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Light-activated binary nucleotide reagent for inactivation of DNA polymerase
Title
Light-activated binary nucleotide reagent for inactivation of DNA polymerase.
Creator
Cornett, Evan, ,, University of Central Florida
Abstract / Description
This work explores a binary reagent approach to increase the specificity of covalent inhibitors. In this approach, two ligand analogs equipped with inert pre-reactive groups specifically bind a target biopolymer. The binding event brings the pre-reactive groups in proximity with each other. The two groups react, generating active chemical intermediates that covalently modify and inactivate the target. In the present study we compare the new approach with the traditional single-component...
Show moreThis work explores a binary reagent approach to increase the specificity of covalent inhibitors. In this approach, two ligand analogs equipped with inert pre-reactive groups specifically bind a target biopolymer. The binding event brings the pre-reactive groups in proximity with each other. The two groups react, generating active chemical intermediates that covalently modify and inactivate the target. In the present study we compare the new approach with the traditional single-component reagent strategy using DNA polymerase from bacteriophage T4 as a model target biopolymer. We report the design and synthesis of two analogs of deoxythymidine triphosphate, a natural DNA polymerase substrate. Together, the analogs function as a binary nucleotide reagent which is activated by light with wavelengths 365 nm and longer. However, the active analog functions as a traditional single component reagent when activated by light with wavelengths at 300 nm and longer. The traditional single-component reagent efficiently inactivated DNA polymerase. However, in the presence of non-target protein the inactivation efficiency is greatly diminished. Under the same conditions, the binary nucleotide reagent also inactivated DNA polymerase, and the inactivation efficiency is not affected by the presence of the non-target protein. Our results validate that a binary approach can be employed to design highly specific covalent inhibitors. The binary reagent strategy might be useful as a research tool for investigation of ligand-protein interactions in complex biological systems and for drug design.
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Date Issued
2012
Identifier
CFE0004366, ucf:49429
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004366
APPLICATION AND COMPARISON OF ACTIVE LEARNING IMPLEMENTATION METHODS IN BIOCHEMISTRY EDUCATION
Title
APPLICATION AND COMPARISON OF ACTIVE LEARNING IMPLEMENTATION METHODS IN BIOCHEMISTRY EDUCATION.
Creator
Thibaut, Dylan, Borgon, Robert, Caranto, Jonathan, University of Central Florida
Abstract / Description
Biochemistry has continued to be one of the most complex and important subjects in science education. The purpose of this research is to investigate active learning implementation methods in a Biochemistry I context to determine the most effective means of preparing current science undergraduates. Two Biochemistry I classes over two semesters were analyzed in this study, with class A using a variable active learning schedule and class B using a consistent active learning schedule. Four...
Show moreBiochemistry has continued to be one of the most complex and important subjects in science education. The purpose of this research is to investigate active learning implementation methods in a Biochemistry I context to determine the most effective means of preparing current science undergraduates. Two Biochemistry I classes over two semesters were analyzed in this study, with class A using a variable active learning schedule and class B using a consistent active learning schedule. Four aspects were analyzed to determine active learning validity: perception of different active learning properties, standardized final exam grades, class grade, and teaching implementation. The consistent schedule of daily active learning in class B showed an increase in mean final exam score by 12.72%, significantly improved mean student grade in the class from a high C to a low B (p= 0.0038), and comparing student perception of active learning data, showed a significant decrease in student desire for passive learning (p= 0.025), increased desire for active learning (p= 0.022), and increased desire for flipped classrooms (p= 0.042) after first experiencing opposite results in the first semester of implementation which had increased desire for passive learning (p= 0.003) and teacher-centric learning (p= 0.026). A variable active learning schedule showed no significant values besides an increase in individual learning desire (p= 0.037) and a marginally significant increase in desire for passive learning (p= 0.053) both in its second semester of implementation. This research supports that a consistent, daily active learning curriculum making up approximately 40-50% of daily instruction is preferable compared to a variable lecture schedule with active learning days in between lecture days in undergraduate Biochemistry I large-class instruction given that professors perform it over multiple semesters.
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Date Issued
2019
Identifier
CFH2000578, ucf:45662
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH2000578
PHYSICAL CHARACTERISTICS OF AN INDIVIDUAL: THE IDENTIFICATION OF BIOMARKERS FOR BIOLOGICAL AGE DETERMINATION
Title
PHYSICAL CHARACTERISTICS OF AN INDIVIDUAL: THE IDENTIFICATION OF BIOMARKERS FOR BIOLOGICAL AGE DETERMINATION.
Creator
Alvarez, Michelle, Ballantyne, Jack, University of Central Florida
Abstract / Description
It is now a matter of routine for the forensic scientist to obtain the genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. Potential contributors of the stain must either be known to investigators (i.e. a developed suspect) or the questioned profile must be searched against a database of DNA profiles such as those maintained in the CODIS National DNA database. However, in those instances where there is no developed suspect and no match is...
Show moreIt is now a matter of routine for the forensic scientist to obtain the genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. Potential contributors of the stain must either be known to investigators (i.e. a developed suspect) or the questioned profile must be searched against a database of DNA profiles such as those maintained in the CODIS National DNA database. However, in those instances where there is no developed suspect and no match is obtained after interrogation of appropriate DNA databases, the DNA profile per se presently provides no meaningful information to investigators, with the notable exception of gender determination. In these situations it would be advantageous to the investigation, if additional probative information could be obtained from the biological stain. A useful biometric that could provide important probative information, and one that may be amenable to molecular genetic analysis, is the biological age of an individual. The ability to provide investigators with information as to whether a DNA donor is a newborn, infant, toddler, child, adolescent, adult, middle-aged or elderly individual could be useful in certain cases, particularly those involving young children such as kidnappings or in providing additional intelligence during terrorist investigations. Currently no validated molecular assays exist for age determination. Biological human ageing can be defined by two distinct processes, degenerative and developmental ageing. The degenerative process of ageing is based on theories which identify an increase or decrease in physiological conditions with increasing age. In contrast, the developmental process of ageing is based on the theory that as individuals increase in chronological age, there will be subtle corresponding molecular based biological changes, each requiring genes to be expressed or silenced, indicative of that particular stage of life. We investigated the degenerative process of chromosomal telomere shortening, as well as the developmental process of gene expression profiling analysis, in an attempt to identify biomarkers of biological age in a self-renewing tissue such as blood. While telomere length analysis was an ineffective method for age determination; gene expression analysis revealed three gene transcripts expressed in an age-dependent physiological manner. These species namely- COL1A2, HBE1 and IGFBP3, were found to be expressed at elevated levels in younger individuals, newborns, or post-pubertal individuals, respectively. The biological process of hemoglobin switching was also investigated for the possibility of determining human age. While experimenting with the potential of using the gamma-hemoglobin chains, as newborn specific gene candidates, we serendipitously discovered four novel truncated transcripts, which we have termed HBG1n1, HBG1n2, HBG2n2 and HBG2n3; whose expression was restricted to whole-blood newborn samples and specific fetal tissues. The molecular origin of these transcripts appears to be at the RNA level, being produced by specific rearrangement events occurring in the standard gamma hemoglobin transcripts (HBG1 and HBG2), which yield these new isoforms that are expressed in a highly regulated tissue specific manner.
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Date Issued
2007
Identifier
CFE0001737, ucf:47297
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0001737
The Owl Sensor: a smart nanostructure for single nucleotide variation analysis
Title
The Owl Sensor: a smart nanostructure for single nucleotide variation analysis.
Creator
Karadeema, Rebekah, Kolpashchikov, Dmitry, Chumbimuni Torres, Karin, Harper, James, University of Central Florida
Abstract / Description
Analysis of single nucleotide variations (SNVs) in DNA and RNA sequences is extensively used in healthcare for detection of genetic mutations and analysis of drug resistant pathogens. Here we developed a nucleic acid sensor able to differentiate between a fully matched analyte and one with a SNV in a wide temperature range of 5(&)deg;C-32(&)deg;C. The sensor, dubbed here the 'Owl Sensor' due to the complex's resemblance to owl eyes, utilizes recent developments in DNA nanotechnology and...
Show moreAnalysis of single nucleotide variations (SNVs) in DNA and RNA sequences is extensively used in healthcare for detection of genetic mutations and analysis of drug resistant pathogens. Here we developed a nucleic acid sensor able to differentiate between a fully matched analyte and one with a SNV in a wide temperature range of 5(&)deg;C-32(&)deg;C. The sensor, dubbed here the 'Owl Sensor' due to the complex's resemblance to owl eyes, utilizes recent developments in DNA nanotechnology and synthetic biology to self-assemble a fluorescent DNA nanostructure called a Double Crossover, or DX Tile, capable of differentiating SNVs in a large temperature range, including ambient temperature. In the presence of fully matched nucleic acid analytes, a stable complex is formed with high fluorescent signal; however in the presence of a single base variation in the analyte, unfavourable helicity results in little-to-no observed complex formation. The novelty of the approach is that selectivity of analyte recognition is, at least in part, determined by the structural rigidity of the entire nanostructure rather than by the stability of analyte-probe hybrid, as is the case for conventional hybridization probes. The rigid nanostructure collapses if a minor imperfection, e.g. if a single-base mispairing, is present. Owl Sensor differentiates fully matched analyte from mismatched in a wide temperature range, with mismatched analyte producing only the background fluorescence, selectivity that is hard to achieve by conventional hybridization probes. Owl Sensor therefore promises to add to the toolbox for diagnosis of genetic disorders and infectious diseases at ambient temperatures.
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Date Issued
2016
Identifier
CFE0006691, ucf:51916
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006691
Biochemical Characterization of Rv2633c from Mycobacterium tuberculosis and the Effects of Mutagenesis on Iron Binding
Title
Biochemical Characterization of Rv2633c from Mycobacterium tuberculosis and the Effects of Mutagenesis on Iron Binding.
Creator
Strickland, Kyle, Self, William, Rohde, Kyle, Davidson, Victor, University of Central Florida
Abstract / Description
Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that is the causative agent of the disease Tuberculosis (TB). TB kills an estimated 1.8 million people annually and roughly one third of the world's population carries Mtb in a dormant state. Drug resistant Mtb strains are on the rise, thus a new method of combating this disease is paramount. Mtb survival inside of macrophages requires overcoming various stressors such as; iron restriction, reactive oxygen species, and hypoxic...
Show moreMycobacterium tuberculosis (Mtb) is a pathogenic bacterium that is the causative agent of the disease Tuberculosis (TB). TB kills an estimated 1.8 million people annually and roughly one third of the world's population carries Mtb in a dormant state. Drug resistant Mtb strains are on the rise, thus a new method of combating this disease is paramount. Mtb survival inside of macrophages requires overcoming various stressors such as; iron restriction, reactive oxygen species, and hypoxic conditions. Mtb employs the use of catalases, nitric oxide reductase, superoxide dismutase, and siderophores to aid in survival. These functions have also been found in a novel group of non-heme diiron binding proteins called hemerythrin-like proteins.The gene Rv2633c encodes a protein with the hemerythrin-like domain and has been shown to be upregulated under acidic or nutrient deficient conditions which coincides with Mtb infection of a macrophage. It has also been shown to be regulated by PhoP, Whib3, and DosR. In this work we expressed the wild type protein and several mutants heterologously in E. coli. The purified proteins were studied via UV-visible spectroscopic analysis, native polyacrylamide gel electrophoresis (native-PAGE) and analyzed for iron content.Our refined expression and purification protocol led to a significant increase in soluble protein with a di-iron cofactor. We found that mutagenesis of 11th amino acid, a histidine, led to the absence of the diiron co-factor. Reduction and autoxidation of protein was also achieved and characterized through UV-visible absorption. Native-PAGE gel analysis indicated only the dimeric form contained iron. This research is the first to produce large quantities of soluble iron laden protein, demonstrate that Rv2663c is capable of both reduction and autoxidation, and show it does not bind oxygen in a functional capacity. This information will enable future studies in protein crystallization, ligand interaction and in vivo studies.
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Date Issued
2019
Identifier
CFE0007729, ucf:52456
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007729