Matching Items (267)
Description
Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet would bring fully hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses ( 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. At the initial experiments at the AMO beamline using 6.9- Å wavelength, Bragg peaks were recorded to 8.5- Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage. Recently, femtosecond X-ray protein nanocrystallography experiments were done at the CXI beamline of the LCLS using 1.3- Å wavelength, and Bragg reflections were recorded to 3- Å resolution; the data are currently being processed. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.
ContributorsHunter, Mark (Author) / Fromme, Petra (Thesis advisor) / Wolf, George (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2011
Description
Filtration for microfluidic sample-collection devices is desirable for sample selection, concentration, preprocessing, and downstream manipulation, but microfabricating the required sub-micrometer filtration structure is an elaborate process. This thesis presents a simple method to fabricate polydimethylsiloxane (PDMS) devices with an integrated membrane filter that will sample, lyse, and extract the DNA from microorganisms in aqueous environments. An off-the-shelf membrane filter disc was embedded in a PDMS layer and sequentially bound with other PDMS channel layers. No leakage was observed during filtration. This device was validated by concentrating a large amount of cyanobacterium Synechocystis in simulated sample water with consistent performance across devices. After accumulating sufficient biomass on the filter, a sequential electrochemical lysing process was performed by applying 5VDC across the filter. This device was further evaluated by delivering several samples of differing concentrations of cyanobacterium Synechocystis then quantifying the DNA using real-time PCR. Lastly, an environmental sample was run through the device and the amount of photosynthetic microorganisms present in the water was determined. The major breakthroughs in this design are low energy demand, cheap materials, simple design, straightforward fabrication, and robust performance, together enabling wide-utility of similar chip-based devices for field-deployable operations in environmental micro-biotechnology.
ContributorsLecluse, Aurelie (Author) / Meldrum, Deirdre (Thesis advisor) / Chao, Joseph (Thesis advisor) / Westerhoff, Paul (Committee member) / Arizona State University (Publisher)
Created2011
Description
Mutations in the DNA of somatic cells, resulting from inaccuracies in DNA<br/>replication or exposure to harsh conditions (ionizing radiation, carcinogens), may be<br/>loss-of-function mutations, and the compounding of these mutations can lead to cancer.<br/>Such mutations can come in the form of thymine dimers, N-𝛽 glycosyl bond hydrolysis,<br/>oxidation by hydrogen peroxide or other radicals, and deamination of cytosine to uracil.<br/>However, many cells possess the machinery to counteract the deleterious effects of<br/>such mutations. While eukaryotic DNA repair enzymes decrease the incidence of<br/>mutations from 1 mistake per 10^7 nucleotides to 1 mistake per 10^9 nucleotides, these<br/>mutations, however sparse, are problematic. Of particular interest is a mutation in which<br/>uracil is incorporated into DNA, either by spontaneous deamination of cysteine or<br/>misincorporation. Such mutations occur about one in every 107 cytidine residues in 24<br/>hours. DNA uracil glycosylase (UDG) recognizes these mutations and cleaves the<br/>glycosidic bond, creating an abasic site. However, the rate of this form of DNA repair<br/>varies, depending on the nucleotides that surround the uracil. Most enzyme-DNA<br/>interactions depend on the sequence of DNA (which may change the duplex twist),<br/>even if they only bind to the sugar-phosphate backbone. In the mechanism of uracil<br/>excision, UDG flips the uracil out of the DNA double helix, and this step may be<br/>impaired by base pairs that neighbor the uracil. The deformability of certain regions of<br/>DNA may facilitate this step in the mechanism, causing these regions to be less<br/>mutable. In DNA, base stacking, a form of van der Waals forces between the aromatic<br/>nucleic bases, may make these uracil inclusions more difficult to excise. These regions,<br/>stabilized by base stacking interactions, may be less susceptible to repair by<br/>glycosylases such as UDG, and thus, more prone to mutation.
ContributorsUgaz, Bryan T (Author) / Levitus, Marcia (Thesis director) / Van Horn, Wade (Committee member) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
The goal of this project was to design and create a genetic construct that would allow for <br/>tumor growth to be induced in the center of the wing imaginal disc of Drosophila larvae, the <br/>R85E08 domain, using a heat shock. The resulting transgene would be combined with other <br/>transgenes in a single fly that would allow for simultaneous expression of the oncogene and, in <br/>the surrounding cells, other genes of interest. This system would help establish Drosophila as a <br/>more versatile and reliable model organism for cancer research. Furthermore, pilot studies were <br/>performed, using elements of the final proposed system, to determine if tumor growth is possible <br/>in the center of the disc, which oncogene produces the best results, and if oncogene expression <br/>induced later in development causes tumor growth. Three different candidate genes were <br/>investigated: RasV12, PvrACT, and Avli.
ContributorsSt Peter, John Daniel (Author) / Harris, Rob (Thesis director) / Varsani, Arvind (Committee member) / School of Molecular Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Single cell phenotypic heterogeneity studies reveal more information about the pathogenesis process than conventional bulk methods. Furthermore, investigation of the individual cellular response mechanism during rapid environmental changes can only be achieved at single cell level. By enabling the study of cellular morphology, a single cell three-dimensional (3D) imaging system can be used to diagnose fatal diseases, such as cancer, at an early stage. One proven method, CellCT, accomplishes 3D imaging by rotating a single cell around a fixed axis. However, some existing cell rotating mechanisms require either intricate microfabrication, and some fail to provide a suitable environment for living cells. This thesis develops a microvorterx chamber that allows living cells to be rotated by hydrodynamic alone while facilitating imaging access. In this thesis work, 1) the new chamber design was developed through numerical simulation. Simulations revealed that in order to form a microvortex in the side chamber, the ratio of the chamber opening to the channel width must be smaller than one. After comparing different chamber designs, the trapezoidal side chamber was selected because it demonstrated controllable circulation and met the imaging requirements. Microvortex properties were not sensitive to the chambers with interface angles ranging from 0.32 to 0.64. A similar trend was observed when chamber heights were larger than chamber opening. 2) Micro-particle image velocimetry was used to characterize microvortices and validate simulation results. Agreement between experimentation and simulation confirmed that numerical simulation was an effective method for chamber design. 3) Finally, cell rotation experiments were performed in the trapezoidal side chamber. The experimental results demonstrated cell rotational rates ranging from 12 to 29 rpm for regular cells. With a volumetric flow rate of 0.5 µL/s, an irregular cell rotated at a mean rate of 97 ± 3 rpm. Rotational rates can be changed by altering inlet flow rates.
ContributorsZhang, Wenjie (Author) / Frakes, David (Thesis advisor) / Meldrum, Deirdre (Thesis advisor) / Chao, Shih-hui (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2011
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 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
Description
Natural products that target the DNA of cancer cells have been an important source of knowledge and understanding in the development of anticancer chemotherapeutic agents. Bleomycin (BLM) exemplifies this class of DNA damaging agent. The ability of BLM to chelate metal ions and effect oxidative damage of the deoxyribose sugar moiety of DNA has been studied extensively for four decades. Here, the study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of BLM to effect single-stranded was then extensively characterized on both the 3′ and 5′-arms of the hairpin DNAs. The strongly bound DNAs were found to be efficient substrates for Fe·BLM A5-mediated cleavage. Surprisingly, the most prevalent site of damage by BLM was found to be a 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence and others generally not cleaved by BLM when examined using arbitrarily chosen DNA substrate were found in examining the library of ten hairpin DNAs. In total, 111 sites of DNA damage were found to be produced by exposure of the hairpin DNA library to Fe·BLM A5. Also, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double stranded DNA damage. Adapting methods previously described by the Povirk laboratory, one hairpin was characterized using this method. The results were in accordance with those previously reported.
ContributorsSegerman, Zachary (Author) / Hecht, Sidney M. (Thesis advisor) / Levitus, Marcia (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
This dissertation describes the work on two projects which involves measuring molecular conductance and studying their properties on the nanoscale using various Scanning Tunneling Microscopy (STM) techniques. The first molecule studied was a porphyrin-fullerene moiety known as a molecular Dyad for photovoltaic applications. This project is further divided into two section, the first one involving the characterization of the Dyad monolayers and conductance measurement in the dark. The Dyads are designed to form charge separated states on illumination. The lifetime of the charged states have been measured efficiently but the single-molecule conductance through the molecules have yet to be characterized. The second part of the project describes the set-up of a novel sample stage which enables the study of molecular conductance under illumination. This part also describes the subsequent study of the molecule under illumination and the observation of a unique charge-separated state. It also contains the verification of the presence of this charge-separated using other characterization techniques like transient absorption spectroscopy. The second project described in the dissertation was studying and comparing the predicted rectifying nature of two molecules, identical in every way except for one stereocenter. This project describes the formation of monolayers of the molecule on gold and then studying and analyzing the current-voltage characteristics of the molecules and looking for rectification. Both the molecules proved to be rectifying, one more than the other as predicted by theoretical calculations.
ContributorsBhattacharyya, Shreya (Author) / Lindsay, Stuart (Thesis advisor) / Moore, Ana (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2011
Description
The use of synthetic cathinones or "bath salts" has risen dramatically in recent years with one of the most popular being Methylendioxypyrovalerone (MDPV). Following the temporary legislative ban on the sale and distribution of this compound , a multitude of other cathinone derivatives have been synthesized. The current study seeks to compare the abuse potential of MDPV with one of the emergent synthetic cathinones 4-methylethcathinone (4-MEC), based on their respective ability to lower current thresholds in an intracranial self-stimulation (ICSS) paradigm. Following acute administration (0.1, 0.5, 1 and 2 mg/kg i.p.) MDPV was found to significantly lower ICSS thresholds at all doses tested (F4,35=11.549, p<0.001). However, following acute administration (0.3,1,3,10,30 mg/kg i.p) 4-MEC produced no significant ICSS threshold depression (F5,135= 0.622, p = 0.684). Together these findings suggest that while MDPV may possess significant abuse potential, other synthetic cathinones such as 4-MEC may have a drastically reduced potential for abuse.
ContributorsWegner, Scott Andrew (Author) / Olive, M. Foster (Thesis director) / Presson, Clark (Committee member) / Sanabria, Federico (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Department of Psychology (Contributor)
Created2013-05
Description
Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps.
ContributorsRanjit, Suman (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013