A robotic microscope was designed using a microcontroller to take time-lapse digital photographs of developing salamander embryos. The microcontroller operated three stepper motors to control three-axis movement accurately, and two six mega-pixel digital cameras to capture through-focus time-lapse digital pictures of six views of Ambystoma mexicanum embryos (axolotl, a salamander). The device is designed to take images every five minutes for 80 hours of early development, from fertilization to stage 20, when the neural tube closes to form the brain and spinal column. Techniques to enhance the embryo images were investigated including image fusion to get in-focus views from a stack of images. In the early embryo surface epithelial cells differentiate to form neural tissue and external skin tissue. Observing the whole embryo surface at cellular level will give a better idea of the stress and strain each cell undergoes and what physical forces are involved in cell differentiation.

Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

A robotic microscope was designed using a microcontroller to take time-lapse digital photographs of developing salamander embryos. The microcontroller operated three stepper motors to control three-axis movement accurately, and two six mega-pixel digital cameras to capture through-focus time-lapse digital pictures of six views of Ambystoma mexicanum embryos (axolotl, a salamander). The device is designed to take images every five minutes for 80 hours of early development, from fertilization to stage 20, when the neural tube closes to form the brain and spinal column. Techniques to enhance the embryo images were investigated including image fusion to get in-focus views from a stack of images. In the early embryo surface epithelial cells differentiate to form neural tissue and external skin tissue. Observing the whole embryo surface at cellular level will give a better idea of the stress and strain each cell undergoes and what physical forces are involved in cell differentiation.

Time-lapse seismic is a modern technology for monitoring production-induced changes in and around a hydrocarbon reservoir. Time-lapse (4D) seismic may help locate undrained areas, monitor pore fluid changes and identify reservoir compartmentalization. Despite several successful 4D projects, there are still many challenges related to time-lapse technology. Perhaps the most important are to perform quantitative time-lapse and to model and interpret time-lapse effects in thin layers. The former requires one to quantify saturation and pressure effects on rock elastic parameters. The latter requires an understanding of the combined response of time-lapse effects in thin layers and overcoming seismic vertical resolution limitation. / This thesis presents an integrated study of saturation and pressure effects on elastic properties. Despite the fact that Gassmann fluid substitution is standard practice to predict time-lapse saturation effects, its validity in the field environment rests upon a number of assumptions. The validity of Gassmann equations, ultimately, can only be tested in real geological environments. In this thesis I developed a workflow to test Gassmann fluid substitution by comparing saturated P-wave moduli computed from dry core measurements with those obtained from sonic and density logs. The workflow has been tested on a turbidite reservoir from the Campos Basin, offshore Brazil. The results show good statistical agreement between the P-wave elastic moduli computed from cores using the Gassmann equations and the corresponding moduli computed from log data. This confirms that all the assumptions of the Gassmann theory are adequate within the measurement error and natural variability of elastic properties. These results provide further justification for using the Gassmann theory to interpret time-lapse effects in this sandstone reservoir and in similar geological formations. / Pressure effects on elastic properties are usually obtained by laboratory measurements, which can be affected by core damage. I investigated the magnitude of this effect on compressional-wave velocities by comparing laboratory experiments and log measurements. I used Gassmann fluid substitution to obtain low-frequency saturated velocities from dry core measurements taken at reservoir pressure, thus mitigating the dispersion effects. The analysis is performed for an unusual densely cored well from which 43 cores were extracted over a 45 m thick turbidite reservoir. These computed velocities show very good agreement with the sonic-log measurements. This is encouraging because it implies that core damages that may occur while bringing the core samples to the surface are small and do not adversely affect the measurement of elastic properties on these core samples. Should core damage have affected our measurements, we would have expected a systematic difference between properties measured in situ and on the recovered. This confirms that, for this particular region, the effect of core damage on ultrasonic measurements is less than the measurement error. Consequently, stress sensitivity of elastic properties as obtained from ultrasonic measurements are adequate for quantitative interpretation of time-lapse seismic data. / In some circumstances, stress sensitivity may not be obtained by ultrasonic measurements. Cores may be affected by damage, bias in the plugging process and scale effects and therefore may not be representative of the in situ properties. Consequently it is desirable to obtain this dependence from an alternative method. This other approach ideally should provide the pressure - velocity dependence from an intact rock. Few methods can sample the in situ rock. Seismic, for instance, provides in situ information, but lacks vertical resolution. Well logs, on the other hand, can provide high vertical resolution information, but usually are not available before and after production changes. I propose a method to assess the in situ pressure - velocity dependence using well data. I apply this method to a reservoir made up of sandstone. I used 23 wells drilled and logged in different stages of development of a hydrocarbon field providing rock and fluid properties at different pressures. For each well logged at a specific time, pore pressure, velocity and porosity, among other properties, are known. Pore pressure is accessed from a Repeat Formation Tester (RFT). As a field depletes and new wells are drilled and logged, similar data sets related to different stages of depletion are available. I present an approach expanding Furre et al. (2009) study incorporating porosity and obtaining a three dimensional relationship with velocity and pressure. The idea is to help to capture rock property variability. / Quantitative time-lapse studies require precise knowledge of the response of rocks sampled by a seismic wave. Small-scale vertical changes in rock properties, such as those resulting from centimetre scale depositional layering, are usually undetectable in both seismic and standard borehole logs (Murphy et al., 1984). I present a methodology to assess rock properties by using X-ray computed tomography (CT) images along with laboratory velocity measurements and borehole logs. This methodology is applied to rocks extracted from around 2.8 km depth from offshore Brazil. This improved understanding of physical property variations may help to correlate stratigraphy between wells and to calibrate pressure effects on velocities, for seismic time-lapse studies. / Small scale intra-reservoir shales have a very different response from sands to fluid injection and depletion, and thus may have a strong effect on the equivalent properties of a heterogeneous sandstone reservoir. Since shales have very low permeability, an increase of pore pressure in the sand will cause an increase of confining pressure in the intra-reservoir shale. I present a methodology to compute the combined seismic response for depletion and injection scenarios as a function of net to gross (NTG or sand – shale fraction). This approach is appropriate for modelling time-lapse effects of thin layers of sandstones and shales in repeated seismic surveys when there is no time for pressure in shale and sand to equilibrate. I apply the developed methodology to analyse the sand - shale combined response to typical shale and sandstone stress sensitivities for an oil field located in Campos Basin, Brazil. For a typical NTG of 0.6, there is a difference of approximately 35% in reflection coefficient during reservoir depletion from the expected value if these shales are neglected. Consequently, not considering the small shales intra-reservoir may mislead quantitative 4D studies. / The results obtained in this research are aimed to quantify pressure and saturation effects on elastic properties. New methodologies and workflows have been proposed and tested using real data from South America (Campos Basin) datasets. The results of this study are expected to guide future time-lapse studies in this region. Further investigations using the proposed methodologies are necessary to verify their applicability in other regions.

BACKGROUND Heart disease and renal dysfunction are often mutually reinforcing conditions, although the factors underlying this relationship are not fully understood. Cardiac remodelling resulting from disease is partly caused by the reactivation of developmental programmes. By unravelling the mechanisms that drive kidney development and function, it may be possible to gain novel insight into remodelled kidney states that are linked to disease. In this study, we have investigated the interplay between renal and cardiovascular systems during nephrogenesis at the level of the blood filter. METHODOLOGY Owing to the optical transparency and rapid external development of the embryo, the zebrafish provides a research model for advanced imaging technologies, allowing us to visualise structures located deep within living specimens. Here, we combine deep-tissue live imaging and novel functional assays to study development and function of the pronephric kidney - the first and most basic kidney to form in the embryo. RESULTS Using two-photon excitation microscopy, we have successfully established methodology for performing, deep-tissue, time-lapse imaging in living embryos of the two primary cell types forming the kidney – endothelial and epithelial cells. Fluorescence angiograms were performed using supra-vital dye agents to visualise circulatory flow in relation to the pronephric vasculature and the process of blood-filtration. Observations from live imaging studies, supported by immunostaining, were used to create a comprehensive model of the developing glomerular morphology, and interactions at the endothelial-epithelial cell interface, where glomerular epithelial primordia merge around the vascular component of the pronephric kidney. To investigate renal function, we devised a novel assay of pronephric filtration, by tracking the accumulation of injected fluorescent tracers within the excreted filtrate of embryos. This allowed us to relate our time-lapse observations to maturation of the glomerulus, and the evolution of perm-selective function. Finally, we explored methods of mechanically obstructing blood-flow in order to investigate whether altered hemodynamic forces would influence pronephric development. We found that in those embryos with severely disrupted circulatory flow, the glomerular morphology was affected. CONCLUSIONS In summary, the combination of these techniques has allowed us to visualise the multi-cellular organisation of the pronephric kidney over time, which has previously been limited to primarily fixed-tissue approaches. A detailed model of pronephric development has been developed, which could ultimately be used to dissect the molecular mechanisms underlying embryonic kidney development.

The main goal of this thesis is to characterize workshop processes in a chosen enterprise, analyze them and suggest possible improvements. The cooperation was formed with VSP DATA a.s. company which is situated the town Tábor. This enterprise is specialized in service and repairs. The theoretical part of this thesis was based on studying specialized literature focused on processes, production, services and improvement methods. Main information, characteristics and core activities are provided in introduction of practical part of this work. The necessary information for the next part of thesis was taken from the corporate internal data and from the regulated conversation with several experts of the company. For mapping out the times of the workshop processes was used data from the entire year 2016. This technique helped with analysis of present state and to found out key problems with excessively long time duration of repairs. The suggestion of optimization was focused on reduction of time of processes and attainment of their continuous flow. A certain part of the proposals is the effort to change the current state of workforce. Among recommendations there are 5S method, improvement of sharing experience and implementation of time limit for customer decision-making. To achieve better results of optimization it is necessary to combine partial metod, rather then implement them separately.

Le sujet de cette thèse est l'étude quantitative du rôle du morphogène Fgf8 durant la somitogenèse en utilisant des perturbations spatio-temporelles de sa concentration dans un embryon de poisson zèbre en développement. Mon objectif était d'élucider le rôle que joue Fgf8 dans le modèle du «clock and wavefront» de la somitogenèse . A cet effet, j'ai développé des moyens optiques afin de perturber rapidement sa concentration dans un embryon vivant par un éclairage approprié. J’ai montré que le blocage du Fgf8 endogène (en utilisant un morpholino contre Fgf8) ou sa surexpression (en utilisant une approche de photo-activation développée dans le laboratoire d'accueil) affectaient la taille des somites observées dans un embryon de poisson zèbre à 24 hpf. Pour comprendre la raison de ce changement de taille des somitesj’ai construit un système de vidéomicroscopie à intervalle régulier qui m'a permis de suivre l'évolution parallèle de 20-30 embryons de poisson zèbre en temps réel. Après avoir comparé la période de la segmentation, la vitesse d'allongement de la queue, la vitesse de racourcissement du PSM et la distribution spatiale de MAPK phosphorylé, mes résultats montrent que la sur-expression globale de Fgf8 induit un retard subtil dans la période de segmentation et ralentit la vitesse de déplacement du front d'onde, qui est la cause principale de la variation de taille des somites. / The subject of this thesis is the quantitative study of the role of Fgf8 in somitogenesis using spatio-temporal perturbations of its concentration in a developing zebrafish embryo. My goal was to elucidate the role that Fgf8 plays in the clock and wavefront model of somitogenesis. For that purpose, I have developed optical means to quickly perturb its concentration in a live embryo by an appropriate illumination. I have shown that blocking endogenous Fgf8 (using a morpholino against Fgf8) or inducing over-expression of Fgf8 (using a photo-activable approach developed in the host lab) affected the size of somites observed at 24 hpf in a zebrafish embryo. To address the reason for this change in somite size, I have built a time-lapse microscopy set-up that allowed me to monitor the parallel development of 20-30 zebrafish embryos in real time. After comparing the period of segmentation, the velocity of tail elongation, the speed of PSM shortening, and the MAPK pattern of phosphorylation (the target of Fgf8), my results show that global over-expression of Fgf8 induces a subtle delay in the segmentation period and slows down the posterior moving wavefront velocity, which is the major cause of the change in somite size.

Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

The H+-ATPase rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na+ uptake and H+ extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid-base status. In the present study, a ratiometric imaging technique using the pH indicator dye BCECF was developed to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo. Initial validation experiments demonstrated that HR cells subjected to respiratory acidosis (1% CO2) or metabolic alkalosis (20 mM NH4Cl) exhibited changes in BCECF 513/438 emission ratios which were consistent with the expected effects of these treatments on pHi. Subsequent experiments focussed on the involvement of the two principal apical membrane acid excretory pathways, the Na+/H+ exchanger (isoform NHE3b; zslc9a3.2) and the H+-ATPase (atpv1aa) in pHi regulation. Additionally, the role of HR cell carbonic anhydrase (“CA2-like a”) was investigated because of its presumed role in providing H+ for Na+/H+ exchange and H+-ATPase. To do so, relative HR cell pHi changes were monitored during acid-base challenges in shams and in fish experiencing morpholino gene knockdown of either NHE3b, H+-ATPase or “CA2-like a”. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO2 and the extent of post-CO2 alkalization were altered markedly in fish experiencing knockdown of “CA2-like a”, NHE3b or H+-ATPase. Although there were slight differences among the three knockdown experiments, the typical response was a greater degree of intracellular acidification during CO2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. Knockdown of “CA2-like a”, although presumed to limit H+ availability to NHE3b and H+-ATPase, yielded qualitatively similar results to knockdown of either single H+ excretory pathway. The metabolic alkalosis and subsequent acidification associated with NH4Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid-base regulation in zebrafish HR cells depending on the nature of the acid-base disturbance.

One of the most challenging tasks in the oil industry is the production of reliable reservoir forecast models. Because of different sources of uncertainties the numerical models employed are often only crude approximations of the reality. This problem is tackled by the conditioning of the model with production data through data assimilation. This process is known in the oil industry as history matching. Several recent advances are being used to improve history matching reliability, notably the use of time-lapse seismic data and automated history matching software tools. One of the most promising data assimilation techniques employed in the oil industry is the ensemble Kalman filter (EnKF) because its ability to deal with highly non-linear models, low computational cost and easy computational implementation when compared with other methods. A synthetic reservoir model was used in a history matching study designed to predict the peak production allowing decision makers to properly plan field development actions. If only production data is assimilated, a total of 12 years of historical data is required to properly characterize the production uncertainty and consequently the correct moment to take actions and decommission the field. However if time-lapse seismic data is available this conclusion can be reached 4 years in advance due to the additional fluid displacement information obtained with the seismic data. Production data provides geographically sparse data in contrast with seismic data which are sparse in time. Several types of seismic attributes were tested in this study. Poisson’s ratio proved to be the most sensitive attribute to fluid displacement. In practical applications, however the use of this attribute is usually avoided due to poor quality of the data. Seismic impedance tends to be more reliable. Finally, a new conceptual idea was proposed to obtain time-lapse information for a history matching study. The use of crosswell time-lapse seismic tomography to map velocities in the interwell region was demonstrated as a potential tool to ensure survey reproducibility and low acquisition cost when compared with full scale surface surveys. This approach relies on the higher velocity sensitivity to fluid displacement at higher frequencies. The velocity effects were modeled using the Biot velocity model. This method provided promising results leading to similar RRMS error reductions when compared with conventional history matched surface seismic data.