Mountain Scholar :: Browsing by Author "Krapf, Diego, committee member"

Browsing by Author "Krapf, Diego, committee member"

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Open Access
Biochemical, biophysical and functional characterization of histone chaperones
(Colorado State University. Libraries, 2014) Zhang, Ling, author; Luger, Karolin, advisor; Krapf, Diego, committee member; Nyborg, Jennifer, committee member; van Orden, Alan, committee member; Stargell, Laurie, committee member
Nucleosomes, the basic repeating unit of chromatin, are highly dynamic. Nucleosome dynamics allow for various cellular activities such as replication, recombination, transcription and DNA repair, while maintaining a high degree of DNA compaction. Each nucleosome is composed of 147 bp DNA wrapping around a histone octamer. Histone chaperones interact with histones and regulate nucleosome assembly and disassembly in the absence of ATP. To understand how nucleosome dynamics are regulated, it is essential to characterize the functions of histone chaperones. The first project of my doctoral research focused on the comparison of different nucleosome assembly proteins employing various biochemical and molecular approaches. Nucleosome assembly proteins (Nap) are a large family of histone chaperones, including Nap1 and Vps75 in Saccharomyces cerevisiae, and Nap1 (also Nap1L1), Nap1L2-6 (Nap1-like 2-6, with Nap1L4 being Nap2) and Set in metazoans. The functional differences of nucleosome assembly proteins are thus interesting to explore. We show that Nap1, Nap2 and Set bind to histones with similar and high affinities, but Nap2 and Set do not disassemble non-nucleosomal DNA-histone complexes as efficiently as Nap1. Also, nucleosome assembly proteins do not display discrepancies for histone variants or different DNA sequences. In the second project, we identified Spn1 as a novel histone chaperone and look into new functions of Spn1 on the regulation of chromatin structural states. Spn1 was identified as a transcription regulator that regulates post-recruitment of RNA polymerase II in yeast. We demonstrated that Spn1 is a H3/H4 histone chaperone, a novel finding that was not observed previously. Spn1 also interacts with Nap1, and forms ternary complexes with Nap1 and histones. We also show that Spn1 has chromatin assembly activity and N- and C- terminal domains of Spn1 are required for its histone chaperone properties. At the same time, we had an interesting observation that Spn1 potentially has topoisomerase/nuclease activity, which is dependent on magnesium ions. This activity of Spn1 can also help answer questions raised by in vivo assays related to Spn1, including its correlation with telomere length, the heat sensitivity in the reduction of function yeast strains, and the elongated lifespan in the Spn1ΔNΔC strain. Our studies on the functional comparison of nucleosome assembly proteins revealed their distinct roles in the regulation of nucleosome dynamics. Our findings on the histone chaperone functions and nuclease/topoisomerase activities disclosed new roles of Spn1 in chromatin regulation, by regulating histone-DNA interaction and also maintenance of DNA integrity.
Open Access
Bulk and interface vibrational Raman spectroscopy with coherence modulated optical susceptibilities
(Colorado State University. Libraries, 2010) Wilson, Jesse W., author; Bartels, Randy, advisor; Krapf, Diego, committee member; Marconi, Mario, committee member; Roberts, Jacob, committee member
The effect on an ultrashort probe pulse of an impulsively prepared vibrational coherence is described by effective linear and nonlinear optical susceptibility perturbations. Linear susceptibility perturbations modulate both the amplitude and phase of a probe pulse. Three spectral interferometry methods are described for measuring this phase modulation, geared toward spectral resolution, noise suppression, and rapid data acquisition. Third-order nonlinear interactions perturbations may be used to acquire surface-specific Raman spectra. While second-order spectroscopy is an established surface-specific technique, odd-order methods have been passed over because the signal is generated in the bulk media. We show that through a surface Fresnel modulation, coherence-modulated third harmonic generation can be used to obtain surface-specific vibrational information. Bulk and interface contributions to the vibrational signal are separated by scanning the interface through the focus of the laser beam.
Open Access
Computational approaches to predict drug response to cytotoxic chemotherapy
(Colorado State University. Libraries, 2020) Mannheimer, Joshua D., author; Gustafson, Daniel, advisor; Prasad, Ashok, advisor; Krapf, Diego, committee member; Thamm, Douglas, committee member
Cancer is the second leading cause of death in the United States. Statistically, within a lifetime there is slightly above a one-third chance of developing some form of cancer and a one in five chance of dying from the disease. Thus, it is no hyperbole that the understanding and treatment of cancer is one of the most pressing issues in medical research of the current era. Cytotoxic chemotherapies are a class of anti-cancer drugs that are widely used to treat a number of cancers. While cytotoxic chemotherapies are extremely effective in treating a subset of individuals for some cancers, drug resistance resulting in failure of treatment is a prominent obstacle in many cancer patients. Precision medicine, a novel concept to the 21st century, is the application of disease treatments that are specifically tailored to an individual and the specific attributes of their disease. In oncology, precision medicine particularly refers to the use of gene expression and other biological factors to inform an individual's treatment. Because cancer and its response to treatment result from many complex biological interactions, computational methods have become an essential tool to identify the molecular signatures that are the basis for precision treatment. In this thesis, a systematic analysis of the computational approaches is performed to gain insight necessary for the development of novel computational approaches in precision medicine in cancer. Statistical learning models are a class of computational modeling methods that identify and extrapolate complex patterns from large amounts of data. Specifically, this involves applying statistical learning approaches on in vitro data from cell lines and patient tumor data to predict drug response, particularly for cytotoxic chemotherapies, with an emphasis on understanding the fundamental modeling principles and data attributes driving model performance. The first chapter serves as an introduction to chemotherapy and the advancements that have driven computational approaches to precision applications in cancer. The second chapter serves as a technical introduction to statistical learning models and approaches. In the third chapter a systematic assessment of linear and non-linear modeling approaches are applied to in vitro cell lines panel including the National Cancer Institute's 60 cancer cell lines (NCI60) and cell lines of Genomics of Drug Sensitivity in Cancer (GDSC) to predict drug response in several cytotoxic chemotherapies. With in-depth analysis it is shown that the relationship between tumor tissue histotype and drug response is the major driver of model performance and can be maintained in as little as 250 random genes. The fourth chapter utilizes statistical models to explore the influence of drug induced gene perturbations on drug response models in comparison with basal gene expression. The findings indicate that drug induced changes in gene expression are superior predictors of drug response. Second, it is demonstrated that Boolean network representation of gene interactions show distinct topological differences between drug induced changes in gene expression and basal gene expression. Finally, in the fifth chapter, drug induced gene changes demonstrating high levels of connectivity in the previously developed networks are applied to derive a basal gene expression signature to predict response to combined gemcitabine and cisplatin chemotherapy treatment in patients with bladder cancer. These models show that this derived signature performs better than a random cohort of genes and in some situations genes derived directly from basal gene expression.
Open Access
Cooperative control of mobile sensor platforms in dynamic environments
(Colorado State University. Libraries, 2014) Ragi, Shankarachary, author; Chong, Edwin K. P., advisor; Krapf, Diego, committee member; Luo, J. Rockey, committee member; Oprea, Iuliana, committee member
We develop guidance algorithms to control mobile sensor platforms, for both centralized and decentralized settings, in dynamic environments for various applications. More precisely, we develop control algorithms for the following mobile sensor platforms: unmanned aerial vehicles (UAVs) with on-board sensors for multitarget tracking, autonomous amphibious vehicles for flood-rescue operations, and directional sensors (e.g., surveillance cameras) for maximizing an information-gain-based objective function. The following is a brief description of each of the above-mentioned guidance control algorithms. We develop both centralized and decentralized control algorithms for UAVs based on the theories of partially observable Markov decision process (POMDP) and decentralized POMDP (Dec-POMDP) respectively. Both POMDPs and Dec-POMDPs are intractable to solve exactly; therefore we adopt an approximation method called nominal belief-state optimization (NBO) to solve (approximately) the control problems posed as a POMDP or a Dec-POMDP. We then address an amphibious vehicle guidance problem for a flood rescue application. Here, the goal is to control multiple autonomous amphibious vehicles while minimizing the average rescue time of multiple human targets stranded in a flood situation. We again pose this problem as a POMDP, and extend the above-mentioned NBO approximation method to solve the guidance problem. In the final phase, we study the problem of controlling multiple 2-D directional sensors while maximizing an objective function based on the information gain corresponding to multiple target locations. This problem is found to be a combinatorial optimization problem, so we develop heuristic methods to solve the problem approximately, and provide analytical results on performance guarantees. We then improve the performance of our heuristics by applying an approximate dynamic programming approach called rollout.
Open Access
Data analysis and predictive modeling for synthetic and naturally occurring biological switches
(Colorado State University. Libraries, 2016) Schaumberg, Katherine A., author; Prasad, Ashok, advisor; Medford, June, advisor; Shipman, Patrick, committee member; Antunes, Mauricio, committee member; Krapf, Diego, committee member
Biological switches are biochemical network motifs responsible for determining the chemical state of cells, and are a key part of every biological system. The impact of these biological switches on cell behavior is broad. For example, many diseases such as cancer are thought to be caused by a misregulation of the bio-chemical state in a cell or group of cells. Also cell fates in differentiating stem cells are controlled by biological switches. Because of their general importance the synthetic biology community has also constructed synthetic biological switches in living organisms. While there are different kinds of possible switches, in my thesis I study switches capable of stably generating two unique molecular states, also called bi-stable switches. Here these switches are studied from two perspectives. In Chapters 1-4 I present theoretical and experimental work on analysis of specific circuits that act like biological switches. In Chapter 5 I employ a data mining perspective to identify gene expression signatures of switches that are sensitive to cytotoxic cancer drugs. This dissertation starts with a computational analysis of the effect of leaky promoter expression on bi-stable biological switches. In several biological and synthetic systems gene transcription is never completely off, even when repressed. This residual expression is referred to here as leaky expression. Bi-stable systems would be expected to have some amount of leaky expression in their off state. However, the impact of leaky expression on the functioning and properties of biological switches has not been well studied. To help fill this gap we conducted a theoretical analysis of leaky expression’s effect on biological switches. Two switches, a positive feedback and negative inhibition-based switch were studied. We found that the different circuit topologies showed different advantages in terms of their ability to handle leaky expression. Next this dissertation describes work done in collaboration with the Medford lab at Colorado State University, to construct and characterize a library of genetic plant parts. These parts would later be used in construction of perhaps the first synthetic bi-stable toggle switch in a plant. As part of this study, experiments were designed and conducted for finding the nature of the experimental noise associated with the assays used to test these plant parts. A mathematical normalization was developed to estimate quantitative information on the performance of each part. Validation experiments were done to assess the usefulness of this method for predicting the behavior of stably transformed plants from higher throughput transient assays. In the end a library of over one hundred quantitatively characterized plant parts in both Arabidopsis and Sorghum was constructed. The quantitative parameters of this library of genetic parts were then used in combination with a probabilistic bootstrap method we developed to predict optimal part combinations for construction of a bi-stable switch in Arabidopsis. The dissertation concludes with a study of biological networks in cancer cells from a data mining perspective. A large amount of data exists in the public domain on the sensitivity of cancer cell lines to cytotoxic drugs. Some cancers appear to be in a "sensitive state" while others are in a "resistant state". We would like to be able to know the gene expression signatures of these two states in order to predict cancer drug sensitivity from gene expression data. As a first step towards this goal we assessed the repeatability of predictions between the two standard databases of cancer cell lines, the NCI60 and the GDSC. This lead to identification of a preprocessing method needed to combine data from multiple databases. This was then followed up with the development of a comparative analysis platform. This platform was used to test the accuracy of models designed to predict drug sensitivity, when different model construction methods were used.
Open Access
Data-driven methods for compact modeling of stochastic processes
(Colorado State University. Libraries, 2024) Johnson, Mats S., author; Aristoff, David, advisor; Cheney, Margaret, committee member; Pinaud, Olivier, committee member; Krapf, Diego, committee member
Stochastic dynamics are prevalent throughout many scientific disciplines where finding useful compact models is an ongoing pursuit. However, the simulations involved are often high-dimensional, complex problems necessitating vast amounts of data. This thesis addresses two approaches for handling such complications, coarse graining and neural networks. First, by combining Markov renewal processes with Mori-Zwanzig theory, coarse graining error can be eliminated when modeling the transition probabilities of the system. Second, instead of explicitly defining the low-dimensional approximation, using kernel approximations and a scaling matrix the appropriate subspace is uncovered through iteration. The algorithm, named the Fast Committor Machine, applies the recent Recursive Feature Machine of Radhakrishnan et al. to the committor problem using randomized numerical linear algebra. Both projects outline practical data-driven methods for estimating quantities of interest in stochastic processes that are tunable with only a few hyperparameters. The success of these methods is demonstrated numerically against standard methods on the biomolecule alanine dipeptide.
Open Access
Demonstration of a compact 100 Hz, 0.1 J, diode-pumped picosecond laser
(Colorado State University. Libraries, 2011) Curtis, Alden, author; Rocca, Jorge, advisor; Krapf, Diego, committee member; Yalin, Azer, committee member
In this work I present an all laser diode pumped chirped pulse amplification laser system that is capable of producing 100 mJ laser pulses at 100 Hz repetition rate with durations of under 5 ps. The primary focus of this work consists of the development of two amplification stages that boost the temporally stretched pulses from a few hundred picoJoules to more than 100 mJ. The first amplifier is a Yb:YAG based regenerative amplifier operated at room temperature, which amplifies the pulses by a factor of about 106. The second stage is a multi-pass, Yb:YAG based amplifier, which is operated at cryogenic temperatures, and further amplifies the pulses by a factor of about 100. This is the first time a combination of room temperature and cryogenically cooled Yb:YAG amplifiers has been demonstrated. The room temperature pre-amplifier maintains more bandwidth than in the cryogenic case for increased compressibility. The cryogenic cooling of the power amplifier allows for increased heat dissipation and decreased saturation intensity for efficient operation. The optical efficiency of this amplifier is higher than that of other diode-pumped systems of comparable energy.
Open Access
Development and implementation of near-infrared ultrafast laser sources generated by nonlinear fiber propagation
(Colorado State University. Libraries, 2015) Domingue, Scott R., author; Bartels, Randy, advisor; Krummel, Amber, committee member; Krapf, Diego, committee member; Marconi, Mario, committee member
This dissertation is broken up into three parts: (I) generating high-quality ultrafast pulses around 1060 nm, (II) using the pulses from part (I) to generate pulses around 1300 nm, and (III) analyzing newly developed experimental theories and methods utilizing these pulses for linear and nonlinear microscopy. The majority of the work in this dissertation is choreographing the dance between nonlinear spectral broadening in optical fiber and the associated complexity in accumulated spectral phase. We have developed and employed several systems which manage to accomplish this task quite elegantly due to our technological contributions, producing high-quality pulses with high oscillator-type pulse energies both at 1060 and 1250 nm. In addition to developing some theory and techniques extending current types of nonlinear microscopy, we have as a capstone an experimental microscope cascading several of our primary source and application technologies to conduct an entirely new form of spectroscopic absorption imaging.
Open Access
Differential desensitization of pre- and postsynaptic mu opioid receptors regulating proopiomelanocortin neurons of the arcuate nucleus
(Colorado State University. Libraries, 2017) Pennock, Reagan L., author; Hentges, Shane, advisor; Tamkun, Michael, committee member; Vigh, Jozsef, committee member; Krapf, Diego, committee member
The mu opioid receptor (MOR) is the primary target of powerful opiate analgesics such as morphine and codeine. Repeated use of opiates, as may occur in patients with chronic pain, leads to the development of tolerance to the drugs' analgesic effects and may result in the development of dependence. This reduces the effectiveness of opiate-based treatments over extended periods of time, and can result in withdrawal when such a treatment is terminated. Many years of study have been dedicated to understanding the processes that lead to the development of tolerance, as an understanding of the mechanisms underlying tolerance could lead the development of novel therapeutic strategies that prolong the efficacy of opioid-based pain treatments. One particular area of focus has been on acute desensitization of the MOR. Studies of acute desensitization, defined as the loss of receptor function that occurs in the seconds to minutes following activation with an agonist, largely focus on the attenuation of desensitization of desensitization-susceptible MORs found on the somato-dendritic region of neurons in various parts of the nervous system. In these studies, we will focus on characterizing desensitization-resistant MORs located on the axon terminal region of GABAergic neurons that form synapses with hypothalamic proopiomelanocortin (POMC) neurons. Activation of presynaptic MORs, as well as other Gαi/o-coupled GPCRs located on presynaptic terminals, results in an inhibition of GABA release, which causes a subsequent inhibition of the amplitude or frequency of inhibitory postsynaptic currents (IPSCs). Our findings demonstrate that apparent resistance to desensitization by presynaptic MORs, measured as a sustained inhibition of IPSC amplitude or frequency, cannot be explained by a large receptor reserve, nor can desensitization become detectable after chronic treatment with the opiate morphine. It was also found that resistance to desensitization is a common, but not universal, property of Gαi/o-coupled G-protein coupled receptors located on presynaptic terminals. Comparison of desensitization-resistant MORs with desensitization-susceptible GABAB receptors revealed that both populations of receptors have similar receptor-effector coupling, and that resistance or susceptibility to desensitization is unaffected by experimental conditions that isolate either Ca2+-independent spontaneous release or Ca2+-dependent synchronous release. These findings provide evidence that resistance or susceptibility to desensitization is not dependent on particular receptor-effector coupling, and is likely receptor delimited. The previous findings suggest that resistance to desensitization by the MOR may be conferred by altered physical properties of presynaptic receptors relative to their postsynaptic counterparts. A likely way that these physical differences could manifest would be through differential mobility of pre- and postsynaptic receptors. To provide proof of principle that such measurements can be made, single-particle tracking of MORs containing an N-terminal FLAG tag was performed the AtT20 cell line. MOR diffusion was measured before and after activation with a maximal, desensitizing concentration of the full MOR agonist DAMGO. In the absence of DAMGO, FLAG-MORs could be found in either a mobile or immobile state. After ten minutes in the presence of DAMGO the fraction of immobile FLAG-MORs was increased, but both mobile and immobile receptors were still present. Because ten minutes in a maximal concentration of DAMGO is sufficient to cause MOR desensitization to reach a maximum and for the internalization of most desensitized receptors to occur, the findings demonstrate that steady-state signaling of the MOR may be maintained by both mobile and immobile receptors. These findings provide a basis for future studies comparing the mobility of pre- and postsynaptic MORs in neurons, as well as determining the role of mobile and immobile MORs in signaling pathways recruited by the receptor.
Open Access
Dynamics of protein interactions with new biomimetic interfaces: toward blood-compatible biomaterials
(Colorado State University. Libraries, 2019) Hedayati, Mohammadhasan, author; Kipper, Matt J., advisor; Krapf, Diego, committee member; Reynolds, Melissa, committee member; Bailey, Travis, committee member
Nonspecific blood protein adsorption on the surfaces is the first event that occurs within seconds when a biomaterial comes into contact with blood. This phenomenon may ultimately lead to significant adverse biological responses. Therefore, preventing blood protein adsorption on biomaterial surfaces is a prerequisite towards designing blood-compatible artificial surfaces.
Open Access
Electrodiffusion on the surface of bilayer membranes
(Colorado State University. Libraries, 2012) Swager, Melissa R., author; Zhou, Yongcheng, advisor; Liu, Jiangguo, committee member; Krapf, Diego, committee member
The cell memebrane is of utmost importance in the transportation of nutrients to the cell which are needed for survival. The magnitude of this is the inspiration for our study of the lipid bilayer which forms the cell membrane. In this paper we present a continuum model of electrodiffusion of lipids on the surface of bilayer membranes. Offering three derivations of the surface electrodiffusion equation, and proofs for the existence and uniqueness of the solution. A method for calculating integration constants using slotboom variables is emloyed. The development of a linear surface finite element method to solve the surface electrodiffusion equation is presented. Numerical simulations implementing the model are also given. The stability of the model is analyzed and a stability scheme using Streamline Upwind Petrov-Galerkin equations is applied. We test our code for robustness using other examples and a complex mesh. The implementation is validated by comparing with the known solution for the equations.
Open Access
Electrophysiological analysis of Kv2 channel regulation by non-canonical and canonical mechanisms
(Colorado State University. Libraries, 2020) Maverick, Emily E., author; Tamkun, Michael, advisor; Amberg, Gregory, committee member; Krapf, Diego, committee member; Tsunoda, Susan, committee member; Vigh, Jozsef, committee member
Kv2 channels are the most abundant voltage-gated potassium channels in the mammalian nervous system and entire body. These channels regulate action potential ﬁring and apoptosis via their canonical conducting functions. However, Kv2 channels also play a non-conducting role in the cells in which they are expressed. Speciﬁcally, they form junctions between the endoplasmic reticulum and plasma membranes, and these junctions regulate a myriad of cellular process. Several studies have now shown that many Kv2.1 channels expressed on the plasma membranes of mammalian cells do not respond canonically to changes in membrane voltage. Instead of opening to allow potassium efﬂux, the pores of these non-canonical channels are locked in a non-conducting state. This state has likely evolved to prevent electrical paralysis that would otherwise be conferred upon cells expressing high levels of completely functional Kv2 channels. The mechanism bringing about the non-conducting state of Kv2.1 channels is unknown. The work described in the ﬁrst part of this dissertation was carried out with the ultimate goal of revealing the mechanism of the Kv2.1 channel non-conducting state. I describe an improved, all-electrophysiological method to quantify the numbers of nonconducting Kv channels expressed in heterologous systems. I validate this approach by measuring the fraction of non-conducting Kv2.1 channels that arise when expressed in HEK293 cells. I go on to use this approach to show evidence for a non-conducting state in the second Kv2 isoform, Kv2.2, for the ﬁrst time. I ﬁnd that like Kv2.1, the Kv2.2 nonconducting state is dependent on the density of channels in the membrane. Surprisingly, I also ﬁnd that two Shaker-related channels, Kv1.4 and Kv1.5 also show density dependence in the fraction of channels that conduct. These results suggest that the mechanism underlying the non-conducting state is more common than we thought, and I discuss hypotheses that should be tested in the future. In the last part of this dissertation I describe the effects of the assembly of Kv2 channels with a newly discovered family of Kv β subunits, the AMIGOs. The experiments in this portion of the dissertation focus on each AMIGO's ability to modulate canonical, conducting Kv2 channels, as well as Kv2's ability to alter AMIGO trafﬁcking and localization. I ﬁnd that both Kv2.1 and Kv2.2 promote AMIGO trafﬁcking to the plasma membrane and alter their localization there. I also ﬁnd that while all three AMIGO isoforms promote Kv2 channel opening, AMIGO2 confers an additional stabilizing effect on the open state by slowing inactivation and deactivation. In all, the work in this dissertation expands on our current understanding of Kv channel function. These ﬁndings should guide future experiments to probe both canonical and non-canonical functions of Kv channels.
Open Access
Exploiting noise, non-linearity, and feedback to differentially control multiple different cells using a single optogenetic input
(Colorado State University. Libraries, 2023) May, Michael P., author; Munsky, Brian, advisor; Stasevich, Tim, advisor; Krapf, Diego, committee member; Shipman, Patrick, committee member
Motivated by Maxwells-Demon, we propose and solve a cellular control problem in which the exploitation of stochastic noise can break symmetry between two cells and allow for specific control of multiple cells using a single input signal. We find that a new type of noise-exploiting controllers are effective and can remain effective despite coarse approximations to the model's scale or extrinsic noise in key model parameters, and that these controllers can retain performance under substantial observer-actuator time delays. We also demonstrate how SIMO controllers could drive two-cell systems to follow different trajectories with different phases and frequencies by using a noise-exploiting controller. Together, these findings suggest that noise-exploiting control should be possible even in the case where models are approximate, and where parameters are uncertain. Having demonstrated the potential of noise-enhanced feedback control through computational modeling, we have also begun the next steps toward automating microscopy to implement this potential in experimental practice. Specifically, we demonstrate a new integrated pipeline to automate the image collection including: (i) quickly search in two-dimensions to find fields of view with cells of desired phenotypes, (ii) targeted collection of three-dimensional image data for these chosen fields of view, and (iii) streamlined processing of the collected images for rapid segmentation, spot detection and tracking, and cell/spot phenotype quantification.
Open Access
Extreme ultraviolet laser ablation mass spectrometer for molecular imaging at the nanoscale
(Colorado State University. Libraries, 2018) Kuznetsov, Ilya, author; Menoni, Carmen, advisor; Rocca, Jorge, committee member; Crick, Dean, committee member; Bernstein, Elliot, committee member; Krapf, Diego, committee member
The demand for high-precision analytical instrumentation in modern science and technology is exploding. The quality of questions to be answered sets the requirements for a given piece of technology. The type of analytical instrumentation that enables users to unambiguously identify, quantify, and map the chemical structure of a solid is imaging mass spectrometry (IMS). Most common commercially available instruments include desorption electrospray ionization (DESI), matrix-assisted laser desorption and ionization (MALDI), and secondary ion mass spectrometry (SIMS) as well as their derivatives. Each of these methods possesses a set of capabilities that define its use for one or another research task. None of them, however, enables scientists to map a solid's molecular composition in three dimensions at the nanoscale. We have developed an extreme ultraviolet laser ablation time-of-flight mass spectrometer (EUV TOF) that relies on sample probing by a 46.9 nm wavelength laser. In this work, the unique interaction of EUV light with matter was experimentally assessed and compared to SIMS TOF. It was found that the spatial resolution can be as small as 80 nm in molecular and atomic analysis in organic and inorganic materials respectively. Depth resolution is as high as 20 nm as measured on an organic bilayer. Sensitivity of the EUV TOF reaches ~0.02 amol, which is estimated to be 20× better than that of SIMS TOF in the sample of the amino acid alanine. Sensitivity in other units—sample utilization efficiency (SUE)—was found to be similar to SIMS TOF when assessed by means of detecting trace actinides in a glass matrix. It was shown that it can be further improved by means of post-ablation ionization (PI) with a secondary UV laser source. Using vacuum ultraviolet (VUV) laser light can increase the mass range of molecular detection. For instance, an intact cholesterol molecule was first detected by EUV TOF operating in VUV PI mode. This approach opens a range of opportunities to use the technique for biological studies. EUV TOF is capable to image chemical composition. This capability is demonstrated by imaging the 3D nanoscale spatial distribution of low mass fragments in a single mycobacterium. With additional instrumental modifications, it will be possible to achieve sub-cellular imaging of the molecular structure of a single microorganism without the need for using externally applied ionization-promoting matrix. Such capabilities may help to steer the development of new drugs in pharmacology and identify the signature isotope pattern of the miniscule bits of material examined by nuclear scientists.
Open Access
High resolution optical analysis of Nav1.6 localization and trafficking
(Colorado State University. Libraries, 2015) Akin, Elizabeth Joy, author; Tamkun, Michael, advisor; Amberg, Gregory, advisor; Di Pietro, Santiago, committee member; Krapf, Diego, committee member; Tsunoda, Susan, committee member
Voltage-gated sodium (Naᵥ) channels are responsible for the depolarizing phase of the action potential in most nerve cell membranes. As such, these proteins are essential for nearly all functions of the nervous system including thought, movement, sensation, and many other basic physiological processes. Neurons precisely control the number, type, and location of these important ion channels. The density of Naᵥ channels within the axon initial segment (AIS) of neurons can be more than 35-fold greater than that in the somatodendritic region and this localization is vital to action potential initiation. Dysfunction or mislocalization of Naᵥ channels is linked to many diseases including epilepsy, cardiac arrhythmias, and pain disorders. Despite the importance of Naᵥ channels, knowledge of their trafficking and cell-surface dynamics is severely limited. Research in this area has been hampered by the lack of modified Naᵥ constructs suitable for investigations into neuronal Naᵥ cell biology. This dissertation demonstrates the successful creation of modified Naᵥ1.6 cDNAs that retain wild-type function and trafficking following expression in cultured rat hippocampal neurons. The Naᵥ1.6 isoform is emphasized because it 1) is the most abundant Naᵥ channel in the mammalian brain, 2) is involved in setting the action potential threshold, 3) controls repetitive firing in Purkinje neurons and retinal ganglion cells, 4) and can contain mutations causing epilepsy, ataxia, or mental retardation. Using single-molecule microscopy techniques, the trafficking and cell-surface dynamics of Naᵥ1.6 were investigated. In contrast to the current dogma that Naᵥ channels are localized to the AIS of neurons through diffusion trapping and selective endocytosis, the experiments presented here demonstrate that Naᵥ1.6 is directly delivered to the AIS via a vesicular delivery mechanism. The modified Naᵥ1.6 constructs were also used to investigate the distribution and cell-surface dynamics of Naᵥ1.6. Somatic Naᵥ1.6 channels were observed to localize to small membrane regions, or nanoclusters, and this localization is ankyrinG independent. These sites, which could represent sites of localized channel regulation, represent a new Naᵥ localization mechanism. Channels within the nanoclusters appear to be stably bound on the order of minutes to hours, while non-clustered Naᵥ1.6 channels are mobile. Novel single-particle tracking photoactivation localization microscopy (spt-PALM) analysis of Naᵥ1.6-Dendra2 demonstrated that the nanoclusters can be modeled as energy wells and the depth of these interactions increase with neuronal age. The research presented in this dissertation represents the first single-molecule approaches to any Naᵥ channel isoform. The approaches developed during the course of this dissertation research will further our understanding of Naᵥ1.6 cell biology under both normal and pathological conditions.
Open Access
Investigating new protein components of the endocytic machinery in Saccharomyces cerevisiae
(Colorado State University. Libraries, 2016) Farrell, Kristen, author; Di Pietro, Santiago, advisor; Bamburg, James, committee member; Krapf, Diego, committee member; Tamkun, Michael, committee member
Clathrin-mediated endocytosis is an essential eukaryotic process which allows cells to control membrane lipid and protein content, signaling processes, and uptake of nutrients among other functions. About 60 proteins have been identified that compose the endocytic machinery in Saccharyomes cerevisiae, or budding yeast. Clathrin-mediated endocytosis is highly conserved between yeast and mammals in terms of both protein content and timing of protein arrival. First, there is an immobile phase in which clathrin and other coat components concentrate at endocytic sites. Second, another wave of proteins assembles about 20 seconds before localized actin polymerization. Third, a fast mobile stage of endocytosis occurs coinciding with local actin polymerization and culminates with vesicle scission. Fourth, most coat proteins disassemble from the internalized vesicle. Despite the knowledge of so many endocytic proteins, gaps still remain in the complete understanding of the endocytic process. We attempt to fill some of these gaps with a screen of the yeast GFP library for novel endocytic-related proteins using confocal fluorescence microscopy. We identified proteins colocalizing with RFP-tagged Sla1, a clathrin adaptor that serves as a well-known marker of endocytic sites. Ubx3 and Tda2, two unstudied proteins, were selected for further investigation based on high degree of colocalization with Sla1. Ubx3 shows fluorescent patch dynamics similar to an endocytic coat protein. Ubx3 is dependent on clathrin for patch lifetime and binds clathrin via a W-box, the first identification of this clathrin binding motif in a non-mammalian species. Uptake assays performed in a knockout strain of Ubx3 display a reduction in both bulk endocytosis by fluorescent dye Lucifer Yellow and cargo-specific endocytosis by methionine transporter Mup1. The Ubx3 knockout cells also show a significant increase in lifetime of early endocytic protein Ede1, and removing its UBX domain alone results in similar defects to the Ubx3 knockout. The endocytic defect may be due to lack of recruitment of ubiquitin regulator AAA ATPase Cdc48 to the endocytic site. Inactivation of Cdc48 reduces Lucifer yellow uptake to minimal levels and causes aggregates of early endocytic protein Ede1-GFP. This is the first identification of a UBX domain-containing protein in clathrin-mediated endocytosis. Tda2 appears at the tail end of each endocytic site, suggesting a function in late stage endocytosis. A Tda2 knockout strain shows similar reductions in bulk and cargo dependent endocytosis through Lucifer yellow and Mup1 uptake assays. Tda2 appears unaffected by clathrin disruption, but is no longer recruited to the endocytic site when cells are treated with the actin depolymerizing agent LatA, suggesting it is associated with the actin cytoskeleton. A crystal structure of Tda2 reveals it is a homolog of mammalian dynein light chain TcTex-1. Tda2 is associated with a larger protein complex in the cytosol but does not co-purify with dynein and is unaffected by addition of the microtubule depolymerizing drug Nocodazole. Tda2 has similar localization to actin capping proteins Cap1/2 which localize to the plus end of actin filaments near the plasma membrane. Tda2 deletion increases Cap1 patch lifetime but reduces its fluorescent intensity. Aim21 fluorescent intensity at endocytic sites is reduced to half without Tda2. When Aim21 is deleted, Tda2 is no longer recruited to endocytic sites and the large Tda2-containing complex is no longer present in the cytosol. Tda2 is a newly identified component of the actin cytoskeleton in stable complex with Aim21. This is the first identification of a TcTex type dynein light chain in yeast and the first dynein light chain associated with clathrin-mediated endocytosis. Thus, we have identified two novel components of endocytic machinery by screening the yeast GFP library. The successful identification of previously uncharacterized endocytic proteins indicates the unique advantages of the GFP library screening. Many previous screens for endocytic proteins rely on the yeast knockout library or cargo accumulation, which have many disadvantages. The GFP library screening method has potential for use with other cellular processes that have distinct cellular localizations and established fluorescent markers. The GFP library also has potential for use in a screen for cargo proteins dependent on clathrin-mediated endocytosis. Additionally, more proteins of the endocytic machinery may be further characterized from the list of Sla1-colocalizing proteins identified in our screen.
Open Access
Live-cell imaging uncovers the relationship between histone acetylation, RNA polymerase II phosphorylation, transcription, and chromatin dynamics
(Colorado State University. Libraries, 2023) Saxton, Matthew Neeley, author; Stasevich, Timothy J., advisor; Nishimura, Erin O., committee member; Hansen, Jeffrey, committee member; Krapf, Diego, committee member
Living cells are capable of turning a one dimensional strand of nucleic acids into a functional polypeptide. A host of steps and factors are involved in the process of transcription and translation, and understanding each of them is necessary for comprehending and characterizing life. While new technologies and assays have expanded our understanding of eukaryotic transcription, there is still much to be learned. In particular, single-molecule microscopy provides a powerful and versatile platform for studying the genesis of RNA with unparalleled spatiotemporal resolution (Chapter 1). First, we characterize the timing, kinetics, and occupancy of phosphorylated RNA polymerase II (RNAP2) using a single-copy HIV-1 reporter system. This work provides strong evidence for clusters of phosphorylated, initiating RNAP2 which is spatially separated from bursty, downstream RNA synthesis. It is found that RNAP2-Ser5-phosphorylation (Ser5ph) precedes RNA output by ~1 minute, and RNAP2 arrives at the locus in a phosphorylated state (Chapter 2). Then, we examine the spatial correlation between H3K27 acetylation and Ser5ph in living cells on the course of minutes to hours. Contrary to expectations based upon ChIP data, we find that the two signals are in fact spatially separated. This argues for a functional separation between transcriptional poising and initiation, likely aiding bursty behavior. Next, the dynamics of single chromatin-incorporated nucleosomes in the context of H3K27 acetylation and transcription initiation is determined with super-resolution single-molecule imaging. The physical movement of chromatin inside of H3K27ac and RNAP2-Ser5ph enriched regions is found to be significantly different, despite both marks being traditionally associated with transcriptionally active chromatin. (Chapter 3). Much of this work utilizes bead-loading in order to introduce proteins and DNA into living cells. A simple, effective, and cheap procedure, bead-loading is a highly effective and versatile technique that is generally underutilized. To facilitate communication of this process, a detailed protocol is included (Chapter 4). While this culmination of work furthers our understanding of cellular genetic expression and eukaryotic transcription, it also introduces many new questions that are promising areas of study. Fortunately, the combination of imaging technology and knowledge developed here provides promising new fronts for studying transcription in living cells (Chapter 5).
Open Access
Measurements of electron-ion collision rates and Rydberg atom populations in ultracold plasmas by using short electric field pulses
(Colorado State University. Libraries, 2017) Chen, Wei-Ting, author; Roberts, Jacob, advisor; Robinson, R. Steve, committee member; Harton, John, committee member; Krapf, Diego, committee member
Ultracold plasmas are good tools for studying fundamental plasma physics. In particular, these plasmas are well-suited to study so-called strong coupling physics the physics of plasmas where nearest-neighbor Coulomb interactions become large enough to cause spatial correlations and break assumptions. An ultracold plasma makes such a good tool because it is it is free of interactions with neutral atoms, and has a well controlled and tunable initial conditions. The UCPs in this work were created from the photoionization of cold 85Rb atoms. The experiments described in this thesis are focused on the measurements of damping of electron center-of-mass oscillations. We developed a method that uses two short electric field pulses to map the temporal profile of the oscillation amplitude. We found that the damping of such oscillations can result from dephasing which is a collisionless mechanism or from electron-ion collisions or a combination of both. Thus, we separate the study of two pulse measurements into two parts. The first part of the two short electric field pulse measurement is about the measurements and modeling of in the collisionless damping regime. The second part will focus on the regime where the damping is dominated by electron-ion collisions where we not only observed strong coupling influence on electron-ion collision rates, but also saw break down of one or more standard assumptions used in plasma physics calculations. Rydberg atoms can be formed in ultracold plasmas through three-body recombination process. Our setup was capable of measuring Rydberg atoms in a energy range above the bottleneck energy. We measured the Rydberg populations at different temperatures, and our preliminary results agree well with a parameter-free calculation. However, there are some unexplained parts of our measurements on early time Rydberg populations. This means more studies are needed in the future in order to interpret our results and make use of them. Future work includes measurements of the strong coupling influence on electron-ion collision rates in a magnetized ultracold plasma, measurement of Rydberg population below the bottleneck energy, a detailed study of evaporations in ultracold plasmas.
Open Access
Modeling local pattern formation on membrane surfaces using nonlocal interactions
(Colorado State University. Libraries, 2015) Adkins, Melissa, author; Zhou, Yongcheng, advisor; Krapf, Diego, committee member; Liu, James, committee member; Mueller, Jennifer, committee member
The cell membrane is of utmost importance in the transportation of nutrients and signals to the cell which are needed for survival. The magnitude of this is the inspiration for our study of the lipid bilayer which forms the cell membrane. It has been recently accepted that the lipid bilayer consists of lipid microdomains (lipid rafts), as opposed to freely moving lipids. We present two lipid raft models using the Ginzburg-Landau energy with addition of the electrostatic energy and the geodesic curvature energy to describe the local pattern formation of these lipid rafts. The development and implementation of a C⁰ interior penalty surface finite element method along with an implicit time iteration scheme will also be discussed as the optimal solution technique.
Open Access
Probing molecular kinetics using higher-order fluorescence correlation spectroscopy
(Colorado State University. Libraries, 2019) Abdollah-Nia, Farshad, author; Gelfand, Martin P., advisor; Van Orden, Alan, advisor; Krapf, Diego, committee member; Prasad, Ashok, committee member; Roberts, Jacob, committee member
Fluorescence correlation spectroscopy (FCS) is a powerful tool in the time-resolved analysis of non-reacting or reacting molecules in solution, based on fluorescence intensity fluctuations. However, conventional (second-order) FCS alone is insufficient to measure all parameters needed to describe a reaction or mixture, including concentrations, fluorescence brightnesses, and forward and reverse rate constants. For this purpose, correlations of higher powers of fluorescence intensity fluctuations can be calculated to yield additional information from the single-photon data stream collected in an FCS experiment. To describe systems of diffusing and reacting molecules, considering cumulants of fluorescence intensity results in simple expressions in which the reaction and the diffusion parts factorize. The computation of higher-order correlations in experiments is hindered by shot noise and common detector artifacts, the effects of which become worse with increasing order. We introduce a technique to calculate artifact-free higher-order correlation functions with improved time resolution, and without any need for modeling and calibration of detector artifacts. The technique is formulated for general multi-detector experiments and verified in both two-detector and single-detector configurations. Good signal-to-noise ratio is achieved down to 1 μs in correlation curves up to order (2,2). Next, we demonstrate applications of the technique to analyze systems of fast and slow reactions. As an example of slow- or non-reacting systems, the technique is applied to resolve two-component mixtures of labeled oligonucleotides. Then, the protonation reaction of fluorescein isothiocyanate (FITC) in phosphate buffer is analyzed as an example of fast reactions (relaxation time < 10 μs). By reference to an (apparent) non-reacting system, the simple factorized form of cumulant-based higher-order correlations is exploited to remove the dependence on the molecular detection function (MDF). Therefore, there is no need to model and characterize the experimental MDF, and the precision and the accuracy of the technique are enhanced. It is verified that higher-order correlation analysis enables complete and simultaneous determination of number and brightness parameters of mixing or reacting molecules, the reaction relaxation time, and forward and reverse reaction rates. Finally, we apply the technique to analyze the conformational dynamics of DNA hairpins. Previous FCS measurements of DNA hairpin folding dynamics revealed at least three conformational states of the DNA are present, distinguished by the brightness of fluorescent dye-quencher labels. Rapid fluctuations between two of the states occurred on time scales observable by FCS. A third state that was static on the FCS time scale was also observed. We show that conventional FCS alone cannot uniquely distinguish the conformational states or assign their roles in the observed mechanism. The additional information offered by higher-order FCS makes it possible (i) to uniquely identify the static and rapidly-fluctuating states; and (ii) to directly measure the brightnesses and populations of all three observed states. The rapid fluctuations occurring on the FCS time-scale are due to a reversible reaction between the two lowest brightness levels, attributed to the folded and random-coil conformations of the DNA. The third state, which is the brightest, is attributed to spatially extended unfolded conformations that are isolated from the more compact conformations by a substantial energy barrier. These conformations attain a maximum equilibrium population of nearly 10% near physiological temperatures and salt concentrations.