Methods for the analysis of multi-scale cell dynamics from fluorescence microscopy images

La embriogénesis es el proceso mediante el cual una célula se convierte en un ser un vivo. A lo largo de diferentes etapas de desarrollo, la población de células va proliferando a la vez que el embrión va tomando forma y se configura. Esto es posible gracias a la acción de varios procesos genéticos, bioquímicos y mecánicos que interaccionan y se regulan entre ellos formando un sistema complejo que se organiza a diferentes escalas espaciales y temporales. Este proceso ocurre de manera robusta y reproducible, pero también con cierta variabilidad que permite la diversidad de individuos de una misma especie. La aparición de la microscopía de fluorescencia, posible gracias a proteínas fluorescentes que pueden ser adheridas a las cadenas de expresión de las células, y los avances en la física óptica de los microscopios han permitido observar este proceso de embriogénesis in-vivo y generar secuencias de imágenes tridimensionales de alta resolución espacio-temporal. Estas imágenes permiten el estudio de los procesos de desarrollo embrionario con técnicas de análisis de imagen y de datos, reconstruyendo dichos procesos para crear la representación de un embrión digital. Una de las más actuales problemáticas en este campo es entender los procesos mecánicos, de manera aislada y en interacción con otros factores como la expresión genética, para que el embrión se desarrolle. Debido a la complejidad de estos procesos, estos problemas se afrontan mediante diferentes técnicas y escalas específicas donde, a través de experimentos, pueden hacerse y confrontarse hipótesis, obteniendo conclusiones sobre el funcionamiento de los mecanismos estudiados. Esta tesis doctoral se ha enfocado sobre esta problemática intentando mejorar las metodologías del estado del arte y con un objetivo específico: estudiar patrones de deformación que emergen del movimiento organizado de las células durante diferentes estados del desarrollo del embrión, de manera global o en tejidos concretos. Estudios se han centrado en la mecánica en relación con procesos de señalización o interacciones a nivel celular o de tejido. En este trabajo, se propone un esquema para generalizar el estudio del movimiento y las interacciones mecánicas que se desprenden del mismo a diferentes escalas espaciales y temporales. Esto permitiría no sólo estudios locales, si no estudios sistemáticos de las escalas de interacción mecánica dentro de un embrión. Por tanto, el esquema propuesto obvia las causas de generación de movimiento (fuerzas) y se centra en la cuantificación de la cinemática (deformación y esfuerzos) a partir de imágenes de forma no invasiva. Hoy en día las dificultades experimentales y metodológicas y la complejidad de los sistemas biológicos impiden una descripción mecánica completa de manera sistemática. Sin embargo, patrones de deformación muestran el resultado de diferentes factores mecánicos en interacción con otros elementos dando lugar a una organización mecánica, necesaria para el desarrollo, que puede ser cuantificado a partir de la metodología propuesta en esta tesis. La metodología asume un medio continuo descrito de forma Lagrangiana (en función de las trayectorias de puntos materiales que se mueven en el sistema en lugar de puntos espaciales) de la dinámica del movimiento, estimado a partir de las imágenes mediante métodos de seguimiento de células o de técnicas de registro de imagen. Gracias a este esquema es posible describir la deformación instantánea y acumulada respecto a un estado inicial para cualquier dominio del embrión. La aplicación de esta metodología a imágenes 3D + t del pez zebra sirvió para desvelar estructuras mecánicas que tienden a estabilizarse a lo largo del tiempo en dicho embrión, y que se organizan a una escala semejante al del mapa de diferenciación celular y con indicios de correlación con patrones de expresión genética. También se aplicó la metodología al estudio del tejido amnioserosa de la Drosophila (mosca de la fruta) durante el cierre dorsal, obteniendo indicios de un acoplamiento entre escalas subcelulares, celulares y supracelulares, que genera patrones complejos en respuesta a la fuerza generada por los esqueletos de acto-myosina. En definitiva, esta tesis doctoral propone una estrategia novedosa de análisis de la dinámica celular multi-escala que permite cuantificar patrones de manera inmediata y que además ofrece una representación que reconstruye la evolución de los procesos como los ven las células, en lugar de como son observados desde el microscopio. Esta metodología por tanto permite nuevas formas de análisis y comparación de embriones y tejidos durante la embriogénesis a partir de imágenes in-vivo. ABSTRACT The embryogenesis is the process from which a single cell turns into a living organism. Through several stages of development, the cell population proliferates at the same time the embryo shapes and the organs develop gaining their functionality. This is possible through genetic, biochemical and mechanical factors that are involved in a complex interaction of processes organized in different levels and in different spatio-temporal scales. The embryogenesis, through this complexity, develops in a robust and reproducible way, but allowing variability that makes possible the diversity of living specimens. The advances in physics of microscopes and the appearance of fluorescent proteins that can be attached to expression chains, reporting about structural and functional elements of the cell, have enabled for the in-vivo observation of embryogenesis. The imaging process results in sequences of high spatio-temporal resolution 3D+time data of the embryogenesis as a digital representation of the embryos that can be further analyzed, provided new image processing and data analysis techniques are developed. One of the most relevant and challenging lines of research in the field is the quantification of the mechanical factors and processes involved in the shaping process of the embryo and their interactions with other embryogenesis factors such as genetics. Due to the complexity of the processes, studies have focused on specific problems and scales controlled in the experiments, posing and testing hypothesis to gain new biological insight. However, methodologies are often difficult to be exported to study other biological phenomena or specimens. This PhD Thesis is framed within this paradigm of research and tries to propose a systematic methodology to quantify the emergent deformation patterns from the motion estimated in in-vivo images of embryogenesis. Thanks to this strategy it would be possible to quantify not only local mechanisms, but to discover and characterize the scales of mechanical organization within the embryo. The framework focuses on the quantification of the motion kinematics (deformation and strains), neglecting the causes of the motion (forces), from images in a non-invasive way. Experimental and methodological challenges hamper the quantification of exerted forces and the mechanical properties of tissues. However, a descriptive framework of deformation patterns provides valuable insight about the organization and scales of the mechanical interactions, along the embryo development. Such a characterization would help to improve mechanical models and progressively understand the complexity of embryogenesis. This framework relies on a Lagrangian representation of the cell dynamics system based on the trajectories of points moving along the deformation. This approach of analysis enables the reconstruction of the mechanical patterning as experienced by the cells and tissues. Thus, we can build temporal profiles of deformation along stages of development, comprising both the instantaneous events and the cumulative deformation history. The application of this framework to 3D + time data of zebrafish embryogenesis allowed us to discover mechanical profiles that stabilized through time forming structures that organize in a scale comparable to the map of cell differentiation (fate map), and also suggesting correlation with genetic patterns. The framework was also applied to the analysis of the amnioserosa tissue in the drosophila’s dorsal closure, revealing that the oscillatory contraction triggered by the acto-myosin network organized complexly coupling different scales: local force generation foci, cellular morphology control mechanisms and tissue geometrical constraints. In summary, this PhD Thesis proposes a theoretical framework for the analysis of multi-scale cell dynamics that enables to quantify automatically mechanical patterns and also offers a new representation of the embryo dynamics as experienced by cells instead of how the microscope captures instantaneously the processes. Therefore, this framework enables for new strategies of quantitative analysis and comparison between embryos and tissues during embryogenesis from in-vivo images.

A resource range invariance rule for optimal offspring size predicts patterns of variability in parental phenotypes

Previous analysis of the rules regarding how much more a female should invest in a litter of size C rather than producing a litter with one more offspring revealed an invariance relationship between litter size and the range of resources per offspring in any litter size. The rule is that the range of resources per offspring should be inversely proportional to litter size. Here we present a modification of this rule that relates litter size to the total resources devoted to reproduction at that litter size. The result is that the range of resources devoted to reproduction should be the same for all litter sizes. When parental phenotypes covary linearly with resources devoted to reproduction, then those traits should also show equal ranges within each litter size category (except for litters of one). We tested this prediction by examining the range in body size (=total length) of female mosquito fish (Gambusia hubbsi) at different litter sizes. Because resources devoted to reproduction may take many forms (e.g., nest defense), this prediction may have broad applicability.

Analysis of the colorimetric properties of goniochromatic colors using the MacAdam limits under different light sources

Technological innovation in all areas has led to the appearance in recent years of new metallic and pearlescent materials, yet no exhaustive studies have been conducted to assess their colorimetric capabilities. The chromatic variability of these special-effect pigments may largely be due to the three-dimensional effect of their curved shapes and orientations when they are directionally or diffusely illuminated. Our study examines goniochromatic colors using the optimal colors (MacAdam limits) associated with normal colors (photometric scale of relative spectral reflectance from 0 to 1) under certain conventional illuminants and other light sources. From a database of 91 metallic and interference samples and using a multi-gonio-spectrophotometer, we analyzed samples with lightness values of more than 100 and others with lightness values of less than 100, but with higher chromaticities than optimal colors, which places them beyond the MacAdam limits. Our study thus demonstrates the existence of chromatic perceptions beyond the normal solid color associated with these materials and independent of the light source. The challenge for future research, therefore, is to replicate and render these color appearances in current and future color reproduction technologies for computer graphics.

Bulk sediment element analysis of sediment cores GeoB10065-9 and GeoB10065-7, offshore northwest Sumba Island, Indonesia

The Australian-Indonesian monsoon has a governing influence on the agricultural practices and livelihood in the highly populated islands of Indonesia. However, little is known about the factors that have influenced past monsoon activity in southern Indonesia. Here, we present a ~6000 years high-resolution record of Australian-Indonesian summer monsoon (AISM) rainfall variations based on bulk sediment element analysis in a sediment archive retrieved offshore northwest Sumba Island (Indonesia). The record suggests lower riverine detrital supply and hence weaker AISM rainfall between 6000 yr BP and ~3000 yr BP compared to the Late Holocene. We find a distinct shift in terrigenous sediment supply at around 2800 yr BP indicating a reorganization of the AISM from a drier Mid Holocene to a wetter Late Holocene in southern Indonesia. The abrupt increase in rainfall at around 2800 yr BP coincides with a grand solar minimum. An increase in southern Indonesian rainfall in response to a solar minimum is consistent with climate model simulations that provide a possible explanation of the underlying mechanism responsible for the monsoonal shift. We conclude that variations in solar activity play a significant role in monsoonal rainfall variability at multi-decadal and longer timescales. The combined effect of orbital and solar forcing explains important details in the temporal evolution of AISM rainfall during the last 6000 years. By contrast, we find neither evidence for volcanic forcing of AISM variability nor for a control by long-term variations in the El Niño-Southern Oscillation (ENSO).

Preliminary analysis of regional-precipitation periodicity /

"September 1980."

Analysis of the Hydrogeological Conditions and the Effects of Development on the Hydraulic Head at Redmond Ridge East

Redmond Ridge East (RRE) is a large-scale master plan community in East King County, WA. In this report, I evaluate the spatial variability of the Quaternary Advance Outwash (Qva) at RRE and the time-series data for 16 water wells with the intent to better understand groundwater below the RRE area. I investigate changes between pre- and post-development conditions through the determination of temporal changes in annual water level, annual water level fluctuations, hydraulic head response to precipitation, and ambient drainage of the aquifer. I also perform a basic analysis of the annual aquifer recharge and a determination for the storage through the implementation of the water table fluctuation (WTF) method. Associated Earth Sciences (AESI) was tasked with monitoring the geological and environmental impacts during the development of RRE and collected the data I use in this report. AESI involvement in monitoring began in 1998 and extends to the present. Sixteen wells were identified in the RRE area with adequate temporal data to conduct the analysis. A comparison of the well logs and aquifer testing data allowed local variations in the Qva to be mapped. The WTF was used to determine a range of reasonable specific yield values for locations where the Qva was unconfined. Yearly average of the seasonal water level high and lows, and the fluctuations were quantified. Temporal relationships were established through linear regression. The average water level was found to be increasing in some locations, and the corresponding fluctuations were found to decrease. However, no clear change between pre- and post-development was observed. The response of hydraulic head to precipitation was investigated through an analysis of hydrographs for ten wells. Periods of consistent response and the corresponding precipitation during each period were delineated. A linear relationship between precipitation and water level change was determined. The threshold precipitation under which there is a positive response in the hydraulic head was established. No observable changes were apparent between pre- and post-development conditions. The ambient drainage for the Qva was calculated using recessional periods on the hydrograph. The transmissivity of Qva varies with thickness of the overlying lodgment till and thickness of the Qva, itself. Water level fluctuations observed in the Qva are consistent with regional observations. Localized areas in the Qva display the large 10 foot fluctuations and these anomalies are likely due to a combination of the local variability in the storativity as well as the concentration and channeling of water due to geographical variations in the Qva and the overlying topography. All trends seen in the RRE area remained relatively constant through time. There was no evidence showing an effect of development on the hydraulic head at RRE. This implies that the style and distribution of infiltration has not changed as a result of development, and that any measures in place are properly mitigating the effects of development on the RRE region.

Causes of variability in light absorption by particles in snow at sites in Idaho and Utah.

This file accompanies “NAmer2014SnowBC_Dohertyetal_v1.xlsx”, which contains data on black carbon (BC) and other light-absorbing particles in snow in Utah and Idaho, for samples collected January-March 2014 in Jan/Feb 2013 and 2014 in Utah. Data are available as an Excel file with headers, or as a comma-separated data file, with no headers. There is one entry per layer of snow sampled. All entries (other than column titles in the .xlsx) are numeric. Detailed information on our measurements can be found in a series of publications, as given below.  Description of the instrument and method used to make the measurements: Grenfell, T. C., S. J. Doherty, A. D. Clarke, and S. G. Warren, Spectrophotometric determination of absorptive impurities in snow, Appl. Opt., 50(14), pp.2037-2048, 2011.  Summary and discussion of dataset “NAmer2014SnowBC_Dohertyetal.xlsx”, including maps of sample locations: Doherty, S. J., D. A. Hegg, P. K. Quinn, J. E. Johnson, J. P. Schwarz, C. Dang and S. G. Warren, Causes of variability in light absorption by particles in snow at sites in Idaho and Utah, J. Geophys. Res. Atmos., 121, doi:10.1002/2015JD024375, 2016. Note that the measurement and analysis techniques used to produce these data were also used in a broad Arctic survey (2006-2010) of BC and other light-absorbing particles snow, as reported here: Doherty, S. J., S. G. Warren, T. C. Grenfell, A. D. Clarke, and R. E. Brandt: Light-absorbing impurities in Arctic snow, Atmos. Chem. Phys., 10, 11647-11680, doi:10.5194/acp-10-11647-2010, 2010. http://www.atmos-chem-phys.net/10/11647/2010/acp-10-11647-2010.html

Population pharmacokinetic analysis of mycophenolic acid in renal transplant recipients following oral administration of mycophenolate mofetil

Aim To develop a population pharmacokinetic model for mycophenolic acid in adult kidney transplant recipients, quantifying average population pharmacokinetic parameter values, and between- and within-subject variability and to evaluate the influence of covariates on the pharmacokinetic variability. Methods Pharmacokinetic data for mycophenolic acid and covariate information were previously available from 22 patients who underwent kidney transplantation at the Princess Alexandra Hospital. All patients received mycophenolate mofetil 1 g orally twice daily. A total of 557 concentration-time points were available. Data were analysed using the first-order method in NONMEM (version 5 level 1.1) using the G77 FORTRAN compiler. Results The best base model was a two-compartment model with a lag time (apparent oral clearance was 271 h(-1), and apparent volume of the central compartment 981). There was visual evidence of complex absorption and time-dependent clearance processes, but they could not be successfully modelled in this study. Weight was investigated as a covariate, but no significant relationship was determined. Conclusions The complexity in determining the pharmacokinetics of mycophenolic acid is currently underestimated. More complex pharmacokinetic models, though not supported by the limited data collected for this study, may prove useful in the future. The large between-subject and between-occasion variability and the possibility of nonlinear processes associated with the pharmacokinetics of mycophenolic acid raise questions about the value of the use of therapeutic monitoring and limited sampling strategies.

Reflections on the statistical analysis of personality and norms in war, peace, and prejudice: Are deviant minorities the problem?

The compelling quality of the Global Change simulation study (Altemeyer, 2003), in which high RWA (right-wing authoritarianism)/high SDO (social dominance orientation) individuals produced poor outcomes for the planet, rests on the inference that the link between high RWA/SDO scores and disaster in the simulation can be generalized to real environmental and social situations. However, we argue that studies of the Person × Situation interaction are biased to overestimate the role of the individual variability. When variables are operationalized, strongly normative items are excluded because they are skewed and kurtotic. This occurs both in the measurement of predictor constructs, such as RWA, and in the outcome constructs, such as prejudice and war. Analyses of normal linear statistics highlight personality variables such as RWA, which produce variance, and overlook the role of norms, which produce invariance. Where both normative and personality forces are operating, as in intergroup contexts, the linear analysis generates statistics for the sample that disproportionately reflect the behavior of the deviant, antinormative minority and direct attention away from the baseline, normative position. The implications of these findings for the link between high RWA and disaster are discussed.

Kinematic analysis of jaw function in children following traumatic brain injury

Primary objective: To investigate jaw movements in children following traumatic brain injury (TBI) during speech using electromagnetic articulography (EMA). Methods and procedures: Jaw movements of two non-dysarthric children ( aged 12.75 and 13.08 years) who had sustained a TBI were recorded using the AG-100 EMA system (Carstens Medizineletronik) during word-initial consonant productions. Mean quantitative kinematic parameters and coefficient of variation ( variability) values were calculated and individually compared to the mean values obtained by a group of six control children ( mean age 12.57 years, SD 1.52). Main outcomes and results: The two children with TBI exhibited word-initial consonant jaw movement durations that were comparable to the control children, with sub-clinical reductions in speed being offset by reduced distances. Differences were observed between the two children in jaw kinematic variability, with one child exhibiting increased variability, while the other child demonstrated reduced or comparable variability compared to the control group. Conclusions: Possible sub-clinical impairments of jaw movement for speech were exhibited by two children who had sustained a TBI, providing insight into the consequences of TBI on speech motor control development.

Analysis of population pharmacokinetic data using NONMEM and WinBUGS

The aim of this report is to describe the use of WinBUGS for two datasets that arise from typical population pharmacokinetic studies. The first dataset relates to gentamicin concentration-time data that arose as part of routine clinical care of 55 neonates. The second dataset incorporated data from 96 patients receiving enoxaparin. Both datasets were originally analyzed by using NONMEM. In the first instance, although NONMEM provided reasonable estimates of the fixed effects parameters it was unable to provide satisfactory estimates of the between-subject variance. In the second instance, the use of NONMEM resulted in the development of a successful model, albeit with limited available information on the between-subject variability of the pharmacokinetic parameters. WinBUGS was used to develop a model for both of these datasets. Model comparison for the enoxaparin dataset was performed by using the posterior distribution of the log-likelihood and a posterior predictive check. The use of WinBUGS supported the same structural models tried in NONMEM. For the gentamicin dataset a one-compartment model with intravenous infusion was developed, and the population parameters including the full between-subject variance-covariance matrix were available. Analysis of the enoxaparin dataset supported a two compartment model as superior to the one-compartment model, based on the posterior predictive check. Again, the full between-subject variance-covariance matrix parameters were available. Fully Bayesian approaches using MCMC methods, via WinBUGS, can offer added value for analysis of population pharmacokinetic data.

Bayesian population pharmacokinetic analysis of sirolimus

Objective: It is usual that data collected from routine clinical care is sparse and unable to support the more complex pharmacokinetic (PK) models that may have been reported in previous rich data studies. Informative priors may be a pre-requisite for model development. The aim of this study was to estimate the population PK parameters of sirolimus using a fully Bayesian approach with informative priors. Methods: Informative priors including prior mean and precision of the prior mean were elicited from previous published studies using a meta-analytic technique. Precision of between-subject variability was determined by simulations from a Wishart distribution using MATLAB (version 6.5). Concentration-time data of sirolimus retrospectively collected from kidney transplant patients were analysed using WinBUGS (version 1.3). The candidate models were either one- or two-compartment with first order absorption and first order elimination. Model discrimination was based on computation of the posterior odds supporting the model. Results: A total of 315 concentration-time points were obtained from 25 patients. Most data were clustered at trough concentrations with range of 1.6 to 77 hours post-dose. Using informative priors, either a one- or two-compartment model could be used to describe the data. When a one-compartment model was applied, information was gained from the data for the value of apparent clearance (CL/F = 18.5 L/h), and apparent volume of distribution (V/F = 1406 L) but no information was gained about the absorption rate constant (ka). When a two-compartment model was fitted to the data, the data were informative about CL/F, apparent inter-compartmental clearance, and apparent volume of distribution of the peripheral compartment (13.2 L/h, 20.8 L/h, and 579 L, respectively). The posterior distribution of the volume distribution of central compartment and ka were the same as priors. The posterior odds for the two-compartment model was 8.1, indicating the data supported the two-compartment model. Conclusion: The use of informative priors supported the choice of a more complex and informative model that would otherwise have not been supported by the sparse data.

Probabilistic cost benefit analysis of generation investment in a deregulated electricity market

A new methodology is proposed for the analysis of generation capacity investment in a deregulated market environment. This methodology proposes to make the investment appraisal using a probabilistic framework. The probabilistic production simulation (PPC) algorithm is used to compute the expected energy generated, taking into account system load variations and plant forced outage rates, while the Monte Carlo approach has been applied to model the electricity price variability seen in a realistic network. The model is able to capture the price and hence the profitability uncertainties for generator companies. Seasonal variation in the electricity prices and the system demand are independently modeled. The method is validated on IEEE RTS system, augmented with realistic market and plant data, by using it to compare the financial viability of several generator investments applying either conventional or directly connected generator (powerformer) technologies. The significance of the results is assessed using several financial risk measures.

Latest developments in the microarray analysis of human brain mRNA

Microarrays are used to monitor the expression of thousands of gene transcripts. This technique requires high-quality RNA, which can be extracted from a variety sources, including autopsy brain tissue. Most nucleic acids and proteins are reasonably stable post mortem. However, their abundance and integrity can exhibit marked intraand inter-subject variability, so care must be taken when comparisons between case-groups are made. We will review issues associated with the sampling of RNA from autopsy brain tissue in relation to various ante- and post-mortem factors.