- ItemOpen AccessUsing finite state projection and Fisher information to improve single-cell experiment design to gain better understanding of DUSP1 transcription dynamics(Colorado State University. Libraries, 2023) Cook, Joshua A., author; Munsky, Brian, advisor; Chong, Edwin, committee member; Ghosh, Soham, committee memberMany recent studies have combined fluorescent biochemical labels, single-cell microscopy, and discrete stochastic modeling to understand and predict how organisms react to environmental changes to control gene expression. The experimental data used in these studies is often collected using intuitively-designed applications of techniques such as single-cell immunnocytochemistry (ICC) to measure protein expression and transport or single-molecule Fluorescence in situ Hybridization (smFISH) to measure the number and position of transcribed mRNA. Once collected, these single-cell data are then analyzed using discrete stochastic models, often based on the framework of the Chemical Master Equation (CME), which can be solved using the Finite State Projection (FSP) algorithm. Unfortunately, these experiments can be expensive and labor intensive to perform, primarily due to long imaging and image analysis times, and it is not clear how these experiments must be designed to obtain the most information when their results are later analyzed using the FSP techniques. The recently discovered Finite State Projection based Fisher information Matrix (FSP-FIM) provides a potential and practical solution to this experiment design challenge by providing direct estimates for how well any potential experiment should be expected to constrain parameters for a given model or set of models. In this report, we examine this challenge of experiment design in the situation where multiple different types of experiments (i.e., ICC and smFISH) are possible, for different time points, for different numbers of measurements per time point, for different environmental inputs, and for different assumed models and combinations of unknown parameters. We extend the previous FSP-FIM theory to address these multiple challenges, and we introduce new computational tools in the form of advances to the Stochastic System Identification Toolkit (in Mathworks Matlab) that allow users to easily and efficiently compute the FSP and FIM for each of these circumstances. Using experimental smFISH data, we demonstrate the effectiveness of the FSP tools to quantitatively reproduce the single-cell transcription dynamics of the Dual Specific Phosphatase 1 (DUSP1) gene under stimulation by Dexamethasone (Dex), and we show how the FSP-FIM can be used to design optimal combinations of ICC and smFISH to further improve quantification of this gene regulatory process, including predicting the optimal allocation of measurement times to obtain the most amount of information from each experiment. To probe the generality of our results, these FSP and FSP-FIM analyses are conducted for different models, under different assumptions on known and unknown parameters, and under different drug dosage regimens. The approach developed in this work is expected to have substantial impact on how computational models can be employed to improve the selection and design of future single-cell experiments.
- ItemOpen AccessStress, structure, and function of the embryonic heart(Colorado State University. Libraries, 2023) Gendernalik, Alex L., author; Bark, David, advisor; Garrity, Deborah, committee member; Puttlitz, Christian, committee member; Heyliger, Paul, committee memberEmbryonic heart development is a complex process that requires the coordination of hemodynamic stress and tissue morphogenesis. Improperly timed or distorted mechanical cues can cause reverberating malformations that result in congenital heart defects (CHDs) or embryo death. CHDs occur in ~1% of live births. Only ~20% have a known genetic origin. Altered mechanical signaling or hemodynamics is likely a common contributor to CHD prevalence. Significant research using animal models has shown that altered hemodynamics causes varied malformations. Mechanical properties describe the underlying tissue architecture, which dictates how the heart transmits and reacts to stress. This work aims to quantify and map the mechanical properties to understand how they direct the formation of specific heart structures and function. Furthermore, we seek to understand how mechanical properties change in response to altered hemodynamics. We hypothesize that mechanical properties indicate regions of eventual structure formation, are sensitive to altered hemodynamics, and dictate the pumping method that the heart uses to drive blood flow. We test this hypothesis through three aims. In aim 1, we describe a novel technique in which we use controlled pressurization to deform the embryonic zebrafish heart. We measure deformation in two-dimensions and identify constitutive models of the embryonic heart tissue. Finite element analysis is used to validate our findings in three dimensions. In this aim, we establish that controlled pressurization is a valid technique for inducing measured deformation of the heart. Through this, we determine that the zebrafish myocardial stiffness is on the order of 10 kPa. In aim 2, we further develop our pressurization technique by measuring deformation in three dimensions using confocal microscopy. Furthermore, we use a morpholino antisense oligonucleotide (MO), gata1, to alter embryonic zebrafish heart hemodynamics by blocking development of red blood cells, thus decreasing the viscosity and arterial pressure based on Poiseuille's law. Upon mapping strain in three dimensions, we find that strain throughout the heart is variable, with specific regions of low and high strain from 2 to 3 days post-fertilization (dpf). Low arterial pressure in gata1 MO embryos resulted in significantly increased strain compared to controls, indicating that altered hemodynamics cause altered mechanical properties in the developing embryonic heart. In aim 3, we seek to determine if the zebrafish early embryonic heart tube drives blood flow through peristalsis or impedance-type pumping. We attempt to directly induce impedance pumping by cannulating the atrial inlet of the heart tube after halting contractions and applying a controlled pressure pulse. Additionally, we use precisely controlled temperatures to increase heart rate, thus increasing arterial pressure. As temperature is increased, we use high speed imaging to analyze the contractile motion and resulting blood flow in the tube heart. Furthermore, we describe a previously unknown response whereby the traveling endocardial closure shortens with increased arterial pressure. In this aim, we fail to find evidence of impedance-type pumping but cannot preclude it contributes to blood flow. In summary, our pressurization technique can be used to map strain in the zebrafish embryonic heart; altering hemodynamics by reducing arterial pressure results in decreased stiffness of the embryonic heart myocardium, and endocardial closure length in the embryonic zebrafish heart tube shortens as arterial pressure and heart rate increase.
- ItemEmbargoNon-ionizing tomographic imaging modalities for bedside lung monitoring(Colorado State University. Libraries, 2023) Vieira Pigatto, Andre, author; Mueller, Jennifer L., advisor; Wilson, Jesse, committee member; Rezende, Marlis, committee member; Wang, Zhijie, committee memberThe need for an accurate and non-ionizing imaging modality for pulmonary assessment of patients undergoing mechanical ventilation due to respiratory failure has increased due to COVID. The ability to quickly detect the development of pathologies at an early stage is highly desirable and could help reduce the incidence of complications. It is also clear that mechanical ventilation can cause ventilator-induced lung injuries, which can be avoided by adequately optimizing the positive end-expiratory pressure to induce alveolar recruitment while preventing hyperinflation. Here, I will explore two non-ionizing pulmonary imaging systems that could be used as monitoring systems in the intensive care unit: Ultrasound Computed Tomography (USCT) and Electrical Impedance Tomography (EIT). The most comprehensive part of this research is the development of a Low-Frequency USCT system, which was motivated by recent studies demonstrating that acoustic waves transmitted at frequencies between 10 kHz and 750 kHz penetrate the lungs and may be useful for thoracic imaging. A novel transducer based on Tonpilz was designed, characterized, and calibrated through vibrational, electrical, and acoustic measurements, and a flexible belt that holds up to 32 transducers was constructed. A Verasonics Vantage 64 Low-frequency Research Ultrasound system was programmed to collect data by transmitting and receiving signals at frequencies of 125 and 156 kHz. The data collection and processing algorithms were developed in MATLAB, and the system was tested on phantom and vertebrate animal experiments; image reconstructions were conducted using a Time-Of-Flight algorithm. As a secondary study, SMA-1, COVID, and regular patients were imaged and analyzed using EIT technology; these results are shown through journal and conference articles presented in the Appendix A and C of this document.
- ItemEmbargoInvestigation of environmental factors on the intranuclear landscape of mesenchymal stromal cells(Colorado State University. Libraries, 2022) Kaonis, Samantha, author; Ghosh, Soham, advisor; Johnstone, Brian, committee member; Popat, Ketul, committee member; Dow, Steven, committee memberMesenchymal stromal cells (MSC), also known as mesenchymal stem cells, are popular candidates for tissue engineering and regenerative medicine. They can differentiate into many tissue types, and they can also help in regeneration through their trophic and immunomodulatory properties. Despite being investigated thoroughly for the last four decades and being under clinical trial in more than a thousand FDA approved studies, their application in clinics is very limited. One of the most important challenges in using MSC is that after harvesting from the patient, they need to be expanded to millions of cells for successful clinical outcomes. During this process, MSCs lose their differentiation potential, and trophic and immunomodulatory properties. In this thesis, I investigated the potential mechanisms of how environmental factors cause the MSC to divert from their phenotype during the expansion process. Subsequently, I intervened these mechanisms to achieve high quality MSCs without compromising the number of cells, i.e., their proliferation potential. Specifically, I investigated how two critical biophysical factors - mechanical stiffness and oxygen concentration of the MSC environment affects the cell phenotype and function through mechanisms involving epigenetic modifiers, transcription factors, and the chromatin architecture. First, the regulation of mechanics-induced population heterogeneity in MSCs was examined. Plastic culture and fibrotic conditions post-transplantation experienced by the MSC is completely different from the natural biomechanical niche of the MSC. Accordingly, the role of the mechanical environment has been shown to be a critical determinant of MSC gene expression and function. In this study, we report that human bone marrow-derived primary MSC population becomes phenotypically heterogenous when they experience an abnormal mechanical environment, compared to their native environment. Using a newly developed technique to quantify the heterogeneity, we provide evidence of phenotypical heterogeneity of MSC through high-resolution imaging and image analysis. Additionally, we provide mechanistic insight into the origin of such substrate mechanics-driven heterogeneity, which is further determined by the cell-cell mechanical communication through the substrate. In the second study, we investigated how the chromatin architecture and epigenetic landscape changes in MSCs by the substrate mechanical stiffness, thus causing a shift from the MSC phenotype. Using high-resolution confocal microscopy and advanced image analysis we identified the key epigenetic drivers in the mechanical stiffness mediated chromatin organization changes. Subsequently, we targeted several components of a proposed mechanobiological pathway to achieve MSCs with higher growth factor secretion without compromising their proliferation. The outcome of these studies might provide mechanism-driven design principles to the molecular, cellular and tissue engineering researchers for the rational design of MSC culture conditions and scaffolds, thus improving their functional outcome. Finally, the effect of oxygen concentration on MSC proliferation and performance wereexplored. Culture under physiological oxygen concentration (physioxia) can increase the proliferation of MSCs through a pathway initiated by the stabilization of the hypoxia-inducible factor-1 (HIF-1). Stabilized HIF-1α translocates into the nucleus, triggering the transcription of target genes conducive to MSC activity and proliferation. However, stabilized HIF-1α also triggers the p21 pathway causing cell cycle arrest, decreasing the MSC proliferation thereby limiting the beneficial effect of physioxia. Maintaining low oxygen conditions can be challenging, especially at a large scale, so rational exploitation and selective manipulation of such pathways through biochemical means has the potential to culture MSCs easily at scale. In this work, we created a mathematical model to predict optimal physioxic culture parameters to achieve the highest MSC proliferation. Through analysis of a gene downstream of the HIF-1 pathway, we also compared standard physioxic culture (2% O2) to treatment with deferoxamine mesylate (DFO), a physioxia-mimicking drug. The outcomes of this study might provide the rationale for MSC culture under standard hyperoxic conditions with only a simple addition of a combination of drugs to the culture medium to improve the scalability of MSC culture. Together, the results of the work will identify the mechanistic details of culture environment factors that play a role in determining the phenotype of MSCs during in vitro expansion process. The combination of these techniques to optimize MSC culture in vitro has the potential to resolve the current impediment to the clinical success of MSC therapies.
- ItemEmbargoUncovering details of the electrical properties of cells(Colorado State University. Libraries, 2022) Nejad, Jasmine E., author; Lear, Kevin L., advisor; Tobet, Stuart, committee member; McGrew, Ashley K., committee member; Simske, Steve, committee memberThe electrical properties of cells have long been studied by scientists across many fields, yet there are still major gaps in our understanding of the intrinsic properties of many types of cells, such as parasite eggs, as well as the detailed electrical behavior of excitable cells, such as neurons. This work aims to provide insights into how these properties can be measured and how machine learning can be used to advance our understanding of these phenomena. The first part of this work discusses the development of a microfluidic impedance cytometer for the enumeration and classification of parasite eggs isolated from fecal samples. Current diagnostics in parasitology rely on the manual counting of eggs, cysts, and oocysts on microscope slides that have been isolated from fecal samples. These methods depend on trained technicians with expertise in the preparation of samples and detection of parasites on these slides, which increases cost and turnaround times for diagnosis. This leads many farmers and ranchers to opt to pool fecal samples from multiple animals to save time and labor. In cattle herds, resistance is often due to underdosing, which can be caused by treating all animals to an average weight or treating by the calendar instead of targeted deworming. This blanket use of anthelmintics, or anti-parasitic medication, is leading to concerns about anthelmintic resistance, which would cause major issues in the livestock industry, as well create unforeseen ecological imbalances. The developed microfluidic system provides a proof-of-concept for a microfluidic impedance cytometer capable of measuring the impedance of parasite eggs at multiple frequencies, simultaneously, as each of the eggs passes through a microfluidic channel past a sensing region. This region consists of parallel electrodes on the top and bottom of the channel, allowing for measurement of the voltage across the channel. When an egg passes through, the signal is interrupted, leaving a distinct profile of the electrical properties at each frequency over time. This system shows proof-of-concept of the impedance measurements at 500kHz and 10MHz and provides insights for further exploration of these properties, with the eventual use of machine learning algorithms for discrimination of parasite eggs from debris, and differentiation of parasite genera. The second part of this work discusses machine learning classification of neuronal subtypes based on features extracted from patch-clamp recordings from adult mice, using data acquired from publicly available databases. Classification of neuronal subtypes has been a continuously progressing area of neuroscience, building on advancements in our understanding of the morphology, physiology, and biochemistry of different neurons, and contributing to the accuracy and repeatability of action potential and neuronal circuit models. This work explores the use of k-nearest neighbors, support vector machine, decision tree, logistic regression, and naïve Bayes algorithms for classification of fast-spiking or regular-spiking neurons from the hippocampus or the primary somatosensory cortex. K-nearest neighbors shows the most accurate classification of these groups, using action potential width, amplitude, and onset potential as features (inputs into the algorithm), with the addition of a measure of rapidity (acceleration near action potential onset) showing major increases in classification accuracy. Of the three methods for measuring rapidity, inverse of the full width at half of the maximum of the second derivative of the membrane potential (V̈m) (IFWd2), inverse of the half width at half of the maximum of V̈m (IHWd2), and the slope of the phase plot (V̇m vs. Vm) near AP onset (phase slope), including the phase slope measure of rapidity increased the accuracy to nearly perfect (weighted f1-score > 0.9999). In addition, the use of phase slope and action potential width as the only features for classification produces measures of accuracy, weighted f1-scores, of >0.9996. The results show the value of rapidity in action potential dynamics, the distinct difference between rapidity in APs generated by hippocampal neurons relative to cortical neurons, and low standard deviations for rapidity values in cortical neurons (fast- and regular-spiking). These findings have potential implications for understanding the ion channel dynamics in action potential initiation and propagation, which can improve the modeling of action potentials and neuronal circuits.