Mountain Scholar :: Browsing by Author "Prasad, Ashok, committee member"

Browsing by Author "Prasad, Ashok, committee member"

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Embargo
3D localization of cytoskeleton in mouse spermatids using stochastic optical reconstruction microscopy
(Colorado State University. Libraries, 2022) Sunny, Reshma, author; Krapf, Diego, advisor; Nikdast, Mahdi, committee member; Prasad, Ashok, committee member
It is estimated by the World Health Organization that globally 186 million individuals live with infertility. Studies have shown that cause of male infertility is unknown in 30 to 50% of the cases. Over the last several years teratozoospermias have been investigated and have been backtracked to events in spermatogenesis. The development of the acrosome and the manchette, protein and vesicle transport in spermatids, and sperm head shaping are crucial steps in the formation of healthy sperms. The cytoskeleton in spermatids plays a crucial role in shaping the sperm head. The acroplaxome exerts forces on the nucleus and gives the mammalian sperm head its species-specific shape, and also facilitates the proper attachment of the nuclear cap called the acrosome, containing the enzymes required for sperm penetration of the oocyte. The manchette should be intact and formed properly to have shortened diameter as spermatids differentiate so that it can constrict the base of the nucleus to shape the head, and also facilitate the transport of cargo to the base of the cell. Thus as studies have confirmed, the disruption in the organization of the cytoskeleton is a concern for infertility. Hence it is crucial to learn more about the cytoskeletal structures in spermatids. The goal of this thesis is to 3D localize these structures. The major structures we are interested in are the acroplaxome and the manchette. For this, we use a super-resolution microscopy method called Stochastic Optical Reconstruction Microscopy to image spermatid cytoskeleton. Our experiments confirmed the presence of α-tubulin in the manchette and that of F-actin in the manchette and the acroplaxome, as previously observed by researchers with 2D confocal images. We observed that the manchette reduces in diameter and progresses to the caudal portion of the cell at the later steps of differentiation and that the structure forms completely at step 10 and disassembles after step 14.
Open Access
A pharmacokinetic investigation of chloroquine analogues in cancer autophagy modulation
(Colorado State University. Libraries, 2020) Collins, Keagan P., author; Gustafson, Daniel, advisor; Prasad, Ashok, committee member; Yao, Tingting, committee member; Tham, Douglas, committee member; Thorburn, Andrew, committee member
Hydroxychloroquine (HCQ) is currently being investigated for safety and efficacy as an autophagy inhibitor in Phase I/II cancer clinical trials. It is the only clinically-approved autophagy inhibitor for use in cancer clinical trials in the United States. HCQ is used in combination with other chemotherapeutics to augment their efficacy and has shown moderate success in treating patients with late stage cancers. While HCQ has a good safety index and shows promise as an addition to standard of care treatment regimens, it suffers from several critical pharmacologic shortcomings which we take steps to address herein. The primary issues with usage of HCQ addressed in this work are the metrics used to predict patient tumor concentration of the drug following various dosing regimens. HCQ pharmacokinetics (PK) are highly variable in patients, with no correlation between traditional plasma:tumor concentrations. The first step taken to address this problem is to characterize likely sources of interindividual variability in HCQ PK. To do this a physiologically-based pharmacokinetic (PBPK) model was developed to investigate absorption, distribution, metabolism, and toxicity (ADMET) factors relating to HCQ in a mathematical system representative of the human body. This model was developed based on physiological and biochemical parameters relevant to HCQ ADMET in mice and scaled to represent humans. The model was capable of simulating single and multiple dosing regimens in humans, that would be characteristic of a cancer clinical trial. PBPK modeling addressed variability that would be associated with the macrophysiologic scale, but intrinsic and extrinsic factors on the cellular scale needed to be further defined to strengthen the understand of HCQ PK. To investigate factors that affect cellular uptake and sub-compartment localization of HCQ, a base PK model of lysosomotropic agents like HCQ was applied. Model specific parameters were identified for a panel of four human breast cancer cell lines, and a majority of differences in cellular uptake of the drug could be attributed to differences in the relative lysosomal volume fraction of each cell line. The model was able to characterize HCQ PK under different extracellular pH conditions, and identified a positive-feedback loop, related to transcription factor EB (TFEB) activation. This feedback loop caused the cell to increase its lysosomal volume over time of exposure to HCQ, resulting in a continuous increase of HCQ concentration within the cell. Through sensitivity analysis of the model, acidic extracellular pH was identified as a critical limiting factor of HCQ uptake into cells – which is particularly important as the tumor microenvironment is physiologically acidic. HCQ concentrations in cells cultured in an acidic microenvironment are decreased up to 10-fold, which cannot be overcome without the aid of agents that neutralize this pH. Dimeric analogues of HCQ, Lys05 and DC661, have been reported to maintain potency in acidic conditions and so were investigated in a comparative context to HCQ. Lys05 and DC661 were found to behave similarly to HCQ pharmacokinetically – i.e. highly dependent on the lysosomal profile of the cell. These drugs exhibited similar kinetic uptake curves as HCQ, and also induced the lysosomal biogenesis PK feedback loop. Unlike HCQ, Lys05 and DC661 uptake was not completely inhibited by acidic extracellular pH, and they were able to maintain activity under these conditions. PK of these drugs was characterized in a murine model to investigate their potential as in vivo agents, suggesting they could maintain high concentrations for a longer duration than HCQ. Lys05 and DC661 share many pharmacologic similarities to HCQ, while not sharing significant shortcomings such as inactivity under acidic extracellular conditions suggesting they should be investigated for further application as next generation autophagy inhibitors.
Open Access
Action potential initiation mechanisms: analysis and numerical study
(Colorado State University. Libraries, 2022) Aldohbeyb, Ahmed A., author; Lear, Kevin L., advisor; Vigh, Jozsef, committee member; Prasad, Ashok, committee member; Venayagamoorthy, Karan, committee member
Action potentials (AP) are the unitary elements of information processing in the nervous system. Understanding AP initiation mechanisms is a fundamental step in determining how neurons encode information. However, variation in neuronal response is a characteristic of mammalian neurons, which further complicate the analysis of neuronal firing dynamics. Several studies have associated the variation in AP onset with the type and densities of voltage-gated ion channels, diversity in synaptic inputs, neuron intrinsic properties, cooperative Na+ gating, or AP backpropagation. But the mechanisms that underlie the response variability remain unclear and subject to debate. Even though all these studies tried to answer the same question, the definition of AP onset and rapidity differs between them, highlighting the need for a more systematic and consistent method to quantify AP onset features, and hence analyzing the variation in AP onset. Two novel methods were developed to quantify AP rapidity. The proposed methods have lower relative variation, higher ability to classify neuron types, and higher sensitivity and specificity to voltage-gated Na+ channels parameters than current methods. AP rapidity was used to analyze different factors impacting the AP activation mechanism. However, the prior rapidity quantification methods are subjectively based on the researcher's judgment, which complicates the comparison between different studies. Thus, we proposed a more systematic and consistent method based on the full-width or half-width at half the rising phase peak of the membrane potential's second-time derivative (Vm). First, using an HH-type model, we showed that the peak width methods are sensitive to changes in the Na+ channel parameters and conductance and minimally impacted by changes in the K+ channel parameters compared to the phase slope, the standard quantification method. Second, we compared the peak width methods to the two prior methods, phase slope and error ratio, using recordings from cortical and hippocampal pyramidal neurons, hippocampal PVBCs, and FS cortical neurons found in online databases. The results showed that the new methods have the lowest variation between neurons within a specific type while significantly differentiating several neuron types. Together, the two studies showed that the peak width methods provide another sensitive tool to investigate the mechanisms impacting AP onset dynamics and provide a better tool to study Na+ channels kinetics and AP onset features. A conductance-based model that includes dynamics of ion concentration and cooperative Na+ channels was developed to investigate the mechanisms responsible for observed neuronal response variation. Random response variability has previously been observed in spike trains evoked from individual neurons by the same DC stimulus, but we observed systematic variation. The first APs' in a burst had attributes that were comparable regardless of the stimulus strength, while the subsequent APs' attributes monotonically change during bursts, and the magnitude of change increases with stimulus strength. These two spike train features were observed in three different neuron types (n = 51), indicating a shared mechanism is responsible for the spike train pattern. Various existing computational models fail to replicate the monotonic variation in AP attributes. We proposed incorporating ion concentration dynamics and cooperative gating to account for the missing behavior. A model with dynamic reversal potential but without cooperative Na+ channel gating reproduces the AP attribute's variation during bursts, but not the first APs' attributes. The first APs' attributes were reproduced only in the presence of a fraction of cooperative Na+ channels. Cooperative gating also enhanced the magnitude of modeled variation of some AP attributes to better match the electrophysiological recordings. Therefore, we conclude that changes in ion concentration dynamics could be responsible for the monotonic change in some AP's attributes during normal neuronal firing, and cooperative gating can enhance this effect. Thus, the two mechanisms contribute to the observed variability in neuronal response, especially the variation in AP rapidity.
Open Access
Advances in single-pixel imaging toward biological applications
(Colorado State University. Libraries, 2014) Winters, David G., author; Bartels, Randy, advisor; Marconi, Mario C., committee member; Prasad, Ashok, committee member; Bernstein, Elliot R., committee member
In this work, we discuss two new methods for single-pixel imaging. First, we leverage advances in laser metrology and frequency synthesis to measure small shifts in the center frequency of an optical pulse. Pulses acquire such shifts when probing a transient optical susceptibility, as in impulsive stimulated Raman scattering, which we use to demonstrate the technique. We analyze the limits of this technique with regard to fundamental noise, and predict detection sensitivity in these limiting cases. We then present work on imaging in two dimensions, both x-y and x-z, using single element detectors. We accomplish this by multiplexing spatial frequency projections in time, allowing rapid two dimensional imaging without an imaging detector. As we eliminate the imaging detector, the sensitivity to scattering is dramatically decreased, allowing the method to be used deep in scattering tissue. Results are shown for several geometries and experimental configurations, demonstrating imaging capabilities across a variety of sample types, including fluorescent and biological samples.
Embargo
Anomalous diffusion of mRNA in the cytoplasm of HeLa cells
(Colorado State University. Libraries, 2024) Roessler, Ryan, author; Krapf, Diego, advisor; Stasevich, Tim, committee member; Prasad, Ashok, committee member
Information about the diffusive motion of RNA would provide insights into intracellular structures and functions, as well as gene expression and genetic regulation. We study the motion of individual messenger RNA molecules in the cytoplasm of HeLa cells. RNAs are imaged in live cells via total internal reflection (TIRF) microscopy. In order to visualize individual RNA molecules expressing the MYH9 gene, they were labeled via MS2 stem loops bound to coat proteins tagged with the HaloTag-JF646 fluorophore. We then used single-particle tracking to obtain trajectories of individual molecules. Trajectories were analyzed in terms of their mean-squared displacement (MSD) and power spectral density (PSD). We observed non-ergodic, subdiffusive behavior, with statistics that depend on observation time, i.e., aging. Additionally, we observe stochastic switching between two mobility states with an order of magnitude difference in diffusivity. This switching process is responsible for the aging nature of the system. When compared to the cytoplasmic motion of synthetic nanoparticles, the analysis of RNA trajectories gives rise to discrepancies that raise questions about specific intracellular interactions.
Open Access
Atomic force microscopy: more than surface imaging
(Colorado State University. Libraries, 2020) Bishop, Terrance Tyler, author; Krapf, Diego, advisor; Prasad, Ashok, committee member; Van Orden, Alan, committee member
Atomic Force Microscopy (AFM) is a powerful imaging tool that has capabilities that go beyond the abilities of most other microscopes. Here, three examples of these capabilities were considered. First, the AFM was operated in an image generating mode to determine the surface heterogeneity of polysaccharide membranes. Second, the AFM was used to record force-indentation curves, these curves were fit with a Hertzian model to determine the stiffness of murine smooth muscle cells. Finally a approach for attaching 10 µm and 2 µm polystyrene beads to tip-less AFM cantilevers was proposed, and a viscoelastic contact model was tested to determine the viability of the created probes.
Open Access
Biomechanics of transapical mitral valve implantation
(Colorado State University. Libraries, 2014) Koenig, Evan Kienholz, author; Dasi, Lakshmi Prasad, advisor; Prasad, Ashok, committee member; Orton, Christopher, committee member
Heart disease is the number one killer in the United States. Within this sector, valve disease plays a very important role: Approximately 6% of the entire population has either prolapse or stenosis of the mitral valve and this percentage only increases when looking only at the elderly population. Transapical mitral valve implantation has promised to be a potential therapy for high-risk patients presenting with MR; however it is unclear what the best method of securing a valve within the mitral annulus may be to provide a safe and efficient valve replacement. The objective of this research is to study and understand the underlying biomechanics of fixation of transapical mitral valves within the native mitral annulus. Two different transapical mitral valve prosthesis designs were tested: One valve design has a portion of the leaflets atrialized such that it has a shorter stent height and the valve itself sits within the native annulus, the other design is not atrialized and protrudes further into the left ventricle. The valves were implanted in a left heart simulator to assess leaflet kinematics and hemodynamics using high speed imagery and particle image velocimetry techniques. An in vitro passive beating heart model was then used to assess the two different fixation methods (namely, anchored at the apex vs. anchored at the annulus) with respect to paravalvular regurgitation. Leaflet kinematics and hemodynamics revealed proper leaflet coaptation and acceptable pressure gradients and inflow fillings; however, both designs yielded elevated turbulence stresses within the ventricle. At 60 beats per minute, leaflet opening and closing times were both under 0.1 seconds, max Reynolds shear stresses were between 40 and 60 N/m2 and maximum velocities were approximately 1.4 m/s. Assessment of the different fixation methods during implantation revealed the superiority of the atrialized valve when anchored at the annulus (p<0.05), but showed no such comparison during tethered implantation. In addition to the results of statistical testing, observations show that the importance of the relationship between ventricular stent height and fixation method compared with native anatomy plays an important role in overall prosthesis function regardless of implantation method.
Open Access
Bridgeland stability of line bundles on smooth projective surfaces
(Colorado State University. Libraries, 2014) Miles, Eric W., author; Cavalieri, Renzo, advisor; Achter, Jeff, committee member; Peterson, Chris, committee member; Prasad, Ashok, committee member; Pries, Rachel, committee member
Bridgeland Stability Conditions can be thought of as tools for creating and varying moduli spaces parameterizing objects in the derived category of a variety X. Line bundles on the variety are fundamental objects in its derived category, and we characterize the Bridgeland stability of line bundles on certain surfaces. Evidence is provided for an analogous characterization in the general case. We find stability conditions for P1 × P1 which can be seen as giving the stability of representations of quivers, and we deduce projective structure on the Bridgeland moduli spaces in this situation. Finally, we prove a number of results on objects and a construction related to the quivers mentioned above.
Open Access
Combinatorial structures of hyperelliptic Hodge integrals
(Colorado State University. Libraries, 2021) Afandi, Adam, author; Cavalieri, Renzo, advisor; Shoemaker, Mark, advisor; Adams, Henry, committee member; Prasad, Ashok, committee member
This dissertation explores the combinatorial structures that underlie hyperelliptic Hodge integrals. In order to compute hyperelliptic Hodge integrals, we use Atiyah-Bott (torus) localization on a stack of stable maps to [P1/Z2] = P1 × BZ2. The dissertation culminates in two results: a closed-form expression for hyperelliptic Hodge integrals with one λ-class insertion, and a structure theorem (polynomiality) for Hodge integrals with an arbitrary number of λ-class insertions.
Open Access
Day and night for cyanobacteria: systems and synthetic biology approaches to understanding and engineering Synechocystis sp. PCC 6803 under day/night light cycles
(Colorado State University. Libraries, 2018) Werner, Allison Jean Zimont, author; Peebles, Christie A. M., advisor; Reardon, Kenneth, committee member; Prasad, Ashok, committee member; Heuberger, Adam, committee member
Photosynthetic organisms—including plants, algae, and cyanobacteria—harness sunlight as an energy source to grow, utilizing atmospheric carbon dioxide in the process. This ability can be harnessed for the sustainable production of food, fuels, and chemicals, reducing demand for petrol-based products and overall greenhouse gas emissions. Photosynthetic success rests on the efficient and timely capture of sunlight. Natural day/night cycles subject these organisms to changing energy availability, presenting a fundamental question: How do phototrophs regulate metabolism to thrive under daily and dramatic changes in energy supply? This question has significant impact on the productivity of plants, algae, and cyanobacteria. Cyanobacteria have been extensively engineered for the production of biofuels, polymers, and valuable pigments under continuous-light (CL) laboratory conditions. However, industrial production requires outdoor cultivation under diurnal light/dark (LD) cycles, where yield improvements in engineered strains observed in CL are lost in LD cycles. The success of industrially-productive cyanobacteria biotechnology is limited by the lack of appropriate strain engineering tools and gap in knowledge of photosynthetic metabolism under daily day/night light cycles. The aim of this thesis is therefore to improve the feasibility of cyanobacteria biotechnology in industrially-relevant conditions by integrating aspects of diurnal LD cycles into genetic tools and by expanding the current knowledge of dynamic photosynthetic metabolism. The first part of this thesis presents novel genetic engineering tools which enable light-entrained gene expression under diurnal LD cycles. The tools developed here enable engineering of temporally controlled chemical production under diurnal LD cycles, which we hypothesize will improve yield in outdoor cultivation environments. The second part of this thesis presents time-course characterization of growth and metabolite abundance under realistic diurnal LD cycles. Previous work was limited to on/off patterns of low light and restricted to detecting few metabolites. To expand the realism of light profiles and metabolite scope, a photobioreactor was engineered to supply sinusoidal patterns and intensity of light (sinLD cycles), and a multi-platform mass spectrometry workflow was developed to enable semi-comprehensive metabolite detection. Cyanobacteria growth under realistic diurnal sinLD cycles is presented for the first time, to our knowledge. We observe a short lag phase at the onset of day, followed by cell mass increase during the early day, cell division during afternoon and evening, and slight mass loss overnight. Further, comprehensive metabolite abundance every 30-120 minutes across a 24-hour diurnal sinLD cycle is presented. Insoluble C6 carbohydrates displayed sharp oscillations at the day/night transition; insoluble C5 carbohydrates and glucosamine display these in addition to abundance 're-sets' at the night/day transition. Free amino acids and nucleic acids increase immediately upon transition to light during the lag phase, followed by gradual incorporation into protein during the mass accumulation phase. Metabolites involved in central metabolism did not oscillate to the same extent as other pathways. Accumulation of phosphoenolpyruvate but not pyruvate during the light phase suggests a potential bottleneck. Integration of the metabolomics data into genome-scale metabolic models to perform dynamic flux balance analysis could improve the method by which engineering targets are identified for production in outdoor conditions. Together, this thesis demonstrates the need for revision of the current approach to cyanobacteria strain engineering. More broadly, this work highlights the dynamic nature of photosynthetic metabolism and motivates future investigations into metabolic regulation and metabolic flux under realistic day/night cycles.
Open Access
Design and fabrication of a flow chamber for the study of cell adhesion and hemocompatibilty in dynamic conditions
(Colorado State University. Libraries, 2011) Migita, Kevin, author; Popat, Ketul, advisor; Dasi, Lakshmi Prasad, advisor; Prasad, Ashok, committee member
Cell adhesion is a well characterized condition of both biomaterial and tissue engineering research. It plays a role in biocompatibility and the proliferation, differentiation and viability of seeded cells. With respect to hemocompatibility, platelet adhesion and subsequent activation is a driving factor in the failure of blood contacting medical devices. Platelets aggregates are vital components in the wound healing and foreign body responses and display various forms of adhesion based on blood flow. However, the study of platelet adhesion on implantable tissue engineering scaffolds under dynamic conditions is very limited, particularly with directional flow. A flow chamber which incorporates a tissue engineering scaffold or functionalized biomaterial was designed and fabricated for investigation of flow patterns and cellular adhesion in response to dynamic conditions on these surfaces. The device utilizes a combination of aspects from both tissue engineering bioreactors and microfluidics platforms to result in a flow chamber which provides the directional flow of a perfused flow bioreactor with the advantages of controlling chamber shape and real time monitoring presented by Polydimethylsiloxane microfluidics chambers. Results of fluid flow study in the chamber modeled for laminar and shear gradient simulated flow show the ability of the device to manipulate flow patterns. Dynamic and static studies of platelet adhesion to poly-(ε-caprolactone) flat and electrospun nanofiber surfaces utilizing the flow chamber provide insight into the hemocompatibility of tissue engineering scaffolds in a dynamic flow setting.
Open Access
Development of genetic parts for improved control of translation initiation in Synechocystis sp. PCC 6803 with an application in biofuel production
(Colorado State University. Libraries, 2021) Sebesta, Jacob, author; Peebles, Christie A. M., advisor; Peers, Graham, committee member; Prasad, Ashok, committee member; Reardon, Kenneth, committee member
Metabolic engineering is developing into a field that can change the way we produce a wide variety of valuable chemicals. Many chemicals are already produced in microbial cultures. Metabolic engineering enables us to modify organisms to produce metabolites they don't usually produce, assuming an enzyme can be identified in another organism that catalyzes the formation of that product (or an enzyme can be designed for that task through protein engineering). The distribution of accumulated metabolites can also be altered. There are some cases where metabolites can be accumulated through cultivation practices. Methods of metabolic engineering to overexpress, knockdown, or knockout native enzymes provide additional tools to alter cellular metabolism and drive accumulation of those products. Precise control over gene expression is central to these efforts. To avoid competition with human food crops and the resources need to produce them, cyanobacteria may be utilized for production of valuable chemicals. Through photosynthesis, they can utilize carbon dioxide from geological formations or from industrial waste streams. Since most metabolic engineering has been developed in E. coli and yeast, it was necessary to first adapt the basic methods for use in cyanobacteria. Along with my co-authors Dr. Allison Werner and Dr. Christie Peebles, we reviewed methods for producing genetically modified Synechocystis Sp. PCC6803 (S. 6803). To facilitate the generation of strains with many modifications, we covered the method developed in the Peebles Lab for making markerless selections which remove any antibiotic selection markers. A previous graduate student in the Peebles lab, Stevan Albers, found that strong promoter-ribosome binding site combinations that drove high expression of GFP did not necessarily result in high expression when used to drive expression of a different gene. Therefore, in our work to produce bisabolene in S. 6803 we tested many ribosome binding sites. In addition, we tested five different codon optimizations of the bisabolene synthase to ensure that expression was not prevented by slow translation elongation. We found that the simple measure of the codon adaptation index (CAI) correlated with expression of the five different codon optimizations. Using a thermodynamic model of translation initiation, we designed ten ribosome binding sites to increase bisabolene synthase expression by 10-fold. Only one of those designs actually approached a 10-fold increase, highlighting the need to continue testing several ribosome binding sites to achieve a desired expression level. Since industrial cultivation of cyanobacteria occurs outdoors, subject to natural light:dark cycles, we tested two of the designed strains in light:dark cycles. The strains reached similar bisabolene titers after being exposed to the same amount of total light period as those previously tested in continuous light. Overall, this work increased the highest bisabolene titer reported in cyanobacteria by approximately 10-fold. The need to test many ribosome binding sites limits progress in cyanobacterial metabolic engineering. The research of others suggest that ribosome binding sites interact with coding sequences by forming secondary structures with different free energy of folding. The estimation of the free energy of folding may be inaccurate, and, further, the kinetics of such folding may also be important to translation initiation rates. We tested two different designs to limit the impacts that secondary structures that span either side of the start codon may have on translation initiation rates in both E. coli and S. 6803. Utilization of a 21-nucleotide leader sequence after the start codon to make the sequence context consistent for ribosome binding sites between different coding sequences did not improve the correlation found between the expression of two different reporter genes in either organism. Bicistronic designs use translational coupling between an upstream open reading frame and the gene of interest with a ribosome binding site contained within the upstream open reading frame to re-initiate translation. This design exploits the helicase activity of ribosomes in elongation mode to actively unfold the secondary structure around the start codon of the gene of interest. We expected this activity to reduce the impacts of secondary structure and improve the correlation in expression between two different reporter genes. Intriguingly, the correlation was much improved in E. coli, but not in S. 6803. Together, this dissertation suggests that there are important differences in translation initiation between E. coli and S. 6803. Improved ribosome binding site design for cyanobacteria would facilitate further increases in terpenoid production both by enabling higher expression of heterologous terpenoid synthases and by reducing the number of strains that must be tested to achieve the desired expression level for each enzyme. Future directions suggested by this work include studies of translation initiation mechanisms in cyanobacteria, development of cell-free expression systems to facilitate rapid testing of many different genetic constructs, and further efforts at pathway engineering to increase terpenoid titer and productivity in cyanobacteria.
Open Access
Dynein mutagenesis reveals the molecular basis for dynein regulation in broad spectrum neurological diseases
(Colorado State University. Libraries, 2020) Marzo, Matthew G., author; Markus, Steven M., advisor; Bamburg, James R., committee member; DeLuca, Jennifer, committee member; Prasad, Ashok, committee member
Eukaryotic cells rely on cytoskeletal networks to organize materials, transport organelles, give cells shape, and provide locomotion. The cytoskeleton is comprised of many diverse proteins, and three classes of polymeric protein structures are the actin, microtubule, and intermediate filament networks. The microtubule network, and its associated motors, dynein and kinesin, is of interest to the field of neurological disease, due to the prevalence of mutations in the microtubule network in human disease. To better understand the molecular basis for the diseases caused by de novo dynein mutations, we performed a screen of mutants using budding yeast dynein. The results from our experiments present a platform for the molecular dissection of dynein mutations which can be readily applied to new mutations or precisely explore known mutations. The screen-based approach allowed us to identify a new mechanism of yeast dynein regulation, which is autoinhibition of the dynein motor. We demonstrate that this mechanism regulates dynein activity in cells and functions to limit in vivo motor activity in the cytoplasm. Autoinhibition is regulated by Pac1 in yeast, a Lissencephaly-1 homolog, and we demonstrate that Pac1 operates in the dynein autoinhibition pathway by preventing the "closed" autoinhibited state, thereby promoting "open" dynein. This represents an entirely novel function of Pac1/LIS1, and allows us to further refine our model for cortical offloading.
Open Access
Effects of inlet/outlet locations and influent temperature on hydraulic disinfection efficiency in contact tanks
(Colorado State University. Libraries, 2017) Zhang, Yishu, author; Venayagamoorthy, S. Karan, advisor; Ramirez, Jorge A., committee member; Prasad, Ashok, committee member
This study focuses on understanding the effect of inlet/outlet locations and influent temperature on hydraulic disinfection efficiency of drinking water contact tanks for small systems. Computational fluid dynamics (CFD) simulations of flow and scalar transport in a concrete rectangular tank with three inlet/outlet location configurations were performed. The temperature of the influent into the system was varied in the second part of this study in order explore the effects of temperature gradients on the flow and scalar transport. Hydraulic disinfection efficiencies were computed through the use of residence time distribution (RTD) curves obtained from the CFD simulations and the baffling factor (BF). The physical tank that was used for all tracer tests is located at the Hydraulics Lab at Colorado State University's Engineering Research Center (ERC) in Fort Collins. The rectangular concrete tank was initially constructed with a bottom inlet and top outlet configuration and has a total volume of 1500 gallons. After the CFD simulation results were validated using tracer tests, two principle objectives were investigated using CFD simulations. First, the effect of inlet/outlet locations and their respective sizes were investigated. For a given constant temperature for both the inflow and ambient water in the tank, three inlet/outlet location combinations (i.e. bottom inlet-bottom outlet, bottom inlet-top outlet, and top inlet-bottom outlet) with two different outlet sizes (i.e. 2-in.-diameter and 4-in.-diameter) were modeled using 15 CFD simulations. Both baffled and un-baffled tanks were modeled. The resultsshow that a small modification of the outlet pipe diameter results in minor changes in the baffling factor and hydraulic disinfection efficiency. All adjusted un-baffled tanks (i.e. with the three different inlet/outlet configurations) did not yield any satisfactory disinfection performance due to the severe short circuiting that occurs in the tank. The main finding is that for baffled tanks, the top inlet-bottom outlet configuration performed the best and increased baffling factor by over 30% relative to the bottom inlet-bottom outlet configuration for the baffled tank which is commonly found in praxis. Second, the effect of buoyancy that can occur in disinfection tanks due to drastic temperature differences between the inflow and the ambient water in the contact tank was investigated. Only negatively buoyant conditions were studied in this research. Temperature differences of 0°C, 5°C, 10°C, and 15°C were created by injecting cold inflow to the baffled tanks under two conditions namely: (i) no heat flux condition and (ii) constant wall condition. For the first condition, it was assumed that no heat exchange between tank (and baffle) walls and fluid occurs; while for the second condition, the wall temperature was held constant at 20°C. Both conditions were simulated at different flow rates to capture flow regimes ranging from laminar to turbulent. It was found that the baffling factor varied significantly between laminar, transitional, and turbulent flows. The best hydraulic disinfection efficiency was achieved when the flow was laminar. For no heat flux condition, the effects of the buoyancy increased baffling factor by 57% compared to the base case with no temperature difference. On the other hand, for turbulent flow conditions with a strong temperature difference, the baffling factor reduced by 49% compared to the base case. The constant wall temperature condition produced similar results, but with a smaller change in baffling factor. From a hydrodynamic analysis of the flow fields obtained from CFD simulations, it was concluded that buoyancy could either increase hydraulic disinfection efficiency or decrease it, depending on the flow regime. Hence, care should be exercised to avoid flows in transitional to turbulent regimes because the negative buoyancy could decrease the baffling factor and lead to inadequate microbial deactivation.
Open Access
Electromechanical and curvature-driven molecular flows for lipid membranes
(Colorado State University. Libraries, 2015) Mikucki, Michael, author; Zhou, Yongcheng, advisor; Tavener, Simon, committee member; Liu, Jiangguo, committee member; Prasad, Ashok, committee member
Lipid membranes play a crucial role in sustaining life, appearing ubiquitously in biology. Gaining a quantitative understanding of the flows of lipid membranes is critical to understanding how living systems operate. Additionally, the mechanical properties of lipid membranes make them ideal material for nanotechnology, further motivating a need for accurate computational models. This thesis is organized in three projects that model important features of lipid membranes. First, we define the mechanical energy of vesicle lipid membranes and propose a fast numerical algorithm for minimizing this energy. The mechanical energy is well known, and existing computational techniques for minimizing this energy include solving the Euler-Lagrange equations for axisymmetric shapes or approximating the minimization problem by minimizing over a subspace of membrane configurations. We choose the latter approach, making no restrictive symmetry assumptions. Specifically, we use surface harmonic functions to parameterize the membrane surface, drastically reducing the degrees of freedom compared to similar existing approaches. Numerical equilibrium shapes are presented, including conformations exhibited by red blood cells. The numerical results are verified against analytical values of axisymmetric shapes. Second, we develop the electrostatic potential energy for lipid bilayer membranes in the context of lipid-protein interactions. We extend the electrostatic potential energy of a protein-solvent system to include charged lipids in a protein-membrane-solvent system. Here, we model the bilayer membrane as a continuum with general continuous distributions of lipids charges on membrane surfaces. Key geometrical properties of the membrane surfaces under a smooth velocity field allow us to apply the Hadamard-Zolésio structure theorem of shape calculus, and we compute the electrostatic force on membrane surfaces as the shape derivative of the electrostatic energy functional. Third and finally, we develop the mathematical theory and the computational tools for curvature-driven flow of proteins within lipid membranes. Recently, much attention has been devoted to understanding curvature generating and curvature sensing properties of proteins in vesicle membranes. That is, certain proteins prefer regions of specific curvature and naturally flow to these regions. We develop the mathematical theory for curvature-driven diffusion along these membranes, which involves a variable diffusion coefficient. Finite element and finite difference methods have been used to solve diffusion equations on surfaces, but these methods require costly spatial resolution and adaptive mesh refinements for dynamic membrane surfaces. Instead, we use a phase field model with Fourier spectral methods so that no explicit tracking of the surface is required. Furthermore, the spectral accuracy allows for uniform mesh with no refinement near the boundary. The numerical solution of the diffusion equation and the numerical solution of the membrane shape equation is performed in a consistent framework to allow for the coupling of membrane shape with the curvature-driven surface diffusion. Results which capture the curvature preference are presented.
Embargo
Engineering in practice: from quantitative biology modeling to engineering education
(Colorado State University. Libraries, 2024) Weber, Lisa, author; Munsky, Brian, advisor; Atadero, Rebecca, committee member; Prasad, Ashok, committee member; Reisfeld, Brad, committee member
In quantitative analyses of biological processes, one may use many different scales of models (e.g., spatial or non-spatial, deterministic or stochastic, time-varying or at steady-state) or many different approaches to match models to experimental data (e.g., model fitting or parameter uncertainty/ sloppiness quantification with different experiment designs). These different analyses can lead to surprisingly different results, even when applied to the same data and the same model. In Chapters 2, a variety of modeling approaches that can be utilized in analyzing biological processes are explained, with examples included of how to mathematically represent a system in order to use these various modeling approaches. Many of these mechanistic modeling approaches are demonstrated in Chapter 3 when we use a simplified gene regulation model to illustrate many of the concerns regarding modeling approach differences; these include ODE analyses of deterministic processes, chemical master equation and finite state projection analyses of heterogeneous processes, and stochastic simulations. For each analysis, we consider a time-dependent input signal (e.g., a kinase nuclear translocation) and several model hypotheses, along with simulated single cell data, to illustrate different approaches (e.g., deterministic and stochastic) in the identification of mechanisms and parameters of the same model from the same simulated data. We also explore how uncertainty in parameter space varies with respect to the chosen analysis approach or specific experiment design, and conclude with a discussion of how our simulated results relate to the integration of experimental and computational investigations to explore signal-activated gene expression models in yeast [1] and human cells [2]. Different modeling approaches are used in Chapter 4 to build on the work of Scott, et al. (2018, 2019) [3, 4] to evaluate different model classes for DNA structural conformation changes, including the unwinding/rewinding dynamics of the double-stranded DNA (dsDNA) helical structure and subsequent binding interactions with complementary single-stranded oligonucleotides probes (oligos), in relation to different conditions: temperature, salt concentration, and the level of supercoiling of the DNA molecule. This is done to identify a class of models that best fit the DNA unwinding and subsequent oligo probe binding experimental data as a function of these three conditions. In this work, we demonstrate the use of additional quantitative modeling approaches, including a modified genetic algorithm along with the process of cross validation and Markov Chain Monte Carlo (MCMC) simulations with the Metropolis-Hastings (MH) algorithm [5] to explore parameter space. We also demonstrate many of the challenges that can be encountered when modeling complex biological phenomena with actual experimental data. Although much of the work described in Chapters 2 through 4 may appear to be, on the surface, just the use of various computational methods for biological processes to increase understanding of biological mechanisms, much of it also has a separate purpose. The structure of these works and an underlying aim of much of this work, namely Chapters 2 and 3, is to provide guidance with examples to make these computational approaches more accessible to scientists and engineers. Many of these approaches are included in a quantitative biology (UQ-bio) summer school that has been conducted for the last few years as well. Through the process of developing these works and seeking to make quantitative biology more accessible, a related goal manifested to improve the accessibility of engineering education as a whole, which is addressed in Chapter 5, specifically related to diversity, equity, and inclusion (DEI) in undergraduate engineering education. There have been efforts since Fall 2017 to increase the presence of DEI in the undergraduate CBE education using a bottom up approach. To date, various efforts have been incorporated into the first two years of the CBE program. In Chapter 5, these previous efforts, along with lessons learned, are detailed. A substantial, holistic approach to incorporating DEI throughout the CBE curriculum is proposed, based on a review of recent work by other engineering education researchers, to help the CBE department create a more inclusive educational experience for undergraduate students and better enable students to handle the complex challenges they may face in their careers.
Open Access
Experimental and computational analysis of Caenorhabditis elegans small RNAs
(Colorado State University. Libraries, 2019) Brown, Kristen, author; Montgomery, Tai, advisor; Duval, Dawn, committee member; Prasad, Ashok, committee member; Hess, Ann, committee member
Caenorhabditis elegans contains twenty-five Argonautes, of which, only ALG-1 and ALG-2 are known to interact with microRNAs (miRNAs). ALG-5 belongs to the AGO subfamily of Argonautes that includes ALG-1 and ALG-2, but its role in small RNA pathways is unknown. We analyzed by high-throughput sequencing the small RNAs associated with ALG-5, ALG-1, and ALG-2, as well as changes in mRNA expression in alg-5, alg-1, and alg-2 mutants. We show that ALG-5 defines a distinct branch of the miRNA pathway affecting the expression of genes involved in immunity, defense, and development. In contrast to ALG-1 and ALG-2, which associate with the majority of miRNAs and have general roles throughout development, ALG-5 interacts with only a small subset of miRNAs and is specifically expressed in the germline. alg-5 is required for optimal fertility and mutations in alg-5 lead to a precocious transition from spermatogenesis to oogenesis. Our results provide a near-comprehensive analysis of miRNA-Argonaute interactions in C. elegans and reveal a new role for miRNAs in the germline. The small RNA field has grown rapidly since miRNAs were discovered to be conserved regulators of developmental timing. This growth occurred during a time when high-throughput transcriptomic data from microarrays and next-generation sequencing became widely accessible. As a result, research projects dissecting small RNA pathways often produce sequencing data that can be complex and difficult to perform appropriate data analysis for without specialized or advanced computational knowledge. Many researchers end up only study a subset of small RNAs, outsourcing their analysis, or piecing together a pipeline using tools developed for mRNA sequencing. We aim to reduce this barrier to entry in the field and improve reproducibility by creating an open-source, user-friendly data processing pipeline for small RNA sequencing. To create a simple, reproducible pipeline, we utilized the Common Workflow Language (CWL) and Python, while otherwise minimizing dependencies. The pipeline reads a configuration file and sample sheets that can be easily modified by a user to run the complete analysis from raw fastq file to summary statistics and publication-ready plots. We present AQuATx (automated quantitative analysis of transcript expression) for small RNAs and the analysis of C. elegans germline tissue as an example data set. Our software will allow bench scientists with little to no computational knowledge to easily analyze their small RNA sequencing data. Overall, the final software will be a valuable tool for anyone interested in studying small RNAs.
Open Access
GPU-accelerated computational study of block copolymer self-assembly with advanced polymer theories
(Colorado State University. Libraries, 2024) He, Juntong, author; Wang, Qiang, advisor; Prasad, Ashok, committee member; Bailey, Travis, committee member; Gelfand, Martin, committee member
A high-performance GPU-accelerated software package for self-consistent field (SCF) calculations of block copolymer assembly, PSCF+, has been developed. PSCF+ allows various combinations of chain-connectivity models (including the continuous Gaussian chains, discrete Gaussian chains, and freely jointed chains), non-bonded isotropic pair (including the Dirac δ-function, soft-sphere, dissipative particle dynamics, and Gaussian) potentials and system compressibility (incompressible vs. compressible). The Richardson-extrapolated pseudo-spectral methods, the crystallographic fast Fourier transform, the "slice" algorithm, and the automated calculation-along-a-path are implemented in PSCF+, which not only speed up the SCF calculations and reduce the GPU memory usage significantly, but also make it very efficient in constructing phase diagrams. Given the wide use and great success of SCF calculations in understanding and predicting the self-assembled structures of block copolymer, PSCF+ will be an invaluable computational tool for the polymer community. Using PSCF+, we studied the stability of various Frank-Kasper phases formed by neat diblock copolymer (DBC) A-B melts using the "standard" model and the dissipative particle dynamics chain model and found that in general the SCF phase diagrams of these two models are qualitatively the same but with important differences. We also studied the stability of various Frank-Kasper phases formed by binary DBC blends using the "standard" model and found that the relative stability among the Frank-Kasper phases is dominated by their internal-energy densities. Finally, we performed high-accuracy SCF calculations to study the stability of all known tiling patterns formed by symmetrically interacting ABC miktoarm star triblock terpolymers.
Open Access
Integrating discrete stochastic models with single-cell and single-molecule experiments
(Colorado State University. Libraries, 2019) Fox, Zachary R., author; Munsky, Brian, advisor; Stargell, Laurie, committee member; Wilson, Jesse, committee member; Prasad, Ashok, committee member
Modern biological experiments can capture the behaviors of single biomolecules within single cells. Much like Robert Brown looking at pollen grains in water, experimentalists have noticed that individual cells that are genetically identical behave seemingly randomly in the way they carry out their most basic functions. The field of stochastic single-cell biology has been focused developing mathematical and computational tools to understand how cells try to buffer or even make use of such fluctuations, and the technologies to measure such fluctuations has vastly improved in recent years. This dissertation is focused on developing new methods to analyze modern single-cell and single-molecule biological data with discrete stochastic models of the underlying processes, such as stochastic gene expression and single-mRNA translation. The methods developed here emphasize a strong link between model and experiment to help understand, design, and eventually control biological systems at the single-cell level.
Open Access
Intersections of ψ classes on Hassett spaces of rational curves
(Colorado State University. Libraries, 2018) Sharma, Nand, author; Cavalieri, Renzo, advisor; Peterson, Chris, committee member; Achter, Jeff, committee member; Prasad, Ashok, committee member
Hassett spaces are moduli spaces of weighted stable pointed curves. In this work, we consider such spaces of curves of genus 0 with weights all 1/q , q being a positive integer greater than or equal to 2. These spaces are interesting as they have different universal families and different intersection theory when compared with classical moduli spaces of pointed stable rational curves. We develop closed formulas for intersections of ψ-classes on such spaces. In our main result, we encode the formula for top intersections in a generating function obtained by applying an exponential differential operator to the Witten-potential.