Matching Items (24)
Filtering by

Clear all filters

152636-Thumbnail Image.png
Description
Rapid and reliable separation and analysis of proteins require powerful analytical methods. The analysis of proteins becomes especially challenging when only small sample volumes are available, concomitantly with low concentrations of proteins. Time critical situations pose additional challenges. Due to these challenges, conventional macro-scale separation techniques reach their limitations. While

Rapid and reliable separation and analysis of proteins require powerful analytical methods. The analysis of proteins becomes especially challenging when only small sample volumes are available, concomitantly with low concentrations of proteins. Time critical situations pose additional challenges. Due to these challenges, conventional macro-scale separation techniques reach their limitations. While microfluidic devices require only pL-nL sample volumes, they offer several advantages such as speed, efficiency, and high throughput. This work elucidates the capability to manipulate proteins in a rapid and reliable manner with a novel migration technique, namely dielectrophoresis (DEP). Since protein analysis can often be achieved through a combination of orthogonal techniques, adding DEP as a gradient technique to the portfolio of protein manipulation methods can extend and improve combinatorial approaches. To this aim, microfluidic devices tailored with integrated insulating obstacles were fabricated to create inhomogeneous electric fields evoking insulator-based DEP (iDEP). A main focus of this work was the development of pre-concentration devices where topological micropost arrays are fabricated using standard photo- and soft lithographic techniques. With these devices, positive DEP-driven streaming of proteins was demonstrated for the first time using immunoglobulin G (IgG) and bovine serum albumin. Experimentally observed iDEP concentrations of both proteins were in excellent agreement with positive DEP concentration profiles obtained by numerical simulations. Moreover, the micropost iDEP devices were improved by introducing nano-constrictions with focused ion beam milling with which numerical simulations suggested enhancement of the DEP effect, leading to a 12-fold increase in concentration of IgG. Additionally, concentration of β-galactosidase was observed, which seems to occur due to an interplay of negative DEP, electroosmosis, electrokinesis, diffusion, and ion concentration polarization. A detailed study was performed to investigate factors influencing protein DEP under DC conditions, including electroosmosis, electrophoresis, and Joule heating. Specifically, temperature rise within the iDEP device due to Joule heating was measured experimentally with spatial and temporal resolution by employing the thermosensitive dye Rhodamine B. Unlike DNA and cells, protein DEP behavior is not well understood to date. Therefore, this detailed study of protein DEP provides novel information to eventually optimize this protein migration method for pre-concentration, separation, and fractionation.
ContributorsNakano, Asuka (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2014
152827-Thumbnail Image.png
Description
Ribulose-1, 5-bisphosphate carboxylase oxygenase, commonly known as RuBisCO, is an enzyme involved in carbon fixation in photosynthetic organisms. The enzyme is subject to a mechanism-based deactivation during its catalytic cycle. RuBisCO activase (Rca), an ancillary enzyme belonging to the AAA+ family of the ATP-ases, rescues RuBisCO by facilitating the removal

Ribulose-1, 5-bisphosphate carboxylase oxygenase, commonly known as RuBisCO, is an enzyme involved in carbon fixation in photosynthetic organisms. The enzyme is subject to a mechanism-based deactivation during its catalytic cycle. RuBisCO activase (Rca), an ancillary enzyme belonging to the AAA+ family of the ATP-ases, rescues RuBisCO by facilitating the removal of the tightly bound sugar phosphates from the active sites of RuBisCO. In this work, we investigated the ATP/ADP dependent oligomerization equilibrium of fluorescently tagged Rca for a wide range of concentrations using fluorescence correlation spectroscopy. Results show that in the presence of ADP-Mg2+, the oligomerization state of Rca gradually changes in steps of two subunits. The most probable association model supports the dissociation constants (K_d) of ∼4, 1, 1 μM for the monomer-dimer, dimer-tetramer, and tetramer-hexamer equlibria, respectively. Rca continues to assemble at higher concentrations which are indicative of the formation of aggregates. In the presence of ATP-Mg2+, a similar stepwise assembly is observed. However, at higher concentrations (30-75 µM), the average oligomeric size remains relatively unchanged around six subunits per oligomer. This is in sharp contrast with observations in ADP-Mg2+, where a marked decrease in the diffusion coefficient of Rca was observed, consistent with the formation of aggregates. The estimated K_d values obtained from the analysis of the FCS decays were similar for the first steps of the assembly process in both ADP-Mg2+ and ATP-Mg2+. However, the formation of the hexamer from the tetramer is much more favored in ATP-Mg2+, as evidenced from 20 fold lower K_d associated with this assembly step. This suggests that the formation of a hexameric ring in the presence of ATP-Mg2+. In addition to that, Rca aggregation is largely suppressed in the presence of ATP-Mg2+, as evidenced from the 1000 fold larger K_d value for the hexamer-24 mer association step. In essence, a fluorescence-based method was developed to monitor in vitro protein oligomerization and was successfully applied with Rca. The results provide a strong hint at the active oligomeric structure of Rca, and this information will hopefully help the ongoing research on the mechanistic enzymology of Rca.
ContributorsChakraborty, Manas (Author) / Levitus, Marcia (Thesis advisor) / Angell, Charles (Committee member) / Lindsay, Stuart (Committee member) / Arizona State University (Publisher)
Created2014
153458-Thumbnail Image.png
Description
Biophysical techniques have been increasingly applied toward answering biological questions with more precision. Here, three different biological systems were studied with the goal of understanding their dynamic differences, either conformational dynamics within the system or oligomerization dynamics between monomers. With Cy3 on the 5' end of DNA, the

Biophysical techniques have been increasingly applied toward answering biological questions with more precision. Here, three different biological systems were studied with the goal of understanding their dynamic differences, either conformational dynamics within the system or oligomerization dynamics between monomers. With Cy3 on the 5' end of DNA, the effects of changing the terminal base pair were explored using temperature-dependent quantum yields. It was discovered, in combination with simulations, that a terminal thymine base has the weakest stacking interactions with the Cy3 dye compared to the other three bases. With ME1 heterodimers, the goal was to see if engineering a salt bridge at the dimerization interface could allow for control over dimerization in a pH-dependent manner. This was performed experimentally by measuring FRET between monomers containing either a Dap or an Asp mutation and comparing FRET efficiency at different pHs. It was demonstrated that the heterodimeric salt bridge would only form in a pH range near neutrality. Finally, with DNA processivity clamps, one aim was to compare the equilibrium dissociation constants, kinetic rate constants, and lifetimes of the closed rings for beta clamp and PCNA. This was done using a variety of biophysical techniques but with three as the main focus: fluorescence correlation spectroscopy, single-molecule experiments, and time-correlated single photon counting measurements. The stability of beta clamp was found to be three orders of magnitude higher when measuring solution stability but only one order of magnitude higher when measuring intrinsic stability, which is a result of salt bridge interactions in the interface of beta clamp. Ongoing work built upon the findings from this project by attempting to disrupt interface stability of different beta clamp mutants by adding salt or changing the pH of the solution. Lingering questions about the dynamics of different areas of the clamps has led to another project for which we have developed a control to demystify some unexpected similarities between beta clamp mutants. With that project, we show that single-labeled and double-labeled samples have similar autocorrelation decays in florescence correlation spectroscopy, allowing us to rule out the dyes themselves as causing fluctuations in the 10-100 microsecond timescale.
ContributorsBinder, Jennifer (Author) / Levitus, Marcia (Thesis advisor) / Wachter, Rebekka (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2015
153119-Thumbnail Image.png
Description
The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA

The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA has been reported to define the onset of certain types of cancers.

Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed.

Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before.

Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages.

DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue.
ContributorsRoy, Basab (Author) / Hecht, Sidney M. (Thesis advisor) / Jones, Anne (Committee member) / Levitus, Marcia (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)
Created2014
153344-Thumbnail Image.png
Description
Increasing concentrations of carbon dioxide in the atmosphere will inevitably lead to long-term changes in climate that can have serious consequences. Controlling anthropogenic emission of carbon dioxide into the atmosphere, however, represents a significant technological challenge. Various chemical approaches have been suggested, perhaps the most promising of these is based

Increasing concentrations of carbon dioxide in the atmosphere will inevitably lead to long-term changes in climate that can have serious consequences. Controlling anthropogenic emission of carbon dioxide into the atmosphere, however, represents a significant technological challenge. Various chemical approaches have been suggested, perhaps the most promising of these is based on electrochemical trapping of carbon dioxide using pyridine and derivatives. Optimization of this process requires a detailed understanding of the mechanisms of the reactions of reduced pyridines with carbon dioxide, which are not currently well known. This thesis describes a detailed mechanistic study of the nucleophilic and Bronsted basic properties of the radical anion of bipyridine as a model pyridine derivative, formed by one-electron reduction, with particular emphasis on the reactions with carbon dioxide. A time-resolved spectroscopic method was used to characterize the key intermediates and determine the kinetics of the reactions of the radical anion and its protonated radical form. Using a pulsed nanosecond laser, the bipyridine radical anion could be generated in-situ in less than 100 ns, which allows fast reactions to be monitored in real time. The bipyridine radical anion was found to be a very powerful one-electron donor, Bronsted base and nucleophile. It reacts by addition to the C=O bonds of ketones with a bimolecular rate constant around 1* 107 M-1 s-1. These are among the fastest nucleophilic additions that have been reported in literature. Temperature dependence studies demonstrate very low activation energies and large Arrhenius pre-exponential parameters, consistent with very high reactivity. The kinetics of E2 elimination, where the radical anion acts as a base, and SN2 substitution, where the radical anion acts as a nucleophile, are also characterized by large bimolecular rate constants in the range ca. 106 - 107 M-1 s-1. The pKa of the bipyridine radical anion was measured using a kinetic method and analysis of the data using a Marcus theory model for proton transfer. The bipyridine radical anion is found to have a pKa of 40±5 in DMSO. The reorganization energy for the proton transfer reaction was found to be 70±5 kJ/mol. The bipyridine radical anion was found to react very rapidly with carbon dioxide, with a bimolecular rate constant of 1* 108 M-1 s-1 and a small activation energy, whereas the protonated radical reacted with carbon dioxide with a rate constant that was too small to measure. The kinetic and thermodynamic data obtained in this work can be used to understand the mechanisms of the reactions of pyridines with carbon dioxide under reducing conditions.
ContributorsRanjan, Rajeev (Author) / Gould, Ian R (Thesis advisor) / Buttry, Daniel A (Thesis advisor) / Yarger, Jeff (Committee member) / Seo, Dong-Kyun (Committee member) / Arizona State University (Publisher)
Created2015
153253-Thumbnail Image.png
Description
Chloroform (CHCl3) is an important atmospheric pollutant by its direct health effects as well as by its contribution to photochemical smog formation. Chloroform outgassing from swimming pools is not typically considered a source of atmospheric CHCl3 because swimming pools are scarce compared to other sources. However, large urban areas in

Chloroform (CHCl3) is an important atmospheric pollutant by its direct health effects as well as by its contribution to photochemical smog formation. Chloroform outgassing from swimming pools is not typically considered a source of atmospheric CHCl3 because swimming pools are scarce compared to other sources. However, large urban areas in hot climates such as Phoenix, AZ contain a substantial amount of swimming pools, potentially resulting in significant atmospheric fluxes. In this study, CHCl3 formation potential (FP) from disinfection of swimming pools in Phoenix was investigated through laboratory experiments and annual CHCl3 emission fluxes from swimming pools were estimated based on the experimental data.

Swimming pool water (collected in June 2014 in Phoenix) and model contaminants (Pharmaceuticals and Personal Care Products (PPCPs), Endocrine Disrupting Compounds (EDCs), artificial sweeteners, and artificial human waste products) were chlorinated in controlled laboratory experiments. The CHCl3 production during chlorination was determined using Gas Chromatography-Mass Spectrometry (GC-MS) following solid-phase microextraction (SPME). Upon chlorination, all swimming pool water samples and contaminants produced measureable amounts of chloroform. Chlorination of swimming pool water produced 0.005-0.134 mol CHCl3/mol C and 0.004-0.062 mol CHCl3/mol Cl2 consumed. Chlorination of model contaminants produced 0.004-0.323 mol CHCl3/mol C and 0.001-0.247 mol CHCl3/mol Cl2 consumed. These numbers are comparable and indicate that the model contaminants react similarly to swimming pool water during chlorination. The CHCl3 flux from swimming pools in Phoenix was estimated at approximately 3.9-4.3 Gg/yr and was found to be largely dependent on water temperature and wind speed while air temperature had little effect. This preliminary estimate is orders of magnitude larger than previous estimates of anthropogenic emissions in Phoenix suggesting that swimming pools might be a significant source of atmospheric CHCl3 locally.
ContributorsRose, Christy J (Author) / Herckes, Pierre (Thesis advisor) / Fraser, Matthew (Committee member) / Hayes, Mark (Committee member) / Westerhoff, Paul (Committee member) / Arizona State University (Publisher)
Created2014
149856-Thumbnail Image.png
Description
Nucleosomes are the basic repetitive unit of eukaryotic chromatin and are responsible for packing DNA inside the nucleus of the cell. They consist of a complex of eight histone proteins (two copies of four proteins H2A, H2B, H3 and H4) around which 147 base pairs of DNA are wrapped

Nucleosomes are the basic repetitive unit of eukaryotic chromatin and are responsible for packing DNA inside the nucleus of the cell. They consist of a complex of eight histone proteins (two copies of four proteins H2A, H2B, H3 and H4) around which 147 base pairs of DNA are wrapped in ~1.67 superhelical turns. Although the nucleosomes are stable protein-DNA complexes, they undergo spontaneous conformational changes that occur in an asynchronous fashion. This conformational dynamics, defined by the "site-exposure" model, involves the DNA unwrapping from the protein core and exposing itself transiently before wrapping back. Physiologically, this allows regulatory proteins to bind to their target DNA sites during cellular processes like replication, DNA repair and transcription. Traditional biochemical assays have stablished the equilibrium constants for the accessibility to various sites along the length of the nucleosomal DNA, from its end to the middle of the dyad axis. Using fluorescence correlation spectroscopy (FCS), we have established the position dependent rewrapping rates for nucleosomes. We have also used Monte Carlo simulation methods to analyze the applicability of FRET fluctuation spectroscopy towards conformational dynamics, specifically motivated by nucleosome dynamics. Another important conformational change that is involved in cellular processes is the disassembly of nucleosome into its constituent particles. The exact pathway adopted by nucleosomes is still not clear. We used dual color fluorescence correlation spectroscopy to study the intermediates during nucleosome disassembly induced by changing ionic strength. Studying the nature of nucleosome conformational change and the kinetics is very important in understanding gene expression. The results from this thesis give a quantitative description to the basic unit of the chromatin.
ContributorsGurunathan, Kaushik (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Woodbury, Neal (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2011
150715-Thumbnail Image.png
Description
Most of the sunlight powering natural photosynthesis is absorbed by antenna arrays that transfer, and regulate the delivery of excitation energy to reaction centers in the chloroplast where photosynthesis takes place. Under intense sunlight the plants and certain organisms cannot fully utilize all of the sunlight received by antennas and

Most of the sunlight powering natural photosynthesis is absorbed by antenna arrays that transfer, and regulate the delivery of excitation energy to reaction centers in the chloroplast where photosynthesis takes place. Under intense sunlight the plants and certain organisms cannot fully utilize all of the sunlight received by antennas and excess redox species are formed which could potentially harm them. To prevent this, excess energy is dissipated by antennas before it reaches to the reaction centers to initiate electron transfer needed in the next steps of photosynthesis. This phenomenon is called non-photochemical quenching (NPQ). The mechanism of NPQ is not fully understood, but the process is believed to be initiated by a drop in the pH in thylakoid lumen in cells. This causes changes in otherwise nonresponsive energy acceptors which accept the excess energy, preventing oversensitization of the reaction center. To mimic this phenomenon and get insight into the mechanism of NPQ, a novel pH sensitive dye 3'6'-indolinorhodamine was designed and synthesized which in a neutral solution stays in a closed (colorless) form and does not absorb light while at low pH it opens (colored) and absorbs light. The absorption of the dye overlaps porphyrin emission, thus making energy transfer from the porphyrin to the dye thermodynamically possible. Several self-regulating molecular model systems were designed and synthesized consisting of this dye and zinc porphyrins organized on a hexaphenylbenzene framework to functionally mimic the role of the antenna in NPQ. When a dye-zinc porphyrin dyad is dissolved in an organic solvent, the zinc porphyrin antenna absorbs and emits light by normal photophysical processes. Time resolved fluorescence experiments using the single-photon-timing method with excitation at 425 nm and emission at 600 nm yielded a lifetime of 2.09 ns for the porphyrin first excited singlet state. When acetic acid is added to the solution of the dyad, the pH sensitive dye opens and quenches the zinc porphyrin emission decreasing the lifetime of the porphyrin first excited singlet state to 23 ps, and converting the excitation energy to heat. Under similar experimental conditions in a neutral solution, a model hexad containing the dye and five zinc porphyrins organized on a hexaphenylbenzene core decays exponentially with a time constant of 2.1 ns, which is essentially the same lifetime as observed for related monomeric zinc porphyrins. When a solution of the hexad is acidified, the dye opens and quenches all porphyrin first excited singlet states to <40 ps. This converts the excitation energy to heat and renders the porphyrins kinetically incompetent to readily donate electrons by photoinduced electron transfer, thereby mimicking the role of the antenna in photosynthetic photoprotection.
ContributorsBhushan, Kul (Author) / Gust, Devens (Thesis advisor) / Moore, Ana (Committee member) / Hayes, Mark (Committee member) / Arizona State University (Publisher)
Created2012
150925-Thumbnail Image.png
Description
Polydimethyl siloxane is a commonly used fabrication material for microfluidic devices. However, its hydrophobic nature and protein adsorption on the surface restricts its use for microfluidic applications. Also, it is critical to control the electroosmotic flow for electrophoretic and dielectrophoretic manipulations. Therefore, surface modification of PDMS is essential to make

Polydimethyl siloxane is a commonly used fabrication material for microfluidic devices. However, its hydrophobic nature and protein adsorption on the surface restricts its use for microfluidic applications. Also, it is critical to control the electroosmotic flow for electrophoretic and dielectrophoretic manipulations. Therefore, surface modification of PDMS is essential to make it well suited for bioanalytical applications. In this project, the role of polyethylene oxide copolymers F108 and PLL-PEG has been investigated to modify the surface properties of PDMS using physisorption method. Measuring electroosmotic flow and adsorption studies tested the quality and the long-term stability of the modified PDMS surface. Static and dynamic coating strategies were used to modify the PDMS surface. In static coating, the PDMS surface was incubated with the coating agent prior to the measurements. For dynamic coating, the coating agent was always present in the solution throughout the experiment. F108 and PLL-PEG were equally effective to prevent the protein adsorption under both strategies. However, dynamic coating was more time saving. Furthermore, effective reduction of EOF was observed with F108 coating agent under dynamic conditions and with PLL-PEG coating agent under static conditions. Moreover, PLL-PEG dynamic coatings exhibited reversal of EOF. These important findings could be used to manipulate EOF and suggest optimal coating agent and strategies for PDMS surface treatment by the physisorption method.
ContributorsManchanda, Shikha (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2012
151190-Thumbnail Image.png
Description
Mass spectrometric analysis requires that atoms from the sample be ionized in the gas phase. Secondary ion mass spectrometry achieves this by sputtering samples with an energetic primary ion beam. Several investigations of the sputtering and ionization process have been conducted. Oxygen is commonly used in secondary ion mass spectrometry

Mass spectrometric analysis requires that atoms from the sample be ionized in the gas phase. Secondary ion mass spectrometry achieves this by sputtering samples with an energetic primary ion beam. Several investigations of the sputtering and ionization process have been conducted. Oxygen is commonly used in secondary ion mass spectrometry (SIMS) to increase ion yields, but also can complicate the interpretation of SIMS analyses. An 18O implant in silicon has been used to quantify the oxygen concentration at the surface of sputtered silicon in order to study the dependence on oxygen of several sputtering and depth profile phenomena. The ion yield dependence of trace elements in silicon on the surface oxygen concentration is a function of the ionization potential of the element. The ion yield is high and unaffected by oxygen for elements with low ionization potential and ranges over several orders of magnitude for elements with high ionization potential. Depth resolution in sputter profiles has been shown to be degraded by the presence of oxygen, the mechanism of this effect has been investigated using an 18O implant to quantify oxygen levels and it is shown that the process does not appear to be a consequence of surface oxide formation. Molecular ions are a source of mass interference in SIMS analysis, and multiply charged atomic ion signals might be interference-free due to the possible instability of multiply-charged molecular ions. Sputtered SiH2+, AlH2+, BeH2+, Mo22+ and Mg22+ ions have been observed and appear surprisingly stable. The formation mechanism of some of these species has been explored.
ContributorsSobers, Richard Carlisle, Jr (Author) / Williams, Peter (Thesis advisor) / Hayes, Mark (Committee member) / Petuskey, William (Committee member) / Arizona State University (Publisher)
Created2012