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The Estimation of Germ Line De Novo Mutation Rates of Extended Sets of Y-STR Haplotypes to Aid in the Differentiation of Male Biological Relatives in Criminal Investigations
Title
The Estimation of Germ Line De Novo Mutation Rates of Extended Sets of Y-STR Haplotypes to Aid in the Differentiation of Male Biological Relatives in Criminal Investigations.
Creator
Masker, Nicole, Ballantyne, John, Kolpashchikov, Dmitry, Koculi, Eda, University of Central Florida
Abstract / Description
An important forensic application for Y-chromosome short tandem repeats (Y-STRs) is the identification of male DNA. Since the Y-chromosome is non-recombining and most Y-STRs have slippage mutation rates on the order of 1x10-3 or lower, Y-STR commercial multiplex systems do not yet allow personal individualization. This is problematic in criminal investigations where the persons of interest include males of the same paternal line. In order to obtain discrimination among paternal male relatives...
Show moreAn important forensic application for Y-chromosome short tandem repeats (Y-STRs) is the identification of male DNA. Since the Y-chromosome is non-recombining and most Y-STRs have slippage mutation rates on the order of 1x10-3 or lower, Y-STR commercial multiplex systems do not yet allow personal individualization. This is problematic in criminal investigations where the persons of interest include males of the same paternal line. In order to obtain discrimination among paternal male relatives, it would be necessary to use a larger number of Y-STRs or a selection of Y-loci with higher slippage mutations with the hope of encountering a germline meiotic mutation. In this study, Y-STR loci from three multiplexes were examined: an ultra-high discrimination multiplex of 14 non-core loci; Promega's PowerPlex(&)#174; Y23; and a rapidly mutating 13-locus panel (mutation rates above 1x10-2). Taking the three multiplexes together provides a unique 40-locus Y-STR panel (the (")Masker Set(")), a powerful tool to separate closely related males. The loci were examined for discriminative slippage mutations in pairs of paternally related South Brazilian males: 99 grandfather-grandson, 103 uncle-nephew, and 140 brothers. The goal of this study was to analyze the (")Masker Set(") loci by: describing the characteristics and frequency of germ-line mutations; noting differences in mutation rates between and within loci; determining repeat gain and loss rates; and identifying the most informative loci to differentiate male relatives. Using the (")Masker Set("), pairs of male relatives were distinguished by at least one mutation 53% of the time for grandfather-grandson, 62% for uncle-nephew, and 54% for brothers. The most discriminating Y-STR loci were: DYF387S1, DYF404S1, DYS526B, DYS389II, DYS449, DYS547, DYF399S1, DYS458, DYS576, DYF403S1A, DYS508, DYS612, DYS403S1B, DYS518, and DYS627.
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Date Issued
2016
Identifier
CFE0006837, ucf:51784
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006837
Multi-target high-throughput screening assays for antimicrobial drug discovery
Title
Multi-target high-throughput screening assays for antimicrobial drug discovery.
Creator
Grube, Christopher, Roy, Herve, Chakrabarti, Debopam, Moore, Sean, Koculi, Eda, University of Central Florida
Abstract / Description
The rise of antibiotic resistant microbes (bacteria, fungi, and parasites), combined with the current void of new drugs entering the clinical setting, has created an urgent need for the discovery of new antimicrobials. High-throughput screening (HTS) assays represent a fast and cost-efficient method for identifying new therapeutic compounds and have been the longstanding gold standard for drug discovery. The focus of this dissertation is on the development and implementation of novel...
Show moreThe rise of antibiotic resistant microbes (bacteria, fungi, and parasites), combined with the current void of new drugs entering the clinical setting, has created an urgent need for the discovery of new antimicrobials. High-throughput screening (HTS) assays represent a fast and cost-efficient method for identifying new therapeutic compounds and have been the longstanding gold standard for drug discovery. The focus of this dissertation is on the development and implementation of novel methodologies to increase the throughput of target-based HTS by designing assays that allow multiple drug targets to be probed simultaneously. During my graduate studies, I developed three distinct HTS assays. In each of these assays, drug targets were incorporated into synthetic pathways obeying various reaction topologies (e.g., cyclical, parallel, or linear). Each of these reaction topologies conferred specific advantages and limitations to the individual assays. The first assay reconstitutes the bacterial tRNA-dependent pathway for lipid aminoacylation. This two-step pathway combines a tRNA aminoacylation step catalyzed by an aminoacyl-tRNA synthetase (aaRS), and a transferase step, which transfers the amino acid born by the tRNA onto membrane lipids. aaRSs are essential enzymes in all domains of life and represent longstanding drug targets in pathogenic species. The transferase reaction in the pathway is also an appealing drug target since it impacts the cellular permeability of antibiotics. Inhibitors of this reaction could dramatically increase the efficacy of existing therapeutics. The second assay I developed also targets aaRSs, but utilizes a parallel topology that permits the probing of the synthetic and editing activities of up to four aaRSs simultaneously. The third assay utilizes a linear topology that reconstitutes the entire purine salvage pathway from Plasmodium falciparum. Because parasites are unable to synthesize purines de novo, this pathway represents an appealing target for novel antimalarials. Pilot screens using this assay revealed inhibitors for multiple enzymes in the pathway, validating the design of the system. This body of work aims to shift the current paradigm of single-target systems that have historically dominated the HTS field, toward multi-target designs that can be used to more efficiently screen compound libraries against essential pathways in pathogenic microbes.
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Date Issued
2019
Identifier
CFE0007642, ucf:52469
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007642
Optimization of Molecular Beacon-Based Multicomponent Probes for Analysis of Nucleic Acids
Title
Optimization of Molecular Beacon-Based Multicomponent Probes for Analysis of Nucleic Acids.
Creator
Stancescu, Maria, Kolpashchikov, Dmitry, Clausen, Christian, Koculi, Eda, Balaeff, Alexander, Shuler, Michael, University of Central Florida
Abstract / Description
Detection of single nucleotide substitutions (SNS) in DNA and RNA has a growing importance in biology and medicine. One traditional approach for recognition of SNS takes advantage of hybridization probes that bind target nucleic acids followed by measuring ?Tm, the difference in melting temperatures of matched and mismatched hybrids. The approach enables SNS differentiation at elevated temperatures (usually 40-65oC) often only in a narrow range of (
Show moreDetection of single nucleotide substitutions (SNS) in DNA and RNA has a growing importance in biology and medicine. One traditional approach for recognition of SNS takes advantage of hybridization probes that bind target nucleic acids followed by measuring ?Tm, the difference in melting temperatures of matched and mismatched hybrids. The approach enables SNS differentiation at elevated temperatures (usually 40-65oC) often only in a narrow range of (<)10oC and requires high-resolution melting devices. Here we demonstrate that a specially designed DNA probe (X sensor) can broaden ?Tm from ~10oC to ~16oC and distinguish SNS in the interval of ~5-40oC. Therefore, there is no need for heating or measuring Tm for accurate SNS differentiation. Our data indicate that this wide differentiation range is in part due to the non-equilibrium hybridization conditions. Further we explored the idea that it is possible to improve the performance of an X sensor operable in close to equilibrium conditions by shifting its operability to non-equilibrium conditions. One way to achieve this is to introduce as many as possible structured ligands in analyte's dissociated state. Here we show that by introducing the maximum possible conformational constraints in X probe it is possible to shift its operation to non-equilibrium conditions and to improve its selectivity at temperatures (<)15oC. Thus, this work points towards a new strategy for the design of highly selective hybridization sensors which operate in non-equilibrium conditions at close to room temperature. The X sensors could be utilized in qPCR, microarrays, as well as RNA analysis in living cells and for ambient temperature point-of-care diagnostics. In the last part of this work, X sensors were used in real time detection of PCR products. The sensors were optimized to operate in PCR buffer with optimal Mg2+ concentration. They were able to detect the target amplicon together with nonspecific products. The results presented here suggest that X sensors might be adopted for real time PCR format.
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Date Issued
2015
Identifier
CFE0006009, ucf:51006
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006009
Novel Photodynamic Cancer Therapy Agent and Biochemical Phosphate Sensor Based on Nanomaterials
Title
Novel Photodynamic Cancer Therapy Agent and Biochemical Phosphate Sensor Based on Nanomaterials.
Creator
Fadhel, Alaa, Campiglia, Andres, Belfield, Kevin, Harper, James, Koculi, Eda, Bhattacharya, Aniket, University of Central Florida
Abstract / Description
Biochemical research and clinical studies have revolutionized the field of medicine in both diagnosis and therapy. Researchers in the field of biochemistry and biotechnology are using nanomaterials in different applications to develop devices and materials that offer benefits to both patients and the health care industry. These include biochemical sensors, enzyme encapsulation, biomarkers, and drug delivery improvements for the treatment of cancer. This dissertation focuses on investigating...
Show moreBiochemical research and clinical studies have revolutionized the field of medicine in both diagnosis and therapy. Researchers in the field of biochemistry and biotechnology are using nanomaterials in different applications to develop devices and materials that offer benefits to both patients and the health care industry. These include biochemical sensors, enzyme encapsulation, biomarkers, and drug delivery improvements for the treatment of cancer. This dissertation focuses on investigating two biochemical aspects using nanomaterials; namely therapy and clinical diagnosis.For therapy purposes, Silica nanoparticles were used as drug delivery system to develop a new photodynamic cancer therapy agent photo-acid generator (PAG) that selectively induces necrotic cell death of cancer cells. The developed PAG is oxygen-independent and - when excited at specific wavelengths - drops the pH within the lysosome of cancer cells to produce apoptosis/necrosis. It was specifically designed for in vivo applications and conjugated with synthesized, highly monodispersed silica nanoparticles (Si NPs) functionalized with amine groups via amid links (SiN-NH-PAG). Additional Features include high photo-acid quantum yield, high one-photon (1PA) and two-photon absorption (2PA) with low fluorescence quantum yield. In vivo, confocal microscope studies with HCT-116 (Human colorectal carcinoma) cancer cells showed that photodynamic processes in the presence of PAG were completed under one- photon absorption (1PA) conditions. In these experiments, cells were imaged at 1 min intervals for a total of 4 hours with the aid of Differential Interference Contrast (DIC). Among the photodynamic therapy agents tested via cytotoxicity experiments with the MTS assay, (SiN-NH- PAG) showed the best efficiency to induce cell death. The increased effectiveness of the new agent is probably due to the large number of PAG groups present on the surface of Si NPs.iiLysosome colocalization indicates that PAGs are mainly built in lysosomes. The increase of acidic content inside the lysosome was demonstrated with the aid of the LysoSensor Green probe. The drop in the intralysosomal pH was approximately 0.3 units. This is a desirable outcome as most cells underwent necrosis at pH ? 4.4. For clinical diagnosis purposes, a biochemical sensor was developed for the analysis of phosphate ions in urine samples. Abnormal levels of inorganic phosphate in human urine samples are related to the development of certain types of cancers affecting several organs of the human body, including breast, pancreas, lung and thyroid. The new biochemical sensor is based on the fluorescence energy transfer between a lanthanide luminescent probe [Tb-EDTA]-1 and gold nanoparticles (Au NPs) capped with a Cetyltrimethylammonium bromide (CTAB) micelle. With this approach, it was possible to selectively determine inorganic phosphate (Pi) in urine samples at the micro-molar concentration level. Urine samples collected from healthy, non-smoking individuals showed no interference from concomitants usually found in human urine samples. The simplicity of analysis provides an approach well-suited for (")real-time(") monitoring of phosphate ions. Analysis time is made possible within approximately 10 min per sample.
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Date Issued
2016
Identifier
CFE0006528, ucf:51384
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006528
The role of a highly conserved eubacterial ribosomal protein in translation quality control
Title
The role of a highly conserved eubacterial ribosomal protein in translation quality control.
Creator
Naganathan, Anusha, Moore, Sean, Cole, Alexander, Teter, Kenneth, Roy, Herve, Koculi, Eda, University of Central Florida
Abstract / Description
The process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 (&)#197; from the site of...
Show moreThe process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 (&)#197; from the site of decoding, making it difficult to envision how it might affect decoding and reading frame maintenance. Twenty years after the identification of L9's putative function, there is no mechanism for how a remotely located L9 improves translation fidelity. This mystery makes our picture of translation incomplete. Despite the high conservation of L9 in eubacteria, E.coli lacking L9 does not exhibit any obvious growth defects. Thus, the evolutionary advantage conferred by L9 in bacteria is masked under laboratory conditions. In order to uncover unique L9-dependent conditions, a library of E. coli mutants was screened to isolate those that rely on L9 for fitness. Interestingly, factors found to be synergistic with L9 had no known role in fidelity. Six independent mutants were isolated, each exhibiting a severe growth defect that is partially suppressed in the presence of L9. One class of L9-dependent mutations was present in an essential ribosome biogenesis factor, Der. Der's established function is in the maturation of the large ribosomal subunit. The identified mutations severely impaired the GTPase activity of Der. Interestingly, L9 did not directly compensate for the defective GTPase activity of mutant Der. The second class of L9-dependent mutations was present in EpmA and EpmB, factors required to post-translationally modify elongation factor, EF-P. EF-P's established function is in the translation of poly-proline containing proteins. EF-P deficient cells were nearly inviable in the absence of L9; however, L9 did not directly influence poly-proline translation. Therefore, in each case, L9 improved cell health without altering the activity of either Der or EF-P. Remarkably, the der mutants required only the N domain of L9, whereas the absence of active EF-P required full-length, wild-type L9 for growth complementation. Thus, each mutant class needed a different aspect of L9's unique architecture. In cells lacking either active EF-P or Der, there was a severe deficiency of 70S ribosomes and the indication of small subunit maturation defects, both of which worsened upon L9 depletion. These results strongly suggest that L9 plays a role in improving ribosome quality and abundance under certain conditions.Overall, the genetic screen lead to the discovery that bacteria need L9 when either of two important translation factors (Der or EF-P) is inactivated. This work has characterized the physiological requirement for L9 in each case and offers a new insight into L9's assigned role in translation fidelity.
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Date Issued
2015
Identifier
CFE0005674, ucf:50169
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005674
Structural and Functional Studies of Glycine Riboswitches and Development of Fab Chaperone Assisted RNA Crystallography
Title
Structural and Functional Studies of Glycine Riboswitches and Development of Fab Chaperone Assisted RNA Crystallography.
Creator
Sherman, Eileen, Ye, Jingdong, Kolpashchikov, Dmitry, Koculi, Eda, Harper, James, Self, William, University of Central Florida
Abstract / Description
The glycine riboswitch is a structured RNA found upstream of genes in mRNA transcripts in many bacteria, functioning as a biofeedback gene regulator. Upon binding glycine, a complete RNA transcript including gene sequences is transcribed, effectively turning on gene expression. In an effort to understand the intricacies of its functioning, many mutants of the riboswitch were made and characterized during Ph. D. work, resulting in discovery of a P0 duplex/kink-turn motif involving a few...
Show moreThe glycine riboswitch is a structured RNA found upstream of genes in mRNA transcripts in many bacteria, functioning as a biofeedback gene regulator. Upon binding glycine, a complete RNA transcript including gene sequences is transcribed, effectively turning on gene expression. In an effort to understand the intricacies of its functioning, many mutants of the riboswitch were made and characterized during Ph. D. work, resulting in discovery of a P0 duplex/kink-turn motif involving a few nucleotides upstream of the established glycine riboswitch sequence which changed its ligand binding characteristics (Chapter 1). Previously, the two aptamers of the riboswitch were thought to cooperatively bind glycine, but with the inclusion of this leader sequence which forms a kink turn motif with the linker between the two aptamers, glycine binding in one aptamer no longer requires glycine binding in the other. Furthermore, the Kd from three species tested are now a similar, lower value of about 5 (&)#181;M, indicating authenticity of this new consensus sequence. Glycine binding and interaptamer interaction both enhanced one another in trans aptamer assays. Another discovery from this was a shortened construct including all of aptamer II but only part of aptamer I in which a few specific nucleotides prevented glycine binding in aptamer II (Chapter 2). This may provide insight into the nature of interaptamer interactions in the full switch; addition of an oligonucleotide complimentary to these nucleotides restored glycine binding ability to aptamer II. With future development, this could also be a useful molecular biology tool, using two signals, glycine and an oligonucleotide, to allow gene expression.To precisely understand how any macromolecule functions, a 3D structure, obtainable by x-ray crystallography, is vital. A new technique to accomplish that for RNA, precedented in the protein world, is Fab chaperoned crystallography, which has advantages compared to RNA alone. A phage displayed library of Fabs with reduced codon diversity designed for RNA was created, the YSGR Min library (Chapter 3). Its Fabs had specificities and affinities equal to or greater than previous libraries which were originally created for phage displayed selection against proteins. Fab chaperoned RNA crystallography is currently in progress for the glycine riboswitch; the best resolution thus far is 5.3 (&)#197; (Chapter 4). In addition to providing molecular insight into its gene regulation mechanism, a structure of the glycine riboswitch could be applied for use in structure based drug design of novel antibiotics targeting the riboswitch to disrupt important downstream carbon cycle genes in pathogenic bacteria.
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Date Issued
2014
Identifier
CFE0005549, ucf:50285
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005549