine, along with the observation of disorders associated with abnormal spinal loading, provide support to the theory that applied mechanical loading is crucial in maintaining the health of the intervertebral disc. The encompassing goal of this work was to examine the biological response of the intervertebral disc to changes in the surrounding mechanical environment in a large animal model. Aim 1 utilized an organ culture model to explore the relationship between disc mechanics and biology in needle puncture injury, a commonly used model of experimentally induced disc degeneration, thus providing a possible mechanism for in vivo injury induced disc degeneration models. Aim 2 was to explore the interaction between the amplitude of applied mechanical loading and intervertebral disc cell signaling, also performed in an organ culture model to include cell-matrix signal transduction. Aim 3 addressed frequency and age effects on the IVD response to mechanical stimulation, performed in vitro to control for the effects of varying matrix compositions between old and young animals. Finally, Aim 4 utilized kmeans and fuzzy c-means clustering techniques to reveal patterns in experimental phenotype (determined by gene expression data) and gene response to experimental conditions. The application of biclustering, where the gene responses within experimental phenotypes are clustered to elucidate possible mechanisms for different gene level-responses to experimental conditions, was also accomplished. Finally, the ability for the model to predict the behavior of other genes critical to IVD mechanobiology, or in determining the membership of an unexamined experimental phenotype was explored. Overall, applied dynamic compression was not found to significantly alter disc mechanics, while a disruption in the annulus through needle puncture rapidly decreased the compressive modulus. Changes in disc mechanics may precede biological remodeling, with little evidence of remodeling present without mechanical alteration. Aging, however, crucially impacts disc cell biology, particularly in the nucleus pulposus, and will interact with applied loading to further impact the ability for the intervertebral disc cells to maintain a healthy extracellular matrix.

Cette thèse est consacrée au traitement d'images de bioluminescence chez le petit animal. Ce type d'imagerie, bien qu'utilisé en routine pour la recherche en cancérologie par exemple, présente néanmoins des problèmes liés aux phénomènes de diffusion et d'absorption par les tissus internes à l'animal. Il s'ajoute à cela le bruit du système d'acquisition ainsi que le bruit lié aux rayonnements cosmiques. Ceci influe sur la qualité des images acquises et rend leur exploitation délicate. Le but de cette thèse est de compenser ces effets perturbateurs. Les travaux menés ont abouti à la proposition d'un modèle de formation des images de bioluminescence ainsi qu'à une chaîne de traitement adaptée composée d'une étape de filtrage suivie d'une étape de déconvolution. Après étude de la nature des différents bruits liés à l'acquisition, nous avons mis au point un nouveau filtre médian pour la suppression du bruit impulsionnel aléatoire présent sur les images acquises ; ce filtre représente le premier bloc de la chaîne proposée. Pour l'étape de déconvolution, nous avons mené une étude comparative de différents algorithmes de déconvolution. Cela a conduit à choisir un algorithme de déconvolution aveugle initialisé avec la réponse impulsionnelle estimée du système d'acquisition. Nous avons validé notre approche globale en comparant les résultats à la réalité terrain. Au travers de différents essais cliniques, nous avons montré que le traitement que nous proposons permet une amélioration significative de la mesure des sources bioluminescentes et une meilleure distinction de sources très proches, ce qui représente un apport non négligeable pour les utilisateurs d'images de bioluminescence.

Whether there are sex differences in the kinematic organization of non-reproductive behaviors is rarely addressed. In this thesis, evidence is presented that male and female rats organize their posture and stepping differently during a food protection task, contact righting, skilled reaching, and vertical rearing. Neonatal gonadal steroid exposure can alter sex-typical patterns of movement organization. Whether these differences are due to sex differences in body morphology or central nervous system (CNS) was also addressed using gravid females and tfm males. The results reveal that sex differences in movement are CNS based. Furthermore, the expression and choice of sex-typical patterns of movement can be altered by CNS injury. Finally, evidence is presented that sex differences in movement organization are also present in marsupials and insects. The implications of these results for our understanding of the evolution of sex differences in CNS anatomy and behavior will be discussed.
xvi, 249 leaves : ill. ; 28 cm.

Objetivos: Verificar alterações na mecânica e parâmetros de morfometria pulmonar em um estudo temporal sobre enfisema em um modelo animal. Métodos:Setenta camundongos Balb / c receberam instilação nasal de solução de papaína ou salina e foram estudados no 1º, 3º, 15º, 28º e 40º dias após a instilação. Resistência das vias aéreas (Raw), Resistência do tecido (Gtis) e elastância tecidual (Htis), foram analisados. Intercepto Linear Médio (Lm), a proporção do volume de fibras elásticas e colágenas, macrófagos (MAC), o número de células que expressam MMP12 e a expressão de -isoprostano 8 no parênquima pulmonar, foram medidos. Resultados: Comparando os grupos papaína e solução salina ao longo do tempo, foi observado um aumento de Lm após o dia 28, associado a uma diminuição no Htis e Gtis. Houve um aumento na proporção do volume das fibras colágenas a partir do dia 15 até o dia 40, enquanto a proporção do volume de fibras elásticas foi aumentada somente no dia 40. Analisando o número de macrófagos, houve um aumento no dia 1 e manteve-se crescente até o dia 40. A expressão de MMP12 aumentou a partir do dia 3 até o dia 40. No entanto, a expressão de isoprostano 8 foi maior apenas no dias 1 e 3. Conclusão: Um aumento significativo no intercepto linear médio (Lm) após o dia 28 de instilação de papaína foi associado a uma piora na função pulmonar caracterizando assim o enfisema pulmonar. No entanto, no dia 40, as diferenças na morfometria foram mantidos, mas não houve diferenças na mecânica respiratória. O remodelamento da matriz extracelular observada no parênquima pulmonar no dia 40 poderia explicar estes resultados
Objectives: To verify how mechanical and morphometry parameters change in a temporal study of emphysema in an animal model. Methods: Balb/c mice received either a nasal drop of papain solution (Papa) or saline (Sal) and were studied on the 1st, 3rd, 15th, 28th and 40th days after instillation. We evaluated airway resistance (Raw), tissue damping (Gtis) and tissue elastance (Htis). Using morphometry, we measured mean linear intercept (Lm), volume proportion of elastic and collagen fibers, number of macrophages (MAC), number of cells expressing MMP12 and the expression of 8-isoprostane in parenchyma. Results: Comparing Papain and Saline groups in each time window, we observed an increase in Lm after the 28th day associated to a decrease in Htis and Gtis. The volume proportion of collagen fibers increased from the 15th to the 40th day, while the volume proportion of elastic fibers increased only on the 40th day. Analyzing the macrophages number, there was an increase on the 1st day, and it continued increasing until the 40th day. The expression of MMP12 increased from the 3rd until 40th day. However, the expression of 8-isoprostane increased only on the 1st and the 3rd day. Conclusions: A significant increase in mean linear intercept (Lm) after the 28th day of papain instillation was associated to a worsening in lung function. However, on the 40th day, differences in morphometry maintained but there were no differences in respiratory assessment. The extracellular matrix remodeling observed in lung parenchyma on the 40th day could explain these results

Modelos matemáticos são utilizados como ferramentas na avaliação da mecânica respiratória para a compreensão da fisiologia e patologias do sistema respiratório. A presente pesquisa visou avaliar, através da aplicação de modelos matemáticos, a mecânica respiratória em camundongos submetidos à metacolina. Deu-se ênfase no modelo linear de compartimento único e suas variantes não lineares. Camundongos C57BL/6 (n = 8) foram traqueostomizados, ventilados mecanicamente (flexiVent, SCIREQ, Canadá) e perturbações em volume foram aplicadas para a modelagem do sistema respiratório. O protocolo experimental foi elaborado de forma a se analisar a variação dos parâmetros respiratórios durante a aplicação do agente broncoativo e também se verificou a divisão do sinal quasi-senoidal em expirações e inspirações durante a técnica de oscilação forçada (FOT) com perturbação de frequência 2,5 Hz. Com base nisso, uma rotina computacional própria foi desenvolvida para a análise dos experimentos realizados no ventilador mecânico e foram pesquisadas as vantagens e desvantagens dos modelos matemáticos aplicados. Os resultados demonstraram um aumento no desvio padrão dos parâmetros do modelo linear unicompartimental e suas variantes não lineares após a aplicação do broncoconstritor. Acredita-se que esta grande variação nos parâmetros esteja relacionada com o enrijecimento do parênquima e da heterogeneidade da ventilação pulmonar após a utilização da droga. Devido à correlação dos parâmetros do modelo com a fisiologia ocorrer somente no modelo linear, acredita-se que este ainda é o mais indicado na avaliação da mecânica respiratória e as variantes não lineares seriam indicadas como opção em casos onde o modelo linear é incapaz de realizar ajustes adequados ou para informações complementares.
Mathematical models are used as tools in the assessment of respiratory mechanics for the understanding of the physiology and pathologies of the respiratory system. This study aimed to assess the respiratory mechanics by applying mathematical models in mice subjected to challenges with methacholine. Emphasis was placed on linear single-compartment model and its nonlinear variants. C57BL/6 mice (n = 8) were tracheostomized, mechanically ventilated (flexiVent, SCIREQ, Canada) and disturbances in volume were applied to the modeling of the respiratory system. The experimental protocol was developed in order to analyze the variation of respiratory parameters during the application of the bronchoactive agent. The division of quasisinusoidal signal in expirations and inspirations during the forced oscillation technique (FOT) with frequency perturbation of 2.5 Hz was also observed. Based on that, a proper computational routine was developed in order to analyze the experiments in the mechanical ventilator and the advantages and disadvantages of the applied mathematical models. The results demonstrated an increase in the standard deviation of the linear single-compartment model and its nonlinear variants parameters after the application of bronchoconstrictor. It is believed that this large variation in the parameters relates to the parenchyma stiffening and to the heterogeneity of pulmonary ventilation after the use of the drug. Due to the fact that the correlation between the model parameters and the physiology occurred only in the linear model, it is believed that this is still the most suitable model in the assessment of respiratory mechanics. Nonlinear variations of the single-compartment model would be indicated only as an option, for example, in cases where the linear model is incapable of performing appropriate fits or when additional information about the respiratory system is required.

With the vast array of genetically altered (knockout) mice becoming available there is a need for quantitative, repeatable, and efficient methodologies to characterize the phenotypic consequences of knocking out specific genes. Since knockout animals often have the ability to compensate for a single missing gene, it is important to examine the structural, material and morphological properties to obtain a thorough understanding of the changes occurring. For this project, femurs of knockout mice were first scanned using microcomputed tomography (micro-CT) to obtain high-resolution images of the trabecular bone in the distal femur, as well as cortical bone in the mid-diaphysis. After scanning, the femurs were tested to destruction in four-point bending at the mid-diaphysis about the medial lateral axis of the femur. These methodologies allowed quantification of (1) morphologic properties such as bone volume fraction, trabecular properties and 2nd moment of the area (2) structural properties such as stiffness, maximum load at failure, and post yield deformation and (3) material properties such as bone mineral density, elastic modulus and yield strength. As part of two independent studies, two different knockout mice, cyclooxygenase-2 (COX-2 -/-) and Apolipoprotein E (APOE -/-), were examined for structure-function relationships using these methodologies. COX-2 knockout mice were found to have decreased mineral content in their femurs, and increased post yield deformation. APOE knockout mice at 10 weeks of age had decreased bone mass and structural properties. However, by 40 weeks of age APOE deficient mice caught up to and exceeded the structural properties and bone mass of their wild type counterparts.

A model was developed to account for the static elastic behaviour of the lung tissue strip in terms of distributions of collagen and elastin fibers. Distributions of collagen fiber lengths and elastin fiber stiffnesses were determined by fitting the model to data from dog lung tissue strips. These distributions followed 1/f power-laws for more than 95% of the data. Computer simulations of two dimensional tissue strip models with 1/f distributions of collagen fiber lengths also predicted realistic stress-strain curves. The simulations illustrated the gradual development of geometric and stress heterogeneity throughout the tissue as the collagen fibers were recruited during stretch. This model suggests a mechanistic basis for the shape of the pressure-volume curve of whole lung. It also indicates how this curve may be affected by changes in tissue collagen and elastin similar to the changes occurring in the diseases of pulmonary emphysema and fibrosis. Nonparametric block-structured nonlinear models for describing both the static and dynamic stress-strain behaviour of the lung were applied to dog lung tissue strips and to whole rat lungs in vivo. Both the Wiener and Hammerstein models accounted for more than 99% of the tissue strip data, although the Hammerstein model was more consistently accurate across a range of perturbation amplitudes and operating stresses. Plastic dissipation of energy within the lung tissue strip was estimated at less than 20% of the total dissipation during slow sinusoidal cycling. The Hammerstein model was also the best of those investigated for describing the rat lung data in vivo, although there were dependencies of the model parameters on perturbation amplitude and operating point that indicate that a more complicated model is required for the whole lung. Finally, construction of a fiber recruitment model for the dynamic mechanical behaviour of lung tissue strips was attempted. However accurate reproduction of measured behaviour was no

Les oiseaux et poissons se déplacent dans leur environnement fluide en interagissant avec l'air/eau qui les entoure. Pour des régimes inertiels, les mécanismes de propulsion se basent sur un transfert de quantité de mouvement au fluide; les battements d'ailes ou de nageoires générant un jet dans le sillage de l'animal qui le propulse vers l'avant. Pour les oiseaux comme pour les poissons, les structures utilisées possèdent une certaine flexibilité, et sont donc susceptibles de plier de façon importante. La littérature montre que ces déformations passives peuvent améliorer les performances de propulsion lorsqu'elles sont exploitées de façon constructive. Le détail des mécanismes en jeu reste cependant mal compris. L'objectif de cette thèse est d'étudier, à travers deux modèles biomimétiques, la façon dont une structure battante déformable génère des forces de propulsion. Le premier modèle est une version mécanique simplifiée d'insecte dotée d'ailes flexibles, tandis que le deuxième est un nageur dont le corps élastique reproduit le mouvement d'ondulation d'une anguille. Nous montrons que la façon dont ces systèmes se déforment passivement est déterminante pour leurs performances, et que leur réponse élastique peut être décrite par des modèles théoriques simplifiés d'oscillateurs forcés. Ces modélisations mettent par ailleurs en avant le rôle crucial joué par le frottement fluide quadratique qui s'oppose aux mouvements de battements de la structure. Ce résultat introduit l'idée, un peu contre-intuitive, qu'il peut s'avérer avantageux de dissiper une part de son énergie dans le fluide pour améliorer ses performances.

Tese de Doutoramento em Ciências Veterinárias. Especialidade de Sanidade Animal
ABSTRACT - Antibiotics were a truly innovative option in medical therapy for the treatment of diseases caused by microbial agents, having largely contributed for the decrease levels of human and animal morbidity and mortality. Therefore, the overuse and misuse of these drugs in human clinical therapy and in the veterinary medicine, including animal production, contributed for the emergence and dissemination of antibiotic resistant microorganisms, which are a serious threat to human and animal health, and to the ecosystem. The aim of the present thesis was to search the main acquired antibiotic resistance mechanisms to β-lactams, fluoroquinolones and polymixins in Gram negative bacteria recovered from different animal species and matrices, and to investigate the most important mobile genetic elements involved in the dissemination. Thus, the studies concerning antibiotic susceptibility and molecular characterization were performed in collections of bacterial isolates belonging to Enterobacteriaceae family (mainly Escherichia coli and Salmonella enterica). Both bacterial species were associated to antibiotic resistant determinants of clinical relevance in human and veterinary medicine, namely, blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaCTXM- 32, blaCMY-2, qnrS1, aac(6’)-Ib-cr, mcr-1. The diversity of detected mobile genetic elements, e.g., IncI1, IncF and IncX4 plasmids, insertion sequences ISEcp1, as well as integrons of class 1 and 2, suggest their involvement in the dissemination of resistance genes interspecies, and movement within the bacterial cell. Genomic analysis of two isolates (Morganella morganii and Salmonella Enteritidis), highlighted the potencial of omic technologies, as an additional tool to the phenotypic and genotypic characterization of antibiotic resistance. The results obtained throughout this thesis highlight the importance of the different animal species as reservoirs of antibiotic resistant bacteria. In addition, it was reinforced the need of a permanent research and monitoring of antibiotic resistance in the different ecological niches, and the use of genomic approaches, which had an important role in the understanding of the complex problem represented by the dynamic of antibiotic resistance.
RESUMO - Os antibióticos constituíram uma opção verdadeiramente inovadora na terapêutica medicamentosa para o tratamento de doenças provocadas por agentes microbianos, tendo contribuído largamente para a diminuição das taxas de morbilidade e mortalidade humana e animal. Porém, a utilização abusiva e inadequada destes fármacos na prática clínica humana e na medicina veterinária, incluindo a produção animal, contribuiu para a emergência e disseminação de microrganismos resistentes, os quais constituem uma grave ameaça à saúde humana e animal, e para o ecossistema. A presente dissertação teve como objetivo central investigar os principais mecanismos de resistência adquirida aos antibióticos β-lactâmicos, fluoroquinolonas e polimixinas em bactérias de Gram negativo isoladas de diferentes espécies animais e matrizes, bem como os principais elementos genéticos móveis responsáveis pela sua disseminação. Assim, os estudos de suscetibilidade aos antibióticos e caracterização molecular foram realizados em coleções de estirpes bacterianas pertencentes à família Enterobacteriaceae (maioritariamente Escherichia coli e Salmonella enterica). Ambas as espécies bacterianas estavam associadas a determinantes de resistência de relevância clínica humana e veterinária, nomeadamente blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaCTX-M-32, blaCMY-2, qnrS1, aac(6’)-Ib-cr, mcr-1. A diversidade de elementos genéticos móveis detetados, e.g. plasmídeos IncI1, IncF e IncX4, sequências de inserção ISEcp1, bem como integrões de classes 1 e 2, sugere o seu envolvimento na disseminação de genes de resistência aos antibióticos entre espécies, tal como a sua movimentação dentro da própria bactéria. A análise do genoma de duas estirpes (Morganella morganii e Salmonella Enteritidis) realçou o potencial das tecnologias ómicas, como ferramenta adicional na caracterização fenotípica e genotípica da resistência aos antibióticos. Os resultados obtidos salientam a importância que as várias espécies animais representam como reservatórios de bactérias resistentes aos antibióticos. Foi igualmente reforçada a necessidade de uma permanente investigação e monitorização da resistência aos antibióticos nos vários nichos ecológicos, e do uso de abordagens genómicas, as quais tiveram um papel importante na compreensão do complexo problema que representa a dinâmica da resistência aos antibióticos.
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Au cours du développement préimplantatoire, l'embryon de mammifère forme le blastocyste, qui est la structure fixant l'embryon dans l'utérus. La formation du blastocyste repose en grande partie sur les forces contractiles générées par le cortex d'actomyosine. Chez la souris, sur des échelles de temps de quelques secondes, nous observons des impulsions de contractions d'actomyosine voyageant périodiquement autour du périmètre cellulaire. Nous appelons ce phénomène vagues de contraction corticale périodique (PeCoWaCo), une manifestation fascinante et mal comprise de la contractilité. Dans cette étude, nous profitons du développement lent de l'embryon de souris pour étudier des milliers d'impulsions de contraction. Nous mettons également à profit la robustesse de ce développement pour explorer les propriétés biophysiques des PeCoWaCo pendant les étapes de clivage précédant la morphogenèse précoce des mammifères.Nous constatons que, lors des étapes de clivage, des mouvements périodiques apparaissent occasionnellement au stade zygote et 2-cellules puis deviennent systématiques après le 2ème cycle de divisions de clivage. Fait intéressant, la période des oscillations diminue progressivement de 200s au stade zygote à 80s au stade 8-cellules. Étant donné que les cellules deviennent de plus en plus petites avec des divisions de clivage successives, la taille des cellules pourrait être un déterminant important dans l'initiation et la régulation des PeCoWaCo. Nous manipulons la taille des cellules sur une large gamme de rayons cellulaires (10-40 µm) en utilisant la fragmentation et la fusion des cellules et constatons que l'initiation, la persistance ou les propriétés des PeCoWaCo ne dépendent pas de la taille des cellules. Après la période des PeCoWaCo, on découvre que les tensions de surface des blastomères diminuent progressivement jusqu'au stade de 8-cellules et que l’assouplissement artificiel des cellules augmente prématurément les PeCoWaCo. Par conséquent, lors des étapes de clivage, l’assouplissement cortical réveille la contractilité zygotique avant la morphogenèse préimplantatoire. En plus, en manipulant la contractilité de l'actomyosine à l'aide de mutants et des drogues, nous avons montré que la période des PeCoWaCo peut être contrôlée par la régulation du taux de polymérisation de l'actine filamenteuse et de l'activité motrice de la myosine.Dans l'ensemble, nos résultats sur les aspects biophysiques et moléculaires du PeCoWaCo nous aident à comprendre comment la contractilité de l'actomyosine s'éveille avant la morphogenèse préimplantatoire et comment elle est régulée au niveau mécanique et moléculaire
During pre-implantation development, the mammalian embryo forms the blastocyst, which is the structure embedding the embryo into the uterus. The shaping of the blastocyst relies in large part on contractile forces generated by the actomyosin cortex. In the mouse, on timescales of seconds, we observe pulses of actomyosin contractions traveling periodically around the cell perimeter. We call this phenomenon periodic cortical waves of contraction (PeCoWaCo), a fascinating and poorly understood manifestation of contractility. In this study, we take advantage of the slow development of the mouse embryo to study thousands of contraction pulses and of the robustness of the mouse embryo to size manipulation to explore the biophysical properties of PeCoWaCo during the cleavage stages preceding early mammalian morphogenesis. We find that, during cleavage stages, periodic movements appear occasionally at the zygote and the 2-cell stage and become systematic after the 2nd round of cleavage divisions. Interestingly, the period of oscillations progressively decreases from 200 s at the zygote stage to 80 s at the 8-cell stage. Since cells becomes successively smaller with successive cleavage divisions, cell size could be an important determinant in the initiation and regulation of PeCoWaCo. We manipulate cell size on a broad range of cell radii (10-40 µm) using fragmentation and fusion of cells and find that the initiation, persistence or properties of PeCoWaCo do not depend on cell size. Following the period of PeCoWaCo, we discover that blastomeres gradually decrease their surface tensions until the 8-cell stage and that artificially softening cells enhances PeCoWaCo prematurely. Therefore, during cleavage stages, cortical softening awakens zygotic contractility before preimplantation morphogenesis. In addition, by manipulating actomyosin contractility using mutants and drugs, we showed that the period of PeCoWaCo can be tuned by F-actin polymerization rate and myosin motor activity. Altogether our results on biophysical and molecular aspects of PeCoWaCo help us understand how actomyosin contractility awakens before preimplantation morphogenesis and how it is regulated both mechanically and molecularly
İmplantasyon öncesi gelişim sırasında, memeli embriyosu, embriyoyu rahim içineyerleştiren yapı olan blastosisti oluşturur. Blastosistin şekillendirilmesi büyük ölçüdeaktomiyozin korteks tarafından oluşturulan kasılma kuvvetlerine dayanır. Farede, saniyelikzaman ölçeklerinde, hücre çevresinde periyodik olarak dolaşan aktomiyozin kasılmalarınındarbeleri gözlemlenebilir. Bu fenomene, kasılmanın büyüleyici ve yeterince anlaşılmamış birtezahürü olan periyodik kortikal kasılma dalgaları (periodic cortical waves of contraction:PeCoWaCo) diyoruz. Bu çalışmada, erken memeli morfogenezinden önceki bölünmeaşamaları sırasında PeCoWaCo'nun biyofiziksel özelliklerini keşfetmek ve binlerce kasılmadarbesini inceleyebilmek için fare embriyosunun yavaş gelişiminden ve fare embriyosununboyut manipülasyonuna dayanıklılığından faydalandık.Bölünme aşamaları sırasında, zigotta ve 2 hücreli aşamada periyodik hareketlerinzaman zaman ortaya çıktığını ve ikinci tur bölünmeden sonra bu hareketlerin sistematik halegeldiğini bulduk. İlginç bir şekilde, salınım periyodunun zigot aşamasında 200 saniyeden, 8hücreli aşamada 80 saniyeye sistematik olarak azaldığını gözlemledik. Hücreler ardışıkbölünmeleriyle sürekli küçüldüğünden, hücre boyutu PeCoWaCo'nun başlatılmasında vedüzenlenmesinde önemli bir belirleyici olabilir. Hücreleri geniş bir hücre yarıçapı aralığında(10-40 μm) küçük parçalara bölerek veya birbirleriyle birleştirerek PeCoWaCo'nunbaşlatılmasının, kalıcılığının veya genel özelliklerinin hücre boyutuna bağlı olmadığını bulduk.PeCoWaCo periyodunu takiben, embriyo hücrelerinin zigottan 8 hücreli aşamaya kadar yüzeygerilimini kademeli olarak azalttığını ve yapay olarak korteksleri yumuşatılan hücrelerinPeCoWaCo'yu zamanından önce geliştirdiğini keşfettik. Bu sonuçlarla bölünme aşamalarısırasında, kortikal yumuşama, ilke implantasyon öncesi morfogenezinden önce zigotikkasılmaları uyandırdığını gösterdik. Ayrıca, genetik mutantlar ve kimyasallar kullanarakaktomiyozin kasılmasını manipüle ederek, PeCoWaCo periyodunun F-aktin polimerizasyonhızı ve miyozin motor aktivitesinin düzenlenmesi ile ayarlanabileceğini gösterdik.Sonuç olarak, PeCoWaCo'nun biyofiziksel ve moleküler yönleriyle ilgili bulgularımız,aktomiyosin kontraktilitesinin implantasyon öncesi morfogenezinden önce nasıl uyandığını,ayrıca hem mekanik hem de moleküler olarak nasıl düzenlendiğini anlamamıza yardımcı olur

The papers presented here represented an effort to investigate body-size relationships explicitly, using a variety of different approaches, scales of observation and empirical methods, and addressed issues pertinent to several aspects of food web theory, macroecology, and experimental community ecology. At the scale of the food web, (Paper I) the inclusion of parasites in the Ythan food web challenged previous empirical studies of predator-prey body-size relationships and the assumptions underlying food web models. Specifically body-size was neither the determinant of the trophic hierarchy of feeding links in the web, a key assumption in the cascade model and its variants, nor a constraint on the distribution of feeding links between individuals within the food web. Continuing the food web theme, but using a macroecological approach (Paper II), abundance body-size relationships reported in the literature were shown to be biased. Although the shape of the abundance body-size constraint space in the Ythan was broadly similar to the polyhedral shape reported for entire ecosystems, when the body-sizes of small taxa were included in the data set, the statistical relationship between body-size and abundance differed markedly from those documented for most other terrestrial and aquatic studies. At the scale of the marine invertebrate guild (Papers III and IV), an experimental approach demonstrated that the mechanisms proposed to generate patterns in body-size and patterns of re-assembly in benthic assemblages could not be rigorously supported. Specifically, habitat architecture was not as intimately related to body-size patterns as originally claimed, and the re-assembly of benthic communities was not driven by a directional change in organisms size. In both experiments issues of spatio-temporal scale made it difficult to relate patterns to underlying processes.

Previous work has shown that removal of the midline frontal cortex at seven to ten days of age is followed by recovery of function correlated with apparent spontaneous generation of new tissue in the lesion cavity. The question asked in the present thesis was whether the removal of the regrown tissue in adulthood would block normal function. Rats that received P10 frontal lesions underwent second lesions at P160, and were compared to rats with only P10 or P160 lesions. Rats with P10 + P160 lesions were severely impaired on a spatial learning task, especially relative to the P10 lesion-only rats. In a second experiment, rats with P10 + P160 lesions were given intra-ventricular infusions of a cocktail of three growth factors. The animals with growth factors showed marked behavioral recovery, although there was no cell regeneration. The results of these experiments suggest that filled-in tissue in neonatally lesioned rats is functional.
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L’esquizofrènia és una malaltia mental incapacitant que involucra diversos símptomes cognitius, com un filtratge sensoriomotor deteriorat. El filtratge sensoriomotor es pot mesurar mitjançant la inhibició prepols (IPP) de la resposta d’ensurt. Les investigacions en rosegadors sobre l’impacte d’alteracions cerebrals específiques sobre la IPP han estat molt útils per augmentar el coneixement d’aquesta deficiència bàsica de l’esquizofrènia. Aquests estudis mostren que els dèficits en IPP apareixen conjuntament amb altres símptomes, com l’agitació psicomotora, així com alteracions en el circuit cortico-estriato-pallido-talàmic (CEPT). Específicament, tractaments que alteren l’activitat del còrtex prefrontal medial (CPFm), l’hipocamp (HPC) o el nucli accumbens (NAc) redueixen la IPP. És important destacar que la desregulació cortical del balanç excitació-inhibició (E-I) s’ha proposat com el principal substrat subjacent als símptomes cognitius de l’esquizofrènia. A més, aquests estudis mostren que diversos fàrmacs milloren la IPP, com el neuropèptid oxitocina, el qual s’ha proposat com un antipsicòtic natural alternatiu. D’altra banda, els estudis en humans avaluen l’associació entre diferències naturals en la conducta i canvis neurals. En aquesta Tesi Doctoral, amb la idea d’establir un pont entre els estudis en humans i rosegadors, vam explorar si els dèficits naturals en IPP en rates consanguínies i no consanguínies intactes (i) s’associaven amb diferències en altres conductes relacionades amb l’esquizofrènia; (ii) es relacionaven amb diferències funcionals i estructurals en el circuit CEPT; (iii) s’atenuaven per l’administració d’oxitocina. Els nostres subjectes d’estudi van ser les rates consanguínies Romanes d’alta i baixa evitació (RHA i RLA), i les rates no consanguínies HS. Les RHA mostren menor IPP que les RLA, mentre que les HS es van estratificar en subgrups segons els seus nivells d’IPP. Els experiments plantejats també pretenien augmentar la validesa aparent, de constructe i predictiva dels nostres models animals de característiques rellevants per a l’esquizofrènia (RHA i HS-baixa-PPI). Els nostres resultats mostren que l’exploració incrementada en resposta a la novetat s’associa amb dèficits en IPP en rates HS i Romanes. Per estudiar les associacions cerebrals estructurals i funcionals amb la IPP, vam combinar l’ús de ressonància magnètica estructural i expressió de c-Fos després de la IPP. Vam trobar que la baixa IPP s’associa amb baixa activitat del CPFm en rates Romanes i HS i amb un augment d’activitat en el NAc en rates HS. La baixa IPP s’associa també amb una disminució del volum cerebral del CPFm i l’HPC en rates Romanes i HS. A més, emprant immunofluorescència després de la IPP, vam observar un menor percentatge d’activitat d’interneurones inhibitòries GABAèrgiques (parvalbúmina) al CPFm en rates RHA que en RLA. En relació amb l’administració d’oxitocina, vam trobar que l’oxitocina augmentava la IPP en rates HS i RHA, mentre que no afectava la IPP en les RLA. Els valors constitutius d’expressió del gen CD38 (regulador de l’alliberament d’oxitocina) al CPFm eren més baixos en les RHA que en les RLA, mentre que l’administració d’oxitocina va incrementar l’expressió del gen del receptor de oxitocina (OXTR) en ambdues soques. Aquesta Tesi Doctoral mostra un patró consistent d’alteracions conductuals i neurobiològiques en rates HS-baixa-IPP i RHA que incrementa la seva validesa aparent, de constructe i predictiva com a models animals de característiques relacionades amb l’esquizofrènia. És important destacar que els nostres resultats donen suport a la idea que el filtratge sensoriomotor està modulat per estructures cerebrals superiors (CPFm) i el balanç cortical E-I.
La esquizofrenia es una enfermedad mental incapacitante que involucra varios síntomas cognitivos, como un filtraje sensoriomotor deteriorado. El filtraje sensoriomotor se puede medir mediante la inhibición prepulso (IPP) de la respuesta de sobresalto. Los estudios en roedores que analizan el impacto de alteraciones cerebrales específicas sobre la IPP han sido muy útiles para aumentar el conocimiento sobre esta deficiencia básica de la esquizofrenia. Estos estudios muestran que los déficits en IPP aparecen junto a otros síntomas, como agitación psicomotora, y alteraciones en el circuito cortico-estriato-pallido-talámico (CEPT). Específicamente, tratamientos que aumentan o disminuyen la actividad del córtex prefrontal medial (CPFm), el hipocampo (HPC) o el núcleo accumbens (NAc) reducen la IPP. Es importante destacar que la desregulación cortical en el balance excitación-inhibición (E-I) se ha propuesto como el principal sustrato subyacente a los síntomas cognitivos de la esquizofrenia. Además, estos estudios muestran que la IPP mejora con varios fármacos antipsicóticos, como el neuropéptido oxitocina, el cual se ha propuesto como antipsicótico natural alternativo. A diferencia de los estudios en roedores, los estudios en humanos evalúan la asociación entre diferencias comportamentales naturales (diagnóstico, síntomas) y cambios neurales. En esta Tesis Doctoral, nos propusimos contribuir a establecer un puente entre los estudios en humanos y roedores y, para ello, exploramos si los déficits naturales en IPP en ratas consanguíneas y no-consanguíneas intactas (i) se asociaban con diferencias en otras conductas relacionadas con la esquizofrenia; (ii) se relacionaban con diferencias funcionales y estructurales en el circuito CEPT; (iii) se atenuaban por la administración de oxitocina. Usamos las ratas consanguíneas Romanas de alta y baja evitación (RHA y RLA), y las ratas no consanguíneas del stock heterogéneo HS. Las RHA muestran menor IPP que las RLA, mientras que las HS se estratificaron en subgrupos según su IPP. Los experimentos planteados también pretendían aumentar la validez aparente, de constructo y predictiva de nuestros animales modelo de características relevantes para la esquizofrenia (RHA y HS-baja-IPP). En relación con las asociaciones conductuales, nuestros resultados muestran que la exploración incrementada en respuesta a la novedad se asocia con déficits en IPP en ratas HS y Romanas. Para estudiar las asociaciones cerebrales estructurales y funcionales con la IPP, combinamos el uso de resonancia magnética estructural y expresión de c-Fos después de la IPP. Encontramos que la baja IPP se asocia con baja actividad del CPFm en ratas Romanas y HS y con un aumento de actividad en el NAc en ratas HS. La baja IPP se asocia también con una disminución del volumen cerebral del CPFm e HPC en ratas Romanas y HS. Además, mediante el uso de inmunofluorescencia después de la IPP, encontramos un menor porcentaje de actividad de interneuronas inhibitorias GABAérgicas de tipo parvalbúmina en el CPFm en ratas RHA que en RLA. Respecto a la administración de oxitocina, ésta aumentó la IPP en ratas HS y RHA, mientras que no afectó la IPP en las RLA. De acuerdo con el efecto diferencial de la oxitocina sobre la IPP (RHA>RLA), los valores constitutivos de expresión del gen CD38 (regulador de la liberación de oxitocina) en el CPFm fueron más bajos en ratas RHA que en las RLA, mientras que la administración de oxitocina incrementó la expresión del gen del receptor de oxitocina (OXTR) en ambas cepas. Esta Tesis Doctoral muestra un patrón consistente de alteraciones conductuales y neurobiológicas en ratas HS-baja-IPP y RHA que incrementa su validez aparente, de constructo y predictiva como animales modelo de características relacionadas con la esquizofrenia. Nuestros resultados apoyan la idea de que el filtraje sensoriomotor está modulado por estructuras cerebrales superiores (CPFm) y el balance cortical E-I.
Schizophrenia is a debilitating mental disorder that involves several cognitive symptoms, including sensorimotor gating impairments. Sensorimotor gating can be measured via prepulse inhibition (PPI) of the startle response, in which the magnitude of a startle stimulus is attenuated by the presence of a pre-stimulus of lower intensity. Rodent studies evaluating the impact of brain-site specific manipulations on PPI have been very useful to provide insights into this basic schizophrenia-like deficiency. These studies show that PPI deficits are frequently accompanied by other symptoms, including psychomotor agitation, as well as alterations in the cortico-striatal-pallido-thalamic (CSPT) circuit. In particular, treatments that increase or decrease the activity of the medial prefrontal cortex (mPFC), hippocampus (HPC), or nucleus accumbens (NAc) reduce PPI. In this context, a dysfunctional cortical excitatory-inhibitory balance has been proposed as the main neural substrate for cognitive dysfunction in schizophrenia. Moreover, these studies show that PPI deficits can be improved by several antipsychotic drugs, including the neuropeptide oxytocin, which has been suggested as an alternative natural antipsychotic. In contrast to these rodent studies, human studies evaluate the association between natural behavioral differences (diagnosis, symptoms) and neural changes. Thus, in this Doctoral Dissertation, we aimed to contribute to bridge the gap between human and rodent studies by exploring whether spontaneous deficits in PPI in intact inbred and outbred rats are (i) associated with divergences in other schizophrenia-related behaviors, (ii) related to functional and structural differences in the CSPT circuit, and (iii) attenuated by oxytocin. Our subjects of study were the inbred Roman high-avoidance (RHA) and Low-avoidance (RLA) rats, and the outbred heterogeneous stock (HS) rats. RHA rats show lower PPI than RLAs, while HS rats were stratified in sub-groups according to their PPI levels. The present experiments also aimed to provide further face, construct, and predictive validity to our animal models of schizophrenia-relevant symptoms (RHA and HS Low-PPI rats). Regarding behavioral associations, our results show that increased exploration in response to novelty is associated with deficient PPI in HS and Roman rats. Moreover, a high anxious profile was found in rats with increased PPI, while no associations were seen with compulsive-like behavior. In relation to brain structural and functional associations with PPI, we combined structural magnetic resonance imaging and c-Fos expression after PPI in both HS and Roman rats. Our results indicate that lower PPI is associated with decreased mPFC activity in both Roman and HS rats and with increased NAc shell activity in HS rats. Reduced PPI is also associated with decreased mPFC and HPC volumes in Roman and HS rats. Additionally, using immunofluorescence after PPI, we observed a lower percentage of active inhibitory GABAergic parvalbumin interneurons in RHA than RLA rats. Regarding oxytocin administration, we found that oxytocin increased PPI in HS rats, attenuated PPI deficits in RHA rats, and did not affect PPI in RLAs. Consistent with the differential oxytocin effects on PPI (RHA>RLA), constitutive CD38 gen expression (regulator of oxytocin release) was reduced in the mPFC of RHA rats compared to the RLAs, while oxytocin administration increased oxytocin receptor (OXTR) gen expression in both strains. This Doctoral Dissertation shows a consistent pattern of behavioral and neurobiological abnormalities in the HS-Low-PPI rats and RHA rats that increases the face, construct, and predictive validity of these rats as models of schizophrenia-related features. Importantly, our results support the idea that sensorimotor gating is modulated by forebrain structures and highlight the relevance of the mPFC and the cortical excitatory-inhibitory balance in its regulation.
Universitat Autònoma de Barcelona. Programa de Doctorat en Neurociències

Buňka tvoří složitý biologický systém vystavený mnoha mimobuněčným mechanickým podnětům. Hlubší pochopení jejího mechanického chování je důležité pro charakterizaci její odezvy v podmínkách zdraví i nemoci. Výpočtové modelování může rozšířit pochopení mechaniky buňky, která může přispívat k vytvoření vztahů mezi strukturou a funkcí různých typů buněk v různých stavech. Za tímto účelem byly pomocí metody konečných prvků (MKP) vytvořeny dva bendotensegritní modely buňky v různých stavech: model vznášející se buňky pro analýzu její globální mechanické odezvy, jako je protažení nebo stlačení, a model buňky přilnuté k podložce, který vysvětluje odezvu buňky na lokální mechanické zatížení, jako třeba vtlačování hrotu při mikroskopii atomárních sil (AFM). Oba zachovávají základní principy tensegritních struktur jako je jejich předpětí a vzájemné ovlivnění mezi komponentami, ale prvky se mohou nezávisle pohybovat. Zahrnutí nedávno navržené bendotensegritní koncepce umožňuje těmto modelům brát v úvahu jak tahové, tak i ohybové namáhání mikrotubulů (MTs) a také zahrnout vlnitost intermediálních filament (IFs). Modely předpokládají, že jednotlivé složky cytoskeletu mohou měnit svůj tvar a uspořádání, aniž by při jejich odstranění došlo ke kolapsu celé buněčné struktury, a tak umožňují hodnotit mechanický příspěvek jednotlivých složek cytoskeletu k mechanice buňky. Model vznášející se buňky napodobuje realisticky odezvu síla-deformace během protahování a stlačování buňky a obě odezvy ilustrují nelineární nárůst tuhosti s růstem mechanického zatížení. Výsledky simulací ukazují, že aktinová filamenta i mikrotubuly hrají klíčovou úlohu při určování tahové odezvy buňky, zatímco k její tlakové odezvě přispívají podstatně jen aktinová filamenta. Model buňky přilnuté k podložce dává odezvu síla-hloubka vtlačení ve dvou různých místech odpovídající nelineární odezvě zjištěné experimentálně při AFM. Výsledky simulací ukazují, že pro chování buňky je rozhodující místo vtlačení a její tuhost určují aktinová povrchová vrstva, mikrotubuly a cytoplazma. Navržené modely umožňují cenný vhled do vzájemných souvislostí mechanických vlastností buněk, do mechanické úlohy komponent cytoskeletu jak individuálně, tak i ve vzájemné synergii a do deformace jádra buňky za různých podmínek mechanického zatížení. Tudíž tato práce přispívá k lepšímu pochopení mechaniky cytoskeletu zodpovědné za chování buňky, což naopak může napomáhat ve zkoumání různých patologických podmínek jako je rakovina a cévní choroby.

Skeletal muscle is spatially heterogeneous in muscle fibre type composition and microvascular supply. The capacity to quantify this heterogeneity in skeletal muscle is not routinely performed for it’s a laborious and time consuming technique. We have developed a high throughput data pipeline that utilises the simultaneous immunohistochemical labelling of muscle fibre type and microvascular supply, as an input for a semi-automated analysis software package that allows for the analysis of fine morphometric indices of fibre type composition and the interactions with microvascular supply. We have successfully shown that regional variation in fibre type composition impacts the functional characteristics of a muscle. After successful characterisation of regional heterogeneity in both structure and function we sought to establish their influence in physiological (adaptive) angiogenesis. Utilising animal angiogenic models we have shown that shear stress driven angiogenesis is principally a stochastic response that does not promote improved oxygen delivery when we analyse the spatial heterogeneity of the neovasculature. Conversely, skeletal muscle overload (abluminal stretch of microvasculature) increases the homogeneity of the oxygen supply area of the capillary bed, suggesting a tissue driven angiogenic response that is not evident in shear stress. Spinal cord injury induced rarefaction of the capillary bed attempts to maintain a homogeneous distribution of fibre size and capillary supply. The combination therapy of epidural stimulation and locomotor training can ameliorate the phenotypic change and rarefied capillary bed seen with spinal cord injury to that of intact levels. Endurance and resistance exercise have a largely similar global genomic response following a chronic training regime, which we are able to replicate in animal models of exercise through indirect electrical stimulation. The shear stress and muscle overload driven angiogenic response have distinctly different angiogenic pathways that contain no commonly expressed networks.

The electrophysiologic technique (Reuss, L., Proc. Natl. Acad. Sci. USA 82:6014, 1985) was modified to measure changes in steady-state hepatocyte volume during osmotic stress. Hepatocytes in mouse liver slices were loaded with tetramethylammonium ion (TMA$\sp{+}$) during transient exposure of cells to nystatin. Intracellular TMA$\sp{+}$ activity (a$\sp{\rm i}\sb{\rm TMA}$) was measured with TMA$\sp{+}$-sensitive, double-barreled microelectrodes. Loading hepatocytes with TMA$\sp{+}$ did not change their membrane potential (V$\sb{\rm m}$), and under steady-state conditions a$\sp{\rm i}\sb{\rm TMA}$ remained constant over 4 min in single impalements. Hyperosmotic solutions (50, 100, & 150 mM sucrose added to media) and hyposmotic solutions (sucrose in media reduced by 50 & 100 mM) increased and decreased a$\sp{\rm i}\sb{\rm TMA}$, respectively, which suggested transmembrane water movements. The regression coefficient of the ratio of control a$\sp{\rm i}\sb{\rm TMA}$/experimental a$\sp{\rm i}\sb{\rm TMA}$ versus the relative osmolality of media (experimental mOsm/control mOsm) was -0.34 $\pm$ 0.03, p $<$ 0.001, which is less than expected for a perfect osmometer. Corresponding measurements of V$\sb{\rm m}$ showed that its magnitude increased with hyposmolality and decreased with hyperosmolality. When Ba$\sp{2+}$ (2 mM) was present during hyposmotic stress of 0.66 $\times$ 286 mOsm (control), cell water volume increased by a factor of 1.44 $\pm$ 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.12 $\pm$ 0.02, p $<$ 0.001. Ba$\sp{2+}$ also decreased the hyperpolarization of V$\sb{\rm m}$ due to hyposmotic stress from a factor of 1.62 $\pm$ 0.04 to 1.24 $\pm$ 0.09, p $<$ 0.01. When verapamil (50 $\mu$M) was present during hyposmotic stress of 0.69 $\times$ 292 mOsm (control), cell water volume increased by a factor of 1.42 $\pm$ 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.19 $\pm$ 0.02, p $<$ 0.001. Verapamil also decreased the hyperpolarization of V$\sb{\rm m}$ due to hyposmotic stress from a factor of 1.34 $\pm$ 0.07 to 1.08 $\pm$ 0.08, p $<$ 0.05. Similar results were obtained when exposing hepatocytes to Ca$\sp{2+}$-free medium plus EGTA (5 mM). It was concluded that hepatocytes partially regulate their steady-state volume during hypo- and hyperosmotic stress. However, volume regulation during hyposmotic stress diminished along with hyperpolarization of V$\sb{\rm m}$ in the presence of the K$\sp{+}$-channel blocker, Ba$\sp{2+}$, the Ca$\sp{2+}$-channel blocker, verapamil and the Ca$\sp{2+}$-chelator, EGTA. This indicated that cell calcium and membrane potassium conductance (g$\sb{\rm K}$) were involved in hepatocyte volume regulation mechanism and that variation in V$\sb{\rm m}$ provides an intercurrent, electromotive force for hepatocyte volume regulation.

Atherosclerosis is a complex inflammatory disease which may be triggered by an elevated transport of lipid-carrying macromolecules from the blood into the arterial wall. Its non-uniform distribution has been attributed to local variations in wall shear stress. Intramural stresses and strains are regularly overlooked, despite their heterogeneous distribution and direct effect on vascular cell morphology and function. Stenotic flow models of atherosclerosis inadvertently alter both fluid and solid stresses. Using a tapered flow modifying cuff, wall uptake of plasma macromolecules was investigated in the murine carotid artery. The greatest uptake occurred just upstream, the site which has previously been observed to later develop vulnerable atherosclerotic plaques. Uptake was also elevated, but to a significantly lesser extent, just downstream. Flow simulations and the effects of reversing cuff orientation on wall permeability, indicated a role for solid, as well as fluid stresses. Structural simulations revealed that steep spatial and cyclic stress/strain gradients occur at the cuff margins. When fluid-structure interaction effects are included, transmural pressure and circumferential stress and strain are reduced downstream. Arterial branch sites exhibit large variations in biomechanical factors and a predilection for atherosclerotic lesions. Motivated by the need to accurately map strains around branch openings, a novel method was developed to determine in vivo strain in animal models using vascular corrosion casts. A study focused on the descending thoracic rabbit aorta demonstrated its efficacy. Assessing the anatomical correlation of atherosclerosis with biomechanical localising factors is hindered by spatial autocorrelation, which tends to exaggerate significance, and by the use of aggregate data, which artificially inflates correlation coefficients. A comparison of four statistical tests for assessing spatial correlations highlighted substantial differences in obtained significance when applied to maps of wall shear stress and three putatively related arterial properties. A fifth test specific to aggregate data was proposed and applied. Appropriate application of these techniques will help to establish the relative importance of fluid and solid mechanics in atherogenesis.

The estrogen receptor (ER) is a ligand dependent transcriptional activator that belongs to the steroid/nuclear receptor superfamily. To probe the structure and function of the ER ligand binding domain (LBD), we developed a genetic screen in yeast Saccharomyces cerevisiae using a library of reverse ERs screened with a low affinity estrogen agonist, 2-methoxyestrone. Mutants isolated from this screen demonstrated altered ligand binding and/or transactivation properties. One of these mutants, L536P, showed high levels of constitutive activity in several transiently transfected cells and a HeLa line that stably propagates an estrogen-sensitive reporter gene. This suggests that substitution of a proline at position 536 in the wild type ER (HEGO) induces a reversible conformational change in the region of AF-2 that partially mimics activation of the receptor by hormone binding.
Activation of transcription by the ER requires the recruitment of different classes of coactivators. L536P interacted with coactivators in the absence of hormone and this constitutive interaction can be abolished by antiestrogens. We conclude that constitutive activity of L536P-HEGO is manifested to in part from constitutive coactivator binding. We also demonstrated that different classes of coactivators do not recognize the ER LBD in the same manner and can compete for binding to the ER LBD suggesting that different classes of coactivators recognize distinct but overlapping binding sites. Interestingly, coexpression of RIP140 blocked enhanced transactivation by HEGO observed in the presence of TIF-2, suggesting that RIP140 may play a negative role in ER signaling.
Using a yeast two-hybrid system with the ER LBD as bait, we isolated a novel estrogen receptor cofactor (ERC) that interacts with the ER LBD in a hormone dependent manner. The primary structure of ERC consists of 4 ankyrin repeat domains, 2 LXXLL motifs and an ATP/GTP binding domain. ERC is highly expressed in ovary, testes, and spleen, with moderate levels in heart, brain, and placenta. ERC repressed ER transactivation in several cell lines in the presence of estradiol suggesting that ERC may function as a novel estrogen dependent repressor of the ER. Immunohistochemistry and confocal microscopy localized ERC to the cytoplasm with partial nuclear staining. Recently, synphilin-1, a novel protein that has been implicated in the pathogenesis of Parkinson's disease was isolated and is identical to ERC. A role for ERC in intracellular signaling through a membrane bound ER in the brain is currently being investigated.

This study was conducted to evaluate the effect of season on reproductive parameters in cattle in the temperate climate of the UK. In the first study, reproductive fertility data were collected from a local dairy herd. The results revealed that cows born in autumn were inseminated at an earlier age (P < 0.05) and calved earlier (P < 0.001) than spring and summer born animals. In addition, the conception rate within 90 days after calving was higher (P < 0.05) in autumn calving animals. Conception rate was higher (P < 0.05) when insemination was performed at a temperature range from 7 to 15°C compared with < 7 and >15°C and a temperature humidity index (THI) range from 40 to 59 compared with >60 units (10 days before and 21 days after insemination). In the second study, ovarian tissues were collected from a local abattoir to investigate the effect of season on follicular populations, corpus luteum (CL) development and incidence of multiple ovulations. There were no effects of season on antral follicle count. However, individual and total CL weight was heavier in the autumn. Additionally, season influenced multiple ovulations with a higher incidence (P < 0.05) in summer and autumn compared to winter and spring. The number of follicles >7mm in cows with multiple CLs was higher (P < 0.05) than cows with a single CL. The individual CL weight was heavier in single ovulation cows. However, the total luteal tissue weight and total progesterone (P4) content of luteal tissue was higher in cows with multiple ovulations. The third study investigated the effects of culturing granulosa cells (GCs) under low (5%; physiological oxygen (O2)) conditions rather than traditional culture (20%; atmospheric O2) systems. Granulosa cells from antral follicles were cultured in fibronectin coated plates in M199 for up to 144 hour (h) under physiological (5%) and atmospheric (20%) O2 tension. Melatonin was added at one of four concentrations (0, 20, 200, 2000 pg/ml). The number of viable GCs was greater (P < 0.05) under 5% O2 than 20% O2. Reactive oxygen species (ROS) generation was similar under both physiological and atmospheric O2, but was reduced (P < 0.05) by treatment with melatonin. Oestrogen concentration (P < 0.001) and aromatase activity (P < 0.014) were also influenced by O2 tension in a time dependent manner. Both oestradiol (E2) production and aromatase enzyme activity were maintained for up to 144 h of culture under 5% O2 conditions. Progesterone production was increased under 20% O2 compared with 5% O2 (P < 0.05). Additionally, the expression of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1) mRNA increased (P < 0.05) with time under 20% O2, but remained unchanged at 5% O2. Haemoglobin subunit alpha 1 (HBA) transcript was increased (P < 0.05) under 5% O2 levels. The final study quantified the effect of temperature and melatonin on GC function. Cells were cultured for up to 144 h under 5% O2 tension. Treatments commenced after 48 h of culture and consisted of two incubation temperatures (37.5 vs 40.0°C) and four melatonin treatments. Melatonin increased cell number at high temperature (40.0°C). However, BCL2-associated X protein (BAX) mRNA expression was greater (P < 0.05) in GCs cultured at 40.0°C than at 37.5°C by 144 h. Culture temperature did not affect ROS, but melatonin reduced (P < 0.001) generation of ROS. Oestradiol production increased with time (P < 0.001) and was not affected by temperature. In contrast, high temperature reduced P4 production (P < 0.001) at 144 h of culture. Similarly, the effect of melatonin treatment depended on temperature; melatonin increased P4 production at 37.5C, while reducing P4 at 40.0C. Temperature increased acetylserotonin O-methyltransferase (ASMT) mRNA expression (P < 0.05) though there was no significant effect of temperature and melatonin on tumour protein p53 (P53), HSD3B1, superoxide dismutase (SOD1 and SOD2), HBA and heat shock protein family A (Hsp70) member 1A (HSPA1A) gene expression. The results of this thesis contribute to our understanding of the effects of season on ovarian function and seasonal variation in cattle fertility particular in temperate climate regions where season influenced puberty, conception rate, incidence of multiple ovulations and CL development. In in vitro studies, low O2 (5%) enhance cell proliferation, reduced luteinisation and altered steroidogenesis as well as increasing the expression of HBA mRNA. Culture at higher temperature reduced P4 production and increased apoptotic mRNA while addition of melatonin reduced ROS generation and influenced P4 production. This new approach to culture could offer a valuable system for future investigation of the physiological function of cells in vitro.