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- Creators: Barrett, The Honors College
- Creators: Wang, Xiao
Description
Sensitivity is a fundamental challenge for in vivo molecular magnetic resonance imaging (MRI). Here, I improve the sensitivity of metal nanoparticle contrast agents by strategically incorporating pure and doped metal oxides in the nanoparticle core, forming a soluble, monodisperse, contrast agent with adjustable T2 or T1 relaxivity (r2 or r1). I first developed a simplified technique to incorporate iron oxides in apoferritin to form "magnetoferritin" for nM-level detection with T2- and T2* weighting. I then explored whether the crystal could be chemically modified to form a particle with high r1. I first adsorbed Mn2+ ions to metal binding sites in the apoferritin pores. The strategic placement of metal ions near sites of water exchange and within the crystal oxide enhance r1, suggesting a mechanism for increasing relaxivity in porous nanoparticle agents. However, the Mn2+ addition was only possible when the particle was simultaneously filled with an iron oxide, resulting in a particle with a high r1 but also a high r2 and making them undetectable with conventional T1-weighting techniques. To solve this problem and decrease the particle r2 for more sensitive detection, I chemically doped the nanoparticles with tungsten to form a disordered W-Fe oxide composite in the apoferritin core. This configuration formed a particle with a r1 of 4,870mM-1s-1 and r2 of 9,076mM-1s-1. These relaxivities allowed the detection of concentrations ranging from 20nM - 400nM in vivo, both passively injected and targeted to the kidney glomerulus. I further developed an MRI acquisition technique to distinguish particles based on r2/r1, and show that three nanoparticles of similar size can be distinguished in vitro and in vivo with MRI. This work forms the basis for a new, highly flexible inorganic approach to design nanoparticle contrast agents for molecular MRI.
ContributorsClavijo Jordan, Maria Veronica (Author) / Bennett, Kevin M (Thesis advisor) / Kodibagkar, Vikram (Committee member) / Sherry, A Dean (Committee member) / Wang, Xiao (Committee member) / Yarger, Jeffery (Committee member) / Arizona State University (Publisher)
Created2012
Description
Recombinases are powerful tools for genome engineering and synthetic biology, however recombinases are limited by a lack of user-programmability and often require complex directed-evolution experiments to retarget specificity. Conversely, CRISPR systems have extreme versatility yet can induce off-target mutations and karyotypic destabilization. To address these constraints we developed an RNA-guided recombinase protein by fusing a hyperactive mutant resolvase from transposon TN3 to catalytically inactive Cas9. We validated recombinase-Cas9 (rCas9) function in model eukaryote Saccharomyces cerevisiae using a chromosomally integrated fluorescent reporter. Moreover, we demonstrated cooperative targeting by CRISPR RNAs at spacings of 22 or 40bps is necessary for directing recombination. Using PCR and Sanger sequencing, we confirmed rCas9 targets DNA recombination. With further development we envision rCas9 becoming useful in the development of RNA-programmed genetic circuitry as well as high-specificity genome engineering.
ContributorsStandage-Beier, Kylie S (Author) / Wang, Xiao (Thesis advisor) / Brafman, David A (Committee member) / Tian, Xiao-jun (Committee member) / Arizona State University (Publisher)
Created2018
Description
Synthetic biology is an emerging field which melds genetics, molecular biology, network theory, and mathematical systems to understand, build, and predict gene network behavior. As an engineering discipline, developing a mathematical understanding of the genetic circuits being studied is of fundamental importance. In this dissertation, mathematical concepts for understanding, predicting, and controlling gene transcriptional networks are presented and applied to two synthetic gene network contexts. First, this engineering approach is used to improve the function of the guide ribonucleic acid (gRNA)-targeted, dCas9-regulated transcriptional cascades through analysis and targeted modification of the RNA transcript. In so doing, a fluorescent guide RNA (fgRNA) is developed to more clearly observe gRNA dynamics and aid design. It is shown that through careful optimization, RNA Polymerase II (Pol II) driven gRNA transcripts can be strong enough to exhibit measurable cascading behavior, previously only shown in RNA Polymerase III (Pol III) circuits. Second, inherent gene expression noise is used to achieve precise fractional differentiation of a population. Mathematical methods are employed to predict and understand the observed behavior, and metrics for analyzing and quantifying similar differentiation kinetics are presented. Through careful mathematical analysis and simulation, coupled with experimental data, two methods for achieving ratio control are presented, with the optimal schema for any application being dependent on the noisiness of the system under study. Together, these studies push the boundaries of gene network control, with potential applications in stem cell differentiation, therapeutics, and bio-production.
ContributorsMenn, David J (Author) / Wang, Xiao (Thesis advisor) / Kiani, Samira (Committee member) / Haynes, Karmella (Committee member) / Nielsen, David (Committee member) / Marshall, Pamela (Committee member) / Arizona State University (Publisher)
Created2018
Description
Calcium is the only ion capable of triggering electrical and chemical reactions in cells which are part of essential biomolecular processes, such as gene transcription and ion flux. Calcium homeostasis, the control of concentration levels, is therefore crucial for the proper functioning of cells. For example, cardiomyocytes, the cells that form cardiac muscle, rely on calcium transfer process to produce muscle contraction.
The purpose of this work is to study aspects of calcium homeostasis in the model organism Saccharomyces cerevisiae, common yeast. Using luminometric techniques, the response of the yeast was monitored against a set of changes in the environment calcium abundance. The results indicate a complex response as both increase and decreases of external calcium induce elevations in cytosolic calcium concentrations.
Calcium is transferred across compartments by means of channels. In Saccharomyces cerevisiae, many of them have been identified; Cch1p-Mid1p, Vcx1p, Pmc1p, Pmr1p, and Yvc1p. Their participation in calcium homeostasis is well established. Observations of cytosolic calcium increase after a hypertonic shock are mainly associated with influx of ions from the environment though the Cch1p-Mid1p. This process is generally considered as driven by calcium concentration gradients. However, recent studies have suggested that the plasma membrane channel, Cch1p-Mid1p, may possess more sophisticated regulation and sensory mechanisms. The results of our experiments support these ideas.
We carried out experiments that subjected yeast to multiple shocks: a hypertonic shock followed by either a second hypertonic shock, a hypotonic shock, or a yeast dilution pulse where the solution volume increases by the calcium concentration has only a small change. The cytosolic calcium concentration of a yeast population was monitored via luminometry.
The main result of this study is the observation of an unexpected response to the combination of hypertonic and hypotonic shocks. In this case it was observed that the cytosolic calcium concentration increased after both shocks. This indicates that cytosolic calcium increases are not solely driven by the presence of concentration gradients. The response after the hypotonic pulse arises from more complex mechanisms that may include sensor activity at the membrane channels and the release of calcium from internal storages.
The purpose of this work is to study aspects of calcium homeostasis in the model organism Saccharomyces cerevisiae, common yeast. Using luminometric techniques, the response of the yeast was monitored against a set of changes in the environment calcium abundance. The results indicate a complex response as both increase and decreases of external calcium induce elevations in cytosolic calcium concentrations.
Calcium is transferred across compartments by means of channels. In Saccharomyces cerevisiae, many of them have been identified; Cch1p-Mid1p, Vcx1p, Pmc1p, Pmr1p, and Yvc1p. Their participation in calcium homeostasis is well established. Observations of cytosolic calcium increase after a hypertonic shock are mainly associated with influx of ions from the environment though the Cch1p-Mid1p. This process is generally considered as driven by calcium concentration gradients. However, recent studies have suggested that the plasma membrane channel, Cch1p-Mid1p, may possess more sophisticated regulation and sensory mechanisms. The results of our experiments support these ideas.
We carried out experiments that subjected yeast to multiple shocks: a hypertonic shock followed by either a second hypertonic shock, a hypotonic shock, or a yeast dilution pulse where the solution volume increases by the calcium concentration has only a small change. The cytosolic calcium concentration of a yeast population was monitored via luminometry.
The main result of this study is the observation of an unexpected response to the combination of hypertonic and hypotonic shocks. In this case it was observed that the cytosolic calcium concentration increased after both shocks. This indicates that cytosolic calcium increases are not solely driven by the presence of concentration gradients. The response after the hypotonic pulse arises from more complex mechanisms that may include sensor activity at the membrane channels and the release of calcium from internal storages.
ContributorsMintz, David Anthony (Co-author) / Parker, Augustus (Co-author) / Solis, Francisco (Thesis director) / Marshall, Pamela (Committee member) / School of Mathematical and Natural Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Transient Receptor Potential (TRP) ion channels are a diverse family of nonselective, polymodal sensors in uni- and multicellular eukaryotes that are implicated in an assortment of biological contexts and human disease. The cold-activated TRP Melastatin-8 (TRPM8) channel, also recognized as the human body's primary cold sensor, is among the few TRP channels responsible for thermosensing. Despite sustained interest in the channel, the mechanisms underlying TRPM8 activation, modulation, and gating have proved challenging to study and remain poorly understood. In this thesis, I offer data collected on various expression, extraction, and purification conditions tested in E. Coli expression systems with the aim to optimize the generation of a structurally stable and functional human TRPM8 pore domain (S5 and S6) construct for application in structural biology studies. These studies, including the biophysical technique nuclear magnetic spectroscopy (NMR), among others, will be essential for elucidating the role of the TRPM8 pore domain in in regulating ligand binding, channel gating, ion selectively, and thermal sensitivity. Moreover, in the second half of this thesis, I discuss the ligation-independent megaprimer PCR of whole-plasmids (MEGAWHOP PCR) cloning technique, and how it was used to generate chimeras between TRPM8 and its nearest analog TRPM2. I review steps taken to optimize the efficiency of MEGAWHOP PCR and the implications and unique applications of this novel methodology for advancing recombinant DNA technology. I lastly present preliminary electrophysiological data on the chimeras, employed to isolate and study the functional contributions of each individual transmembrane helix (S1-S6) to TRPM8 menthol activation. These studies show the utility of the TRPM8\u2014TRPM2 chimeras for dissecting function of TRP channels. The average current traces analyzed thus far indicate that the S2 and S3 helices appear to play an important role in TRPM8 menthol modulation because the TRPM8[M2S2] and TRPM8[M2S3] chimeras significantly reduce channel conductance in the presence of menthol. The TRPM8[M2S4] chimera, oppositely, increases channel conductance, implying that the S4 helix in native TRPM8 may suppress menthol modulation. Overall, these findings show that there is promise in the techniques chosen to identify specific regions of TRPM8 crucial to menthol activation, though the methods chosen to study the TRPM8 pore independent from the whole channel may need to be reevaluated. Further experiments will be necessary to refine TRPM8 pore solubilization and purification before structural studies can proceed, and the electrophysiology traces observed for the chimeras will need to be further verified and evaluated for consistency and physiological significance.
ContributorsWaris, Maryam Siddika (Author) / Van Horn, Wade (Thesis director) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Department of English (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Bacteria have been shown to possess a large array of regulatory mechanisms to not just respond to a diverse array of environmental stresses, but to injurious artificial proteins as well. A previous investigation introduced DX, a man-made ATP sequestering protein into Escherichia coli (E. coli) which resulted in the formation of novel endoliposome structures and induced a viable but non-culturable state (VBNC) that was not easily reversed. It was hypothesized that the broadly conserved bacterial stringent response pathway may have been responsible for the observed phenotypic changes. With the goal of unveiling the molecular mechanism behind this novel response, changes in cellular morphology and physiology upon DX expression were assessed in a population of E. coli encoding a dysfunctional relA gene, one of the two genes controlling the induction of the stringent response. It was ultimately shown that RelA directly contributed to cellular filamentation, endoliposome structure formation, and the induction of a VBNC state. While the stringent response has been extensively shown to induce a VBNC state, to our knowledge, relA has not yet been shown to induce filamentation or coordinate the formation of endoliposome structures in bacteria. As the stringent response has been shown to be increasingly involved in antibiotic tolerance, this study provided an exciting opportunity to further characterize this adaptive response pathway to aid in the future development of novel therapeutics. In addition to this, this study continued to highlight that the DX protein may serve one of the first tools to allow for the direct selection of bacteria in a VBNC state by morphologically distinguishing non-culturable cells through cellular filamentation.
ContributorsFrost, Fredrick Charles (Author) / Chaput, John (Thesis director) / Wachter, Rebekka (Committee member) / Korch, Shaleen (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-12
Description
Nucleic acids encode the information required to create life, and polymerases are the gatekeepers charged with maintaining the storage and flow of this genetic information. Synthetic biologists utilize this universal property to modify organisms and other systems to create unique traits or improve the function of others. One of the many realms in synthetic biology involves the study of biopolymers that do not exist naturally, which is known as xenobiology. Although life depends on two biopolymers for genetic storage, it may be possible that alternative molecules (xenonucleic acids – XNAs), could be used in their place in either a living or non-living system. However, implementation of an XNA based system requires the development of polymerases that can encode and decode information stored in these artificial polymers. A strategy called directed evolution is used to modify or alter the function of a protein of interest, but identifying mutations that can modify polymerase function is made problematic by their size and overall complexity. To reduce the amount of sequence space that needs to be samples when attempting to identify polymerase variants, we can try to make informed decisions about which amino acid residues may have functional roles in catalysis. An analysis of Family B polymerases has shown that residues which are involved in substrate specificity are often highly conserved both at the sequence and structure level. In order to validate the hypothesis that a strong correlation exists between structural conservation and catalytic activity, we have selected and mutated residues in the 9°N polymerase using a loss of function mutagenesis strategy based on a computational analysis of several homologues from a diverse range of taxa. Improvement of these models will hopefully lead to quicker identification of loci which are ideal engineering targets.
ContributorsHaeberle, Tyler Matthew (Author) / Chaput, John (Thesis director) / Chen, Julian (Committee member) / Larsen, Andrew (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Transgene expression in mammalian cells has been shown to meet resistance in the form of silencing due to chromatin buildup within the cell. Interactions of proteins with chromatin modulate gene expression profiles. Synthetic Polycomb transcription factor (PcTF) variants have the potential to reactivate these silence transgenes as shown in Haynes & Silver 2011. PcTF variants have been constructed via TypeIIS assembly to further investigate this ability to reactive transgenes. Expression in mammalian cells was confirmed via fluorescence microscopy and red fluorescent protein (RFP) expression in cell lysate. Examination of any variation in conferment of binding strength of homologous Polycomb chromodomains (PCDs) to its trimethylated lysine residue target on histone three (H3K27me3) was investigated using a thermal shift assay. Results indicate that PcTF may not be a suitable protein for surveying with SYPRO Orange, a dye that produces a detectable signal when exposed to the hydrophobic domains of the melting protein. A cell line with inducible silencing of a chemiluminescent protein was used to determine the effects PcTF variants had on gene reactivation. Results show down-regulation of the target reporter gene. We propose this may be due to PcTF not binding to its target; this would cause PcTF to deplete transcriptional machinery in the nucleus. Alternatively, the CMV promoter could be sequestering transcriptional machinery in its hyperactive transcription of PcTF leading to widespread down-regulation. Finally, the activation domain used may not be appropriate for this cell type. Future PcTF variants will address these hypotheses by including multiple Polycomb chromodomains (PCDs) to alter the binding dynamics of PcTF to its target, and by incorporating alternative promoters and activation domains.
ContributorsGardner, Cameron Lee (Author) / Haynes, Karmella (Thesis director) / Stabenfeldt, Sarah (Committee member) / Barrett, The Honors College (Contributor) / Department of Finance (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
Description
One major issue that surgeons face during closed body cavity surgery is fogging of the lens surfaces. The cloudy and opaque lens surface caused by water vapor present in closed body cavities forces the surgeon to repeatedly remove the endoscope, wipe it, and reinsert it back into the patient. This presents several risks such as increased surgery time, greater scarring, and an increased chance of infection. In order to address this issue, the development of the Thin Fluid Film Device (TFFD™) VitreOx™ aims to render the lens surface hydrophilic, whereas it is typically hydrophobic. By creating a hydrophilic polymeric nanomesh, the 3-D water droplets can be trapped to lie flatter, thus resulting in a flatter 2-D sheeting effect. The light can no longer be refracted at different angles off of the 3-dimensional water beads, thus eliminating the opacity of the lens surface.
Two animal trials were performed involving a rat and two pigs in order to prove the efficacy of VitreOx™ in addition to being compared with competitor, Covidien Clearify. A laparoscopy was performed on each animal, and the length of time that the endoscope took to fog was measured post product application. The results of the optimized animal clinical trials involving two Yucatan pigs showed that the scope treated with Covidien’s Clearify began fogging within 8 minutes and continued to do so for the remained of the surgery, as opposed to the scope with VitreOx™ which remained fog free for the full 90-minute procedure. The results proved the efficacy of our product.
The second part of the thesis aimed to optimize HemoClear™, the blood evacuating TFFD™. This was done by testing a higher concentration of 6 mg/mL fibrinogen as compared to previous work. After conducting an experiment designed to mimic closed-body cavity surgery it was determined that the HemoClear™ eliminated fog 67% of the time and evacuated blood with a success of 83%. Future work aims to continue testing at this concentration with variances in mixing and application technique.
Two animal trials were performed involving a rat and two pigs in order to prove the efficacy of VitreOx™ in addition to being compared with competitor, Covidien Clearify. A laparoscopy was performed on each animal, and the length of time that the endoscope took to fog was measured post product application. The results of the optimized animal clinical trials involving two Yucatan pigs showed that the scope treated with Covidien’s Clearify began fogging within 8 minutes and continued to do so for the remained of the surgery, as opposed to the scope with VitreOx™ which remained fog free for the full 90-minute procedure. The results proved the efficacy of our product.
The second part of the thesis aimed to optimize HemoClear™, the blood evacuating TFFD™. This was done by testing a higher concentration of 6 mg/mL fibrinogen as compared to previous work. After conducting an experiment designed to mimic closed-body cavity surgery it was determined that the HemoClear™ eliminated fog 67% of the time and evacuated blood with a success of 83%. Future work aims to continue testing at this concentration with variances in mixing and application technique.
ContributorsSinha, Saloni Agarwal (Author) / Culbertson, Robert (Thesis director) / Herbots, Nicole (Committee member) / Watson, Clarizza (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Exploring Structure and Function of Human Cold Sensing Protein TRPM8 with ROSETTA Comparative Models
Description
Transient Receptor Potential (TRP) channels are a diverse class of ion channels notable as polymodal sensors. TRPM8 is a TRP channel implicated in cold sensation, nociception, and a variety of human diseases, including obesity and cancer. Despite sustained interest in TRPM8 since its discovery in 2001, many of the molecular mechanisms that underlie function are not yet clear. Knowledge of these properties could have implications for medicine and physiological understanding of sensation and signaling. Structures of TRP channels have proven challenging to solve, but recent Cryoelectron microscopy (Cryo-EM) structures of TRPV1 provide a basis for homology-based modeling of TRP channel structures and interactions. I present an ensemble of 11,000 Rosetta computational homology models of TRPM8 based on the recent Cryo-EM apo structure of TRPV1 (PDB code:3J5P). Site-directed mutagenesis has provided clues about which residues are most essential for modulatory ligands to bind, so the models presented provide a platform to investigate the structural basis of TRPM8 ligand modulation complementary to existing functional and structural information. Menthol and icilin appear to interact with interfacial residues in the sensor domain (S1-S4). One consensus feature of these sites is the presence of local contacts to the S4 helix, suggesting this helix may be mechanistically involved with the opening of the pore. Phosphatidylinositol 4,5-bisphosphate (PIP2)has long been known to interact with the C-terminus of TRPM8, and some of the homology models contain plausible binding pockets where PIP2 can come into contact with charged residues known to be essential for PIP2 modulation. Future in silico binding experiments could provide testable hypothesis for in vitro structural studies, and experimental data (e.g. distance constraints from electron paramagnetic resonance spectroscopy [EPR]) could further refine the models.
ContributorsHelsell, Cole Vincent Maher (Author) / Van Horn, Wade (Thesis director) / Wang, Xu (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05