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University of Colorado
1.
Manna, Premashis.
Development and Characterization of Improved Red Fluorescent Protein Variants.
Degree: PhD, Chemistry & Biochemistry, 2018, University of Colorado
URL: https://scholar.colorado.edu/chem_gradetds/238 
► Aequorea victoria-based green fluorescent proteins and their blue, cyan and red counterparts offer unprecedented advantage as biological markers owing to their genetic encodability and…
(more)
▼ Aequorea victoria-based green fluorescent proteins and their blue, cyan and red counterparts offer unprecedented advantage as biological markers owing to their genetic encodability and straightforward expression in different organisms. Fluorescent proteins are characterized with complex photo-kinetics due to the presence of light-induced non-fluorescent or dark states which are responsible for their fluorescence intermittency or 'blinking'. We developed time- and frequency-domain techniques for probing the kinetics involving dark state conversion (DSC) and ground state recovery (GSR) in red fluorescent proteins (RFPs). Ensemble-level DSC and GSR kinetics in FPs were presented in the context of their single molecule fluorescence behaviors. Although significant advancements have been made towards improving the key photo-physical properties of the red fluorescent proteins (RFPs), they continue to perform sub-optimally compared to their green or cyan counterparts. We developed a high-throughput microfluidic screening and sorting platform to generate improved RFP variants by engineering their excited state lifetimes. Using this microfluidic system, mCherry and FusionRed mutants were developed with higher
in-vivo brightness and fusion efficiency. We employed a novel rational design for the enhancement of brightness in RFPs through engineering of their radiative rates and extinction coefficients. This enabled us to produce bright mutants those out-performed the existing red fluorescent proteins.
Advisors/Committee Members: Ralph Jimenez, Amy E. Palmer, Thomas Perkins, Joel Kralj.
Subjects/Keywords: directed evolution; excited state lifetime; microfluidics; protein engineering; radiative lifetime; red fluorescent proteins; Biomedical Engineering and Bioengineering; Biophysics; Physical Chemistry
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APA (6th Edition):
Manna, P. (2018). Development and Characterization of Improved Red Fluorescent Protein Variants. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/238
Chicago Manual of Style (16th Edition):
Manna, Premashis. “Development and Characterization of Improved Red Fluorescent Protein Variants.” 2018. Doctoral Dissertation, University of Colorado. Accessed March 04, 2021.
https://scholar.colorado.edu/chem_gradetds/238.
MLA Handbook (7th Edition):
Manna, Premashis. “Development and Characterization of Improved Red Fluorescent Protein Variants.” 2018. Web. 04 Mar 2021.
Vancouver:
Manna P. Development and Characterization of Improved Red Fluorescent Protein Variants. [Internet] [Doctoral dissertation]. University of Colorado; 2018. [cited 2021 Mar 04].
Available from: https://scholar.colorado.edu/chem_gradetds/238.
Council of Science Editors:
Manna P. Development and Characterization of Improved Red Fluorescent Protein Variants. [Doctoral Dissertation]. University of Colorado; 2018. Available from: https://scholar.colorado.edu/chem_gradetds/238
University of Colorado
2.
LeBlanc, Marc-Andre.
High Precision AFM-Based SMFS of Mechanically Labile Type III Secretion System Effectors.
Degree: PhD, 2018, University of Colorado
URL: https://scholar.colorado.edu/chem_gradetds/244
► Pathogenic bacteria have developed a wide range of tools for circumventing or overcoming the host’s defenses. Over time, these tools have become increasingly complex,…
(more)
▼ Pathogenic bacteria have developed a wide range of tools for circumventing or overcoming the host’s defenses. Over time, these tools have become increasingly complex, allowing bacteria to live and thrive within a wide variety of host environments. One such tool is the Type III Secretion System (T3SS), a needle-like complex that allows bacteria to directly inject proteins, known as effectors, from their cytoplasm into host cells. Once inside host cells, effector proteins have a wide range of effects, from shutting down the host immune response to rearranging the host cytoskeleton to accommodate invading bacteria. Because the T3SS needle presents a narrow channel (< 2 nm), effector proteins must be mechanically unfolded before passing through. Proteins are unfolded by a molecular motor that associates with the base of needle and pulls protein into the channel. While this motor can unfold and secrete many proteins, it is unable to unfold proteins that have high mechanical stability. This indicates a need for effectors to be mechanically labile no matter their function. This may be one of the reasons effectors have very low sequence and structural similarity to other members of their protein super-families. This spurred our investigation into how effectors respond to mechanical force. To investigate effector protein stability, I used atomic force microscopy (AFM) to mechanically unfold the proteins. Here I show that effector proteins of the T3SS unfold at very low force, despite containing a wide variety folds and functions. This supports our hypothesis that to facilitate efficient secretion, effectors evolved to be mechanically labile. Because effector proteins unfold at such low force, it was critical for me to utilize site-specific attachment to both the AFM tip and surface, increasing both the amount of data I could collect and the quality of collected data. Site-specific attachment resulted in a 70-fold improvement in the yield of high quality data, allowing rapid characterization of mechanically labile α-helical proteins. Combining site-specific attachment with modified cantilevers allowed the collection of unfolding data for 5 effector proteins, finding they all unfold at low force (<20 pN), making them some of the most mechanically labile proteins studied to date by AFM-SMFS. Comparing the mechanical stability of effector proteins to their <i>in vivo</i> secretion rates, showed that unfolding force does not always correlate with <i>in vivo</i> secretion rate. However, the distance to the transition state does correlate with <i>in vivo</i> secretion rate. To elucidate how effector proteins have evolved to be efficiently secreted, the mechanical stability of an effector protein, NleC, was compared with a non-secreted homologue, protealysin. While the initial unfolding event of NleC occurs below the detection limit of our AFM platform, the unfolding of an intermediate along the unfolding pathway was measured. When compared to the unfolding of protealysin, the unfolding intermediate of…
Advisors/Committee Members: Marcelo Sousa, Thomas Perkins, Michael Stowell, Joseph Falke, Amy Palmer.
Subjects/Keywords: afm; protein unfolding; single-molecule force spectroscopy; specific attachment; type iii secretion system; Biochemistry; Biophysics
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APA (6th Edition):
LeBlanc, M. (2018). High Precision AFM-Based SMFS of Mechanically Labile Type III Secretion System Effectors. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/244
Chicago Manual of Style (16th Edition):
LeBlanc, Marc-Andre. “High Precision AFM-Based SMFS of Mechanically Labile Type III Secretion System Effectors.” 2018. Doctoral Dissertation, University of Colorado. Accessed March 04, 2021.
https://scholar.colorado.edu/chem_gradetds/244.
MLA Handbook (7th Edition):
LeBlanc, Marc-Andre. “High Precision AFM-Based SMFS of Mechanically Labile Type III Secretion System Effectors.” 2018. Web. 04 Mar 2021.
Vancouver:
LeBlanc M. High Precision AFM-Based SMFS of Mechanically Labile Type III Secretion System Effectors. [Internet] [Doctoral dissertation]. University of Colorado; 2018. [cited 2021 Mar 04].
Available from: https://scholar.colorado.edu/chem_gradetds/244.
Council of Science Editors:
LeBlanc M. High Precision AFM-Based SMFS of Mechanically Labile Type III Secretion System Effectors. [Doctoral Dissertation]. University of Colorado; 2018. Available from: https://scholar.colorado.edu/chem_gradetds/244
University of Colorado
3.
Vera, Carlos D.
Functional Characterization of Disease-Causing Mutations in Human Myosin Heavy Chain Genes.
Degree: PhD, 2018, University of Colorado
URL: https://scholar.colorado.edu/mcdb_gradetds/94
► Biophysical and biochemical imbalance of mechanisms relevant to muscle function, can result in morphological changes to the tissue. While the purpose of activities involving exercise…
(more)
▼ Biophysical and biochemical imbalance of mechanisms relevant to muscle function, can result in morphological changes to the tissue. While the purpose of activities involving exercise is to modify the shape and size of skeletal muscle, and the length of these muscles allows wide ranges of stiffness and stretch to be applied, cardiac tissue is not meant to change much. However, stressful extrinsic factors (poor diet, chemotherapy, etc) or intrinsic factors like inherited mutations in muscle functioning genes can result in a myopathy or a disease of the muscle. In fact, another biological process that requires much compliance of many molecules is embryogenesis. Although the timeline of an embryonic structure is limited, compared to an adult heart and muscle composition, continuous and coordinated movement is essential, but cumulative, prolonged disruptions can be harmful. At the core of muscle biology is the myosin molecule which is a motor protein that hydrolyzes ATP, binds to actin, and the spatial dynamics of its function (contraction-relaxation) alter the length of muscle. Myosin cyclically follows specific steps and undertakes well-defined structural conformations during these events, but mutations can alter the time and stability of any of these aspects. In this thesis I did a comprehensive analysis of the ATPase cycle parameters for both embryonic and cardiac myosin and studied the effects of specific associated or linked mutations have on function. The multiple mutations were in the interest of cataloging common features and defects to identify mechanistic patterns. In a collaborative effort I also used these wet-lab measurements to simulate the cycle using a working kinetic model for the myosin ATPase cycle. We have found distinct differences between three different myopathies that will be discussed in the following chapters.
Advisors/Committee Members: Leslie A. Leinwand, Thomas Perkins, Andreas Hoenger, Michael Stowell, Hector Rodriguez.
Subjects/Keywords: cardiomyopathy; muscle; myosin; embryonic myosin; atpase cycle parameters; Biochemistry; Biophysics; Molecular Biology
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APA (6th Edition):
Vera, C. D. (2018). Functional Characterization of Disease-Causing Mutations in Human Myosin Heavy Chain Genes. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/mcdb_gradetds/94
Chicago Manual of Style (16th Edition):
Vera, Carlos D. “Functional Characterization of Disease-Causing Mutations in Human Myosin Heavy Chain Genes.” 2018. Doctoral Dissertation, University of Colorado. Accessed March 04, 2021.
https://scholar.colorado.edu/mcdb_gradetds/94.
MLA Handbook (7th Edition):
Vera, Carlos D. “Functional Characterization of Disease-Causing Mutations in Human Myosin Heavy Chain Genes.” 2018. Web. 04 Mar 2021.
Vancouver:
Vera CD. Functional Characterization of Disease-Causing Mutations in Human Myosin Heavy Chain Genes. [Internet] [Doctoral dissertation]. University of Colorado; 2018. [cited 2021 Mar 04].
Available from: https://scholar.colorado.edu/mcdb_gradetds/94.
Council of Science Editors:
Vera CD. Functional Characterization of Disease-Causing Mutations in Human Myosin Heavy Chain Genes. [Doctoral Dissertation]. University of Colorado; 2018. Available from: https://scholar.colorado.edu/mcdb_gradetds/94
University of Colorado
4.
Fiedler, Brett.
Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors.
Degree: PhD, Chemistry & Biochemistry, 2017, University of Colorado
URL: https://scholar.colorado.edu/jila_gradetds/2
► Fluorescent biosensors are important measurement tools for in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as…
(more)
▼ Fluorescent biosensors are important measurement tools for
in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as membrane potential. Both the development of these sensors and their implementation in examining cellular heterogeneity requires technology for measuring and sorting cells based on the fluorescence levels before and after chemical or physical perturbations. We developed a droplet microfluidic platform for the screening and separation of cell populations on the basis of the
in vivo response of expressed fluorescence-based biosensors after addition of an exogenous analyte. Screening with this instrument reveals increased heterogeneity in an array of targeted Zn
2+ biosensorsin HeLa cells that helps shed light on the complexities of these sensors in different chemical environments. Subsequently, the instrument is used to screen and assess diversity in a number of HeLa-cell based genetic linker libraries for a family of genetically-encoded Zn
2+ sensors. Progress on sensor characterization is made using time-resolved fluorescence techniques to advance a deeper molecular understanding of these sensors to guide further development.
Advisors/Committee Members: Ralph Jimenez, Amy Palmer, Thomas Perkins, Loren Hough, Robert Parson.
Subjects/Keywords: fluorescence; fret; fluorescent protein; biosensor; microfluidic; cell sorting; Analytical Chemistry; Biophysics; Physical Chemistry
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APA (6th Edition):
Fiedler, B. (2017). Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/jila_gradetds/2
Chicago Manual of Style (16th Edition):
Fiedler, Brett. “Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors.” 2017. Doctoral Dissertation, University of Colorado. Accessed March 04, 2021.
https://scholar.colorado.edu/jila_gradetds/2.
MLA Handbook (7th Edition):
Fiedler, Brett. “Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors.” 2017. Web. 04 Mar 2021.
Vancouver:
Fiedler B. Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors. [Internet] [Doctoral dissertation]. University of Colorado; 2017. [cited 2021 Mar 04].
Available from: https://scholar.colorado.edu/jila_gradetds/2.
Council of Science Editors:
Fiedler B. Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors. [Doctoral Dissertation]. University of Colorado; 2017. Available from: https://scholar.colorado.edu/jila_gradetds/2
University of Colorado
5.
Deacon, John C.
Fast kinetics of human myosin heavy chain contraction in health and disease.
Degree: PhD, 2012, University of Colorado
URL: https://scholar.colorado.edu/mcdb_gradetds/11
► The myosin heavy chain composition of human heart and skeletal muscles is dynamic in health and disease and is known to define the maximum…
(more)
▼ The myosin heavy chain composition of human heart and skeletal muscles is dynamic in health and disease and is known to define the maximum velocity and force generated by contracting muscles. The study of the individual isoforms that comprise this diversity has recently been aided by the development of a recombinant expression system capable of producing functional sarcomeric human myosin motors. The eight primary human sarcomeric myosin isoforms are herein shown to differ by between 1.5- and 4.5-fold in their F-actin-activated ATPase activities. Due to the greatly differing contractile environments in which they function it has been anticipated that the kinetics of the reactions that comprise the contractile cycle vary to an even greater extent. Using pre-steady-state techniques it is possible to determine the kinetics of the steps of myosin contraction. Among the eight isoforms tested, we observe multiple biochemical patterns that differentiate the motors into fast-moving and slow, tension-maintaining categories. Additionally, we have characterized pathological point mutations associated with developmental and cardiac disease. We find that unique patterns of alteration to the reactions of the myosin ATPase cycle characterize each mutation. These alterations are predicted to cause significant disruptions to the reactions governing attachment and detachment between myosin and F-actin. These studies lay the foundation for structure vs. function analysis of pathological myosin mutations and fill an important void in understanding the contributions of the various myosin isoforms to human muscle contraction.
Advisors/Committee Members: Thomas Perkins, Shelley Copley, Michael Stowell, Johannes Rudolph, Leslie Leinwand.
Subjects/Keywords: Kinetics; Muscle; Myosin; Biochemistry; Biophysics; Molecular Biology
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APA (6th Edition):
Deacon, J. C. (2012). Fast kinetics of human myosin heavy chain contraction in health and disease. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/mcdb_gradetds/11
Chicago Manual of Style (16th Edition):
Deacon, John C. “Fast kinetics of human myosin heavy chain contraction in health and disease.” 2012. Doctoral Dissertation, University of Colorado. Accessed March 04, 2021.
https://scholar.colorado.edu/mcdb_gradetds/11.
MLA Handbook (7th Edition):
Deacon, John C. “Fast kinetics of human myosin heavy chain contraction in health and disease.” 2012. Web. 04 Mar 2021.
Vancouver:
Deacon JC. Fast kinetics of human myosin heavy chain contraction in health and disease. [Internet] [Doctoral dissertation]. University of Colorado; 2012. [cited 2021 Mar 04].
Available from: https://scholar.colorado.edu/mcdb_gradetds/11.
Council of Science Editors:
Deacon JC. Fast kinetics of human myosin heavy chain contraction in health and disease. [Doctoral Dissertation]. University of Colorado; 2012. Available from: https://scholar.colorado.edu/mcdb_gradetds/11
University of Colorado
6.
Manhart, Carol Michelle.
Investigating protein-DNA interactions at replication forks by photo-crosslinking.
Degree: PhD, Chemistry & Biochemistry, 2013, University of Colorado
URL: https://scholar.colorado.edu/chem_gradetds/78
► During Okazaki fragment synthesis, the replicase must distinguish single-stranded from duplex DNA in advance of the polymerase to sense completion of a fragment and…
(more)
▼ During Okazaki fragment synthesis, the replicase must distinguish single-stranded from duplex DNA in advance of the polymerase to sense completion of a fragment and trigger release from the lagging strand. A hypothesis in the literature proposes that the τ subunit (of the DnaX complex) directly senses completion of an Okazaki fragment. An alternative model suggests that the polymerase subunit senses conversion of a gap to a nick. I show using a novel phenyldiazirine photo-crosslinker linked to the 5-position of thymidylate that the τ subunit is not in position to distinguish gapped DNA from nicked DNA. The α subunit (the polymerase) is positioned to serve as the processivity sensor. Upon encountering duplex DNA, the polymerase likely changes conformation triggering its release from the lagging strand and the β processivity clamp, modulating its own affinity. Unrepaired replication forks dissociate from the helicase and suffer collapse. PriA recognizes stalled replication forks and initiates interactions to reload the helicase and activate a previously stalled fork. I used a FRET helicase assay to develop a PriA- dependent helicase loading system in E. coli and B. subtilis and to identify a minimal substrate to support a photo-crosslinking study also discussed here. I discovered that PriA's ATPase activity dictates substrate specificity. I also show that PriA serves as a checkpoint protein by blocking the replicase from binding to stalled replication forks distinguishing between an alternative model. SPP1 is a bacteriophage that infects B. subtilis. It encodes its own initiation proteins (origin binding protein, primosomal proteins, helicase, and single-strand binding protein (SSB)) but requires its host's primase and major replicative polymerase to replicate its genome. Both host and phage SSBs can support a reconstituted SPP1 system, but phage SSB does not support a reconstituted B. subtilis system. Using the B. subtilis FRET helicase assay, I show that phage SSB can substitute for the host's SSB in helicase reloading. Therefore the defect in the reconstituted system is not at the level of helicase loading or function and must occur after the helicase is loaded. I also show an absolute requirement on all SPP1 components in helicase reloading, including the origin binding protein (in a non-origin-containing template), which suggests a new role for this protein. In collaboration with Tim Lohman's lab at Washington
University, I have contributed to a study into the functions of the C-terminal tails of SSB. SSB functions as a homotetramer whose four C-terminal tails interact with many other proteins necessary for DNA replication and repair. In an in vivo assay, an SSB variant that has two functional C-terminal tails supports viability in E. coli. An SSB variant that has one C- terminal tail is dominant lethal. In a reconstituted rolling circle E. coli replication system, there is a defect in coupled synthesis that causes a two-fold decrease in lagging strand synthesis relative to the leading strand using the variant…
Advisors/Committee Members: Charles S. McHenry, Robert Kuchta, Robert Batey, Marcelo Sousa, Thomas Perkins.
Subjects/Keywords: Biochemistry
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APA (6th Edition):
Manhart, C. M. (2013). Investigating protein-DNA interactions at replication forks by photo-crosslinking. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/78
Chicago Manual of Style (16th Edition):
Manhart, Carol Michelle. “Investigating protein-DNA interactions at replication forks by photo-crosslinking.” 2013. Doctoral Dissertation, University of Colorado. Accessed March 04, 2021.
https://scholar.colorado.edu/chem_gradetds/78.
MLA Handbook (7th Edition):
Manhart, Carol Michelle. “Investigating protein-DNA interactions at replication forks by photo-crosslinking.” 2013. Web. 04 Mar 2021.
Vancouver:
Manhart CM. Investigating protein-DNA interactions at replication forks by photo-crosslinking. [Internet] [Doctoral dissertation]. University of Colorado; 2013. [cited 2021 Mar 04].
Available from: https://scholar.colorado.edu/chem_gradetds/78.
Council of Science Editors:
Manhart CM. Investigating protein-DNA interactions at replication forks by photo-crosslinking. [Doctoral Dissertation]. University of Colorado; 2013. Available from: https://scholar.colorado.edu/chem_gradetds/78
University of Colorado
7.
Blair, Rebecca.
Using Single Molecule FRET to Study the Mechanisms of DNA Bending by TBP and HMGB1.
Degree: PhD, Chemistry & Biochemistry, 2013, University of Colorado
URL: https://scholar.colorado.edu/chem_gradetds/106
► Defining mechanisms of transcriptional regulation is important for understanding how gene expression is controlled, which is essential to cellular viability. Outlined in this thesis…
(more)
▼ Defining mechanisms of transcriptional regulation is important for understanding how gene expression is controlled, which is essential to cellular viability. Outlined in this thesis are studies that characterize DNA-protein interactions involved in transcriptional regulation. Specifically, we have used single-molecule FRET (smFRET) to investigate how proteins bend DNA. A homebuilt TIRF microscope was assembled for single molecule fluorescence studies, which is described in Chapter 2 of this thesis.
We used smFRET to study the extent and kinetics of DNA bending by the transcription factor TBP (TATA binding protein) with consensus and nonconsensus TATA DNA. TBP bent different TATA sequences to the same, homogenous, bent population. Further, TFIIA did not change the extent of DNA bending by TBP, but increased the stability of the TBP-DNA complex. We found that TBP bent consensus TATA DNA to two different kinetic populations bent to the same extent, but only one kinetic bent population existed when TBP was bound to the nonconsensus sequence. The uniform bending of DNA by TBP was not predicted by previous ensemble studies and provided insight into the mechanism of DNA binding by TBP.
HMGB1 is a nuclear protein that binds and bends DNA to facilitate transcription. It has 3 domains: the A and B box, which bend DNA independently, and an acidic unstructured C-terminal tail. We studied how full length HMGB1 and its different domains bend DNA using smFRET. We determined that the full length HMGB1 protein bent DNA to a similar extent as its individual domains, the A box and the B box. However, removal of the C-terminal tail caused the protein to bend DNA to a greater extent and with more heterogeneity than the full length protein. Further, a truncation that contains the B box and C-terminal tail interacted with DNA less efficiently than the B box alone; this truncation could bend DNA at a high concentration to the same extent as the B box alone. Taken together we propose that the full length HMGB1 protein bends DNA primarily through its A box, while the B box interacts with the C-terminal tail, which prevents its interaction with DNA.
Advisors/Committee Members: Jennifer F. Kugel, Amy Palmer, Arthur Pardi, Thomas Perkins, Robert Kuchta.
Subjects/Keywords: DNA bending; HMGB1; single molecule FRET; TBP; TIRF; Biochemistry; Molecular Genetics
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APA (6th Edition):
Blair, R. (2013). Using Single Molecule FRET to Study the Mechanisms of DNA Bending by TBP and HMGB1. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/106
Chicago Manual of Style (16th Edition):
Blair, Rebecca. “Using Single Molecule FRET to Study the Mechanisms of DNA Bending by TBP and HMGB1.” 2013. Doctoral Dissertation, University of Colorado. Accessed March 04, 2021.
https://scholar.colorado.edu/chem_gradetds/106.
MLA Handbook (7th Edition):
Blair, Rebecca. “Using Single Molecule FRET to Study the Mechanisms of DNA Bending by TBP and HMGB1.” 2013. Web. 04 Mar 2021.
Vancouver:
Blair R. Using Single Molecule FRET to Study the Mechanisms of DNA Bending by TBP and HMGB1. [Internet] [Doctoral dissertation]. University of Colorado; 2013. [cited 2021 Mar 04].
Available from: https://scholar.colorado.edu/chem_gradetds/106.
Council of Science Editors:
Blair R. Using Single Molecule FRET to Study the Mechanisms of DNA Bending by TBP and HMGB1. [Doctoral Dissertation]. University of Colorado; 2013. Available from: https://scholar.colorado.edu/chem_gradetds/106
University of Colorado
8.
Mabry, Joshua Nolan.
Single-Molecule Dynamics at the Solid-Liquid Interface.
Degree: PhD, Chemical & Biochemical Engineering, 2015, University of Colorado
URL: https://scholar.colorado.edu/chbe_gradetds/83
► The overall objective of this work was to develop new analytical methods for characterizing molecular dynamics at the solid-liquid interface. We used single-molecule imaging,…
(more)
▼ The overall objective of this work was to develop new analytical methods for characterizing molecular dynamics at the solid-liquid interface. We used single-molecule imaging, based on total internal reflection fluorescence microscopy, to observe surface-active molecules at silica-aqueous interfaces and leveraged high-throughput computational tools to create massive datasets capturing broad distributions of molecular kinetics and detailed information on surface lateral heterogeneity. We developed novel statistical approaches to learn about the molecular-level physical processes in unprecedented detail. With respect to specific applications, we first demonstrated how single-molecule imaging yields a deeper understanding of chromatographic media. We used a combination of single-molecule observations and macroscopic reversed phase liquid chromatography to characterize the surface activity of a hydrophobic analyte across a range of solution conditions, focusing on how anomalous surface sites affected the adsorption kinetics and retention in the column. The other two projects described in this thesis focused on understanding molecular mobility and structure—important variables to consider in the self-assembly of nanodevices and surface coatings. Building off of our understanding of hydrophobic systems, we carefully studied surface diffusion at the interface of a hydrophobic surface and aqueous solution and found that diffusion could be rationally manipulated by changing the polarity of the solution, although the average diffusive behavior was strongly affected by the prevalence of anomalous surface sites. Finally, we developed a new surface mapping method to correlate the conformation and adsorption behavior of molecular building blocks on surfaces. We characterized alpha-helical peptides on deliberately patterned and nominally uniform surfaces of varying hydrophobicity and found that the peptide conformation and adsorption kinetics were sensitive to microscopic, lateral surface heterogeneity. In all of this work, spatial variations in surface chemistry were observed to profoundly affect surface dynamics with significant impact on self-assembly and chemical separation processes. More generally, we found that surface heterogeneity is seemingly ubiquitous and changes considerably the correct interpretation of ensemble-averaged experiments and molecular simulations.
Advisors/Committee Members: Daniel K. Schwartz, Joel Kaar, Thomas Perkins, Diego Krapf, Charles Musgrave.
Subjects/Keywords: adsorption; hydrophobic; single-molecule; solid-liquid interface; surface diffusion; Physical Chemistry; Process Control and Systems
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APA (6th Edition):
Mabry, J. N. (2015). Single-Molecule Dynamics at the Solid-Liquid Interface. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chbe_gradetds/83
Chicago Manual of Style (16th Edition):
Mabry, Joshua Nolan. “Single-Molecule Dynamics at the Solid-Liquid Interface.” 2015. Doctoral Dissertation, University of Colorado. Accessed March 04, 2021.
https://scholar.colorado.edu/chbe_gradetds/83.
MLA Handbook (7th Edition):
Mabry, Joshua Nolan. “Single-Molecule Dynamics at the Solid-Liquid Interface.” 2015. Web. 04 Mar 2021.
Vancouver:
Mabry JN. Single-Molecule Dynamics at the Solid-Liquid Interface. [Internet] [Doctoral dissertation]. University of Colorado; 2015. [cited 2021 Mar 04].
Available from: https://scholar.colorado.edu/chbe_gradetds/83.
Council of Science Editors:
Mabry JN. Single-Molecule Dynamics at the Solid-Liquid Interface. [Doctoral Dissertation]. University of Colorado; 2015. Available from: https://scholar.colorado.edu/chbe_gradetds/83
University of Colorado
9.
Liu, Minghua.
Study of Sub-cellular Structures upon Low-glucose Starvation in S. Pombe.
Degree: PhD, 2015, University of Colorado
URL: https://scholar.colorado.edu/mcdb_gradetds/35
► Many cells and organisms react to depletion of nutrients with an energy saving program. Here we studied Schizosaccharomyces pombe (S. pombe; fission yeast) cells…
(more)
▼ Many cells and organisms react to depletion of nutrients with an energy saving program. Here we studied
Schizosaccharomyces pombe (
S. pombe; fission yeast) cells and how they respond to nutrient starvation by entering a quiescent state that is characterized by a substantial viscosity increase of the cytoplasm, which we term "cytoplasmic freezing". Recently we found evidence that the transition in viscosity of the cell cytoplasm could be reliably reproduced by starving
S. pombe cells of glucose. Also, there is evidence that septins, a GTP binding protein family, might be involved in generating and maintaining the frozen cytoplasmic state. Here I have confirmed by light microscopy that in the absence of Spn2p the phenomenon of “cytoplasmic freezing” did not happen or occurred much later (Florin, unpublished). Taking advantage of confocal fluorescence microscopy, I have identified the relocation of Spn3p in wild type and
Spn2 deleted
S. pombe cells during exponential growth conditions and compared them to the situation upon up to seven days of glucose starvation. In addition using conventional electron microscopy and cryo-electron microscopy, I have imaged for the first time the formation of filamentous septin in starved
S. pombe cells. Similar septin bundles have been reported for different conditions, but not during the kind of starvation conditions reported here (An et al., 2004). I have further confirmed that absence of
Spn2 relocates Spn3p upon starvation in
S. pombe by immuno-labeling combined with conventional and cryo-electron microscopy. Also, I have observed by confocal microscopy that neither the deletion of
Spn2 nor the "cytoplasmic freezing" state in starved cells affect actin location in
S. pombe. However, actin behaves substantially different before versus after starvation, and during exponential growth its distribution is radically different between wildtype and
Spn2 deletion strains. These significant details can help our understanding of the role of septin during glucose starvation in
S. pombe or even in other organisms in the future. Furthermore, by conventional electron microscopy and cryo-electron microscopy I have demonstrated the shape change and fission of mitochondria from elongating tubular structures to separated small ovals upon glucose starvation in
S. pombe. I have also directly observed that during glucose starvation ribosomes tightly assemble at the outer membrane of mitochondria, which is a phenomenon that has not been shown in detail anywhere and its relation to glucose starvation is not well understood. During all the structural studies, I have also tested conditions of a new method that we named cryo-pickling, loosely in association of pickling vegetables etc. to make them tender and/or to preserve them by incubation with a brine solution. Here we tested whether it will be possible to make frozen-hydrated specimens accessible (e.g. vitrified sections) for antibodies…
Advisors/Committee Members: Mark Winey, Andreas Hoenger, Thomas Perkins, Gia Voeltz, Ernst-Ludwig Florin.
Subjects/Keywords: electron microscopy; fission yeast; light microscopy; mitochondria; septin; Biophysics; Cell Biology
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APA (6th Edition):
Liu, M. (2015). Study of Sub-cellular Structures upon Low-glucose Starvation in S. Pombe. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/mcdb_gradetds/35
Chicago Manual of Style (16th Edition):
Liu, Minghua. “Study of Sub-cellular Structures upon Low-glucose Starvation in S. Pombe.” 2015. Doctoral Dissertation, University of Colorado. Accessed March 04, 2021.
https://scholar.colorado.edu/mcdb_gradetds/35.
MLA Handbook (7th Edition):
Liu, Minghua. “Study of Sub-cellular Structures upon Low-glucose Starvation in S. Pombe.” 2015. Web. 04 Mar 2021.
Vancouver:
Liu M. Study of Sub-cellular Structures upon Low-glucose Starvation in S. Pombe. [Internet] [Doctoral dissertation]. University of Colorado; 2015. [cited 2021 Mar 04].
Available from: https://scholar.colorado.edu/mcdb_gradetds/35.
Council of Science Editors:
Liu M. Study of Sub-cellular Structures upon Low-glucose Starvation in S. Pombe. [Doctoral Dissertation]. University of Colorado; 2015. Available from: https://scholar.colorado.edu/mcdb_gradetds/35
University of Colorado
10.
Edelmaier, Christopher.
Computational Modeling of Mitosis in Fission Yeast.
Degree: PhD, 2018, University of Colorado
URL: https://scholar.colorado.edu/phys_gradetds/237
► Mitosis ensures the proper segregation of chromosomes into daughter cells, which is accomplished by the mitotic spindle. During fission yeast mitosis, chromosomes establish bi-orientation as…
(more)
▼ Mitosis ensures the proper segregation of chromosomes into daughter cells, which is accomplished by the mitotic spindle. During fission yeast mitosis, chromosomes establish bi-orientation as the bipolar spindle assembles, meaning that sister kinetochores become attached to microtubules whose growth was initiated by the two sister poles. This process includes mechanisms that correct erroneous attachments made by the kinetochores during the attachment process. This thesis presents a 3D physical model of spindle assembly in a Brownian dynamics-kinetic Monte Carlo simulation framework and a realistic description of the physics of microtubule, kinetochore, and chromosome dynamics, in order to interrogate the dynamics and mechanisms of chromosome bi-orientation and error correction. We have added chromosomes to our previous physical model of spindle assembly, which included microtubules, a spherical nuclear envelope, motor proteins, crosslinking proteins, and spindle pole bodies (centrosomes). In this work, we have explored the mechanical properties of kinetochores and their interactions with microtubules that achieve amphitelic spindle attachments at high frequency. A minimal physical model yields simulations that generate chromosome attachment errors, but resolves them, much as normal chromosomes do.
Advisors/Committee Members: Meredith D. Betterton, Matthew A. Glaser, Loren Hough, Thomas Perkins, Richard McIntosh.
Subjects/Keywords: computational; fission; mitosis; nonequilibrium; yeast; Biophysics; Cellular and Molecular Physiology; Statistical, Nonlinear, and Soft Matter Physics
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APA (6th Edition):
Edelmaier, C. (2018). Computational Modeling of Mitosis in Fission Yeast. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/phys_gradetds/237
Chicago Manual of Style (16th Edition):
Edelmaier, Christopher. “Computational Modeling of Mitosis in Fission Yeast.” 2018. Doctoral Dissertation, University of Colorado. Accessed March 04, 2021.
https://scholar.colorado.edu/phys_gradetds/237.
MLA Handbook (7th Edition):
Edelmaier, Christopher. “Computational Modeling of Mitosis in Fission Yeast.” 2018. Web. 04 Mar 2021.
Vancouver:
Edelmaier C. Computational Modeling of Mitosis in Fission Yeast. [Internet] [Doctoral dissertation]. University of Colorado; 2018. [cited 2021 Mar 04].
Available from: https://scholar.colorado.edu/phys_gradetds/237.
Council of Science Editors:
Edelmaier C. Computational Modeling of Mitosis in Fission Yeast. [Doctoral Dissertation]. University of Colorado; 2018. Available from: https://scholar.colorado.edu/phys_gradetds/237
University of Colorado
11.
Fiore, Julie L.
Single-Molecule Kinetic and Thermodynamic Studies of Cation-Facilitated RNA Folding: Isolated Tertiary Interactions.
Degree: PhD, Physics, 2011, University of Colorado
URL: https://scholar.colorado.edu/phys_gradetds/180
► RNA functional diversity is coupled with its ability to fold into unique structures, a process that is generally hierarchical – tertiary interactions occur between preformed…
(more)
▼ RNA functional diversity is coupled with its ability to fold into unique structures, a process that is generally hierarchical – tertiary interactions occur between preformed secondary structural elements, e.g., loops and helices. For RNA to fold into compact, biochemically competent shapes, counterion neutralization of the negatively charged-phosphate backbone is required. The objective of this thesis is to investigate the physical principles that dictate how an RNA molecule achieves and maintains its tertiary structure. Toward this end, single-molecule fluorescence resonance energy transfer methods are combined with temperature control to probe the mediation of RNA folding landscapes by cation-facilitated tertiary interactions.
The primary focus of this thesis is kinetic and thermodynamic characterization of the ubiquitous GAAA tetraloop-receptor tertiary interaction using freely diffusing and immobilized single-molecule assays. The apparent first-order rate constants (k
dock and k
undock) for the intramolecular docking/undocking of the tetraloop and receptor are measured as function of monovalent, divalent, and trivalent cation concentration. We observe that the [cation] needed to promote folding is correlated with charge density of the ion, which we interpret in terms of counterion condensation on the RNA. The temperature dependence of k
dock and k
undock are also determined, which yield the standard state and transition state free energies, enthalpies, and entropies for docking and undocking. At physiological conditions, the transition state for tetraloop-receptor docking is early, with its formation rate-limited by an entropic barrier. The overall docking reaction is exothermic and entropically costly, consistent with the large number of hydrogen bonding and base-stacking interactions formed by the tertiary contact. Surprisingly, we reveal an entropic origin of Mg
2+-facilitated RNA folding, which conflicts with the common expectation that increasing [Mg
2+] aids folding by reducing electrostatic repulsions of the RNA backbone. We propose instead that higher [Mg
2+] promotes folding by decreasing the entropic penalty of counterion uptake in the folding transition state and by reducing disorder in the unfolded conformational ensemble. This work elucidates potential RNA folding paradigms, such as early transitions states and an entropic origin of tertiary cooperativity and cation-facilitated folding.
Advisors/Committee Members: David J. Nesbitt, Arthur Pardi, W. Carl Lineberger, Thomas Perkins, Margaret Murnane.
Subjects/Keywords: FRET; RNA folding; singe molecule; tetraloop receptor; Biological and Chemical Physics; Chemistry; Physics
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APA ·
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MLA ·
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CSE |
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APA (6th Edition):
Fiore, J. L. (2011). Single-Molecule Kinetic and Thermodynamic Studies of Cation-Facilitated RNA Folding: Isolated Tertiary Interactions. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/phys_gradetds/180
Chicago Manual of Style (16th Edition):
Fiore, Julie L. “Single-Molecule Kinetic and Thermodynamic Studies of Cation-Facilitated RNA Folding: Isolated Tertiary Interactions.” 2011. Doctoral Dissertation, University of Colorado. Accessed March 04, 2021.
https://scholar.colorado.edu/phys_gradetds/180.
MLA Handbook (7th Edition):
Fiore, Julie L. “Single-Molecule Kinetic and Thermodynamic Studies of Cation-Facilitated RNA Folding: Isolated Tertiary Interactions.” 2011. Web. 04 Mar 2021.
Vancouver:
Fiore JL. Single-Molecule Kinetic and Thermodynamic Studies of Cation-Facilitated RNA Folding: Isolated Tertiary Interactions. [Internet] [Doctoral dissertation]. University of Colorado; 2011. [cited 2021 Mar 04].
Available from: https://scholar.colorado.edu/phys_gradetds/180.
Council of Science Editors:
Fiore JL. Single-Molecule Kinetic and Thermodynamic Studies of Cation-Facilitated RNA Folding: Isolated Tertiary Interactions. [Doctoral Dissertation]. University of Colorado; 2011. Available from: https://scholar.colorado.edu/phys_gradetds/180
University of Colorado
12.
Ozdilek, Bagdeser Akdogan.
Characterizing the RNA Binding Properties of the Intrinsically Disordered FUS Protein and RGG/RG Domains.
Degree: PhD, 2017, University of Colorado
URL: https://scholar.colorado.edu/mcdb_gradetds/77
► Recent developments in the comprehensive identification of the RNA-binding protein (RBP) repertoire has accelerated discovery of new RBPs. According to these studies, 20% of…
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▼ Recent developments in the comprehensive identification of the RNA-binding protein (RBP) repertoire has accelerated discovery of new RBPs. According to these studies, 20% of both known and novel RNA-binding proteins are highly disordered. Moreover, analysis of the human mRNA interactome revealed most disease related mutations are found within intrinsically disordered RNA-binding domains (RBDs). For most of these proteins, their RNA-binding properties are poorly characterized. Thus, deciphering intrinsically disordered RBD-RNA interactions on a molecular scale is essential to understanding their impact upon human physiology and diseases.
RGG/RG (arginine/glycine) domains are the second most common RNA binding domain in the human genome, yet their RNA-binding properties have not been well understood. Proteins containing RGG/RG domains regulate all aspects of RNA metabolism including transcription, processing, nucleocytoplasmic shuttling and translation. Proteins such as Fused in Sarcoma (FUS), Fragile X mental retardation (FMRP) and hnRNP U, bind a majority of the cellular transcriptome such that their recognition of RNAs has been considered to be non-specific or “promiscuous”.
Here, I report a detailed analysis of the RNA-binding characteristics of the RGG/RG domains from FUS, FMRP and hnRNP U. While previous studies of FUS focused on RNA binding by the RRM and zinc finger (ZnF) domains, my analysis showed RNA binding activity is driven by the RGG/RG domains. Further, I observed a strong synergy between the RRM and adjacent RGG/RG domains to achieve RNA binding affinities of the full-length FUS. To better characterize RNA-binding properties of RGG/RG domains, we have analyzed RGG/RG domains of FUS, FMRP and hnRNP U
in vitro against a spectrum of different RNAs with well-defined structural and sequence features. These experiments revealed that RGG/RG domains have different degrees of preference for binding to RNAs but share consistent trends in their selectivity towards RNAs with complex secondary structure. Thus, the binding behavior of RGG domains is best described as “degenerate specificity” reflecting that RGG/RG domains interact with a broad spectrum of RNAs that contain frequently observed sequence/structural elements. This mode of specificity is likely further facilitated by the intrinsically disordered nature of RGG/RG domains that enable them to adopt multiple conformations to adaptively bind RNA.
Advisors/Committee Members: Robert T. Batey, Ravinder Singh, Deborah S. Wuttke, Shelley Copley, Thomas Perkins.
Subjects/Keywords: RNA-binding protein; RGG/RG domains; Biochemistry; Molecular Biology
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APA ·
Chicago ·
MLA ·
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CSE |
Export
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APA (6th Edition):
Ozdilek, B. A. (2017). Characterizing the RNA Binding Properties of the Intrinsically Disordered FUS Protein and RGG/RG Domains. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/mcdb_gradetds/77
Chicago Manual of Style (16th Edition):
Ozdilek, Bagdeser Akdogan. “Characterizing the RNA Binding Properties of the Intrinsically Disordered FUS Protein and RGG/RG Domains.” 2017. Doctoral Dissertation, University of Colorado. Accessed March 04, 2021.
https://scholar.colorado.edu/mcdb_gradetds/77.
MLA Handbook (7th Edition):
Ozdilek, Bagdeser Akdogan. “Characterizing the RNA Binding Properties of the Intrinsically Disordered FUS Protein and RGG/RG Domains.” 2017. Web. 04 Mar 2021.
Vancouver:
Ozdilek BA. Characterizing the RNA Binding Properties of the Intrinsically Disordered FUS Protein and RGG/RG Domains. [Internet] [Doctoral dissertation]. University of Colorado; 2017. [cited 2021 Mar 04].
Available from: https://scholar.colorado.edu/mcdb_gradetds/77.
Council of Science Editors:
Ozdilek BA. Characterizing the RNA Binding Properties of the Intrinsically Disordered FUS Protein and RGG/RG Domains. [Doctoral Dissertation]. University of Colorado; 2017. Available from: https://scholar.colorado.edu/mcdb_gradetds/77
. 
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