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- Title
- STABILITY AND RECOVERY OF RNA IN BIOLOGICAL STAINS.
- Creator
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Setzer, Mindy Eileen, Jack Ballantyne, Dr, University of Central Florida
- Abstract / Description
-
In theory, RNA expression patterns, including the presence and relative abundance of particular RNA species, provide cell and tissue specific information that could be of use to forensic scientists. An mRNA based approach could allow the facile identification of the tissue components present in a body fluid stain and conceivably could supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory. Some of the potential advantages include...
Show moreIn theory, RNA expression patterns, including the presence and relative abundance of particular RNA species, provide cell and tissue specific information that could be of use to forensic scientists. An mRNA based approach could allow the facile identification of the tissue components present in a body fluid stain and conceivably could supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory. Some of the potential advantages include greater test specificity, and the ability to perform simultaneous analysis using a common assay format for the presence of all body fluids of forensic interest. In this report, the recovery and stability of RNA in forensic samples was evaluated by conducting an in-depth study on the persistence of RNA in biological stains. Stains were prepared from blood, saliva, semen, and vaginal secretions, and were exposed to a range of environmental conditions so that the affects of different light sources, temperatures, and environments could be assessed. Using the results from quantitation and sensitivity studies performed with pristine forensic stains, the RNA stability of samples which were collected over a period of 1 day to 1 year for blood, saliva, and vaginal secretion stains and for up to 6 months for semen stains were analyzed. The extent of RNA degradation within each type of body fluid stain was determined using quantitation of total RNA and reverse transcriptase polymerase chain reaction (RT-PCR) with selected housekeeping and tissue-specific genes. The results show that RNA can be recovered from biological stains in sufficient quantity and quality for mRNA analysis. The results also show that mRNA is detectable in samples stored at room temperature for at least one year, but that heat and humidity appear to be very detrimental to the stability of RNA.
Show less - Date Issued
- 2004
- Identifier
- CFE0000077, ucf:46141
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000077
- Title
- DESIGN, CONSTRUCTION, AND CHARACTERIZATION OF THE YSGR MINIMAL CODON FAB LIBRARY FOR CHAPERONE-ASSISTED RNA CRYSTALLOGRAPHY.
- Creator
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Holmes, Sean, Ye, Jingdong, University of Central Florida
- Abstract / Description
-
Of the entire human genome, 90% of all genetic information is transcribed but only a fraction of that subsequent RNA is translated into proteins. RNAs which are not translated into proteins are deemed non-coding RNAs. Little is known about this large category of noncoding RNAs, although they perform a variety of functions within the cell. RNA crystallography is used to study RNA tertiary structure, which gives insight to the function of these non-coding RNAs. However, complications associated...
Show moreOf the entire human genome, 90% of all genetic information is transcribed but only a fraction of that subsequent RNA is translated into proteins. RNAs which are not translated into proteins are deemed non-coding RNAs. Little is known about this large category of noncoding RNAs, although they perform a variety of functions within the cell. RNA crystallography is used to study RNA tertiary structure, which gives insight to the function of these non-coding RNAs. However, complications associated with RNA crystallography arise due to RNA's lack of surface functional group diversity, flexible tertiary structure, and conformational heterogeneity. A novel technique, Chaperone-assisted RNA crystallography (CARC), can greatly improve the success in crystallization of RNA. With this technique, synthetic antibodies called Antigen Binding Fragments (Fabs) are employed as crystallization chaperones to promote the structure elucidation of certain target RNAs. To identify Fabs that complex with RNA molecules of interest, we constructed a randomized library of synthetic antibodies enriched in ligand binding regions with tyrosine, serine, glycine, and arginine residues. This Fab protein library was constructed with a minimal codon design, and then screened against a variety of RNA targets. Several Fabs have been identified and isolated through phage display selection against three RNA targets. These Fabs were expressed and biochemically characterized for their binding affinities and specificities. To date, five Fabs have been identified and they have outstanding capabilities in binding their specific RNA antigen.
Show less - Date Issued
- 2012
- Identifier
- CFH0004167, ucf:44856
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004167
- Title
- Exacerbation of efp- sickness in Escherichia coli by an uncharacterized RNA helicase.
- Creator
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Wingo, Robert, Moore, Sean, Roy, Herve, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
In Escherichia coli, growth is rate-limited by translation capacity. Stalled ribosomes have profound effects on a cell such as altered mRNA abundance, decreased ribosome availability, and an imbalanced proteome. The absence of elongation factor P (EF-P), a universally conserved transpeptidation enhancer, presents an extreme example of this scenario, wherein ribosomes accumulate disproportionately onto messages that are more slowly translated and cell growth becomes notably impaired. We...
Show moreIn Escherichia coli, growth is rate-limited by translation capacity. Stalled ribosomes have profound effects on a cell such as altered mRNA abundance, decreased ribosome availability, and an imbalanced proteome. The absence of elongation factor P (EF-P), a universally conserved transpeptidation enhancer, presents an extreme example of this scenario, wherein ribosomes accumulate disproportionately onto messages that are more slowly translated and cell growth becomes notably impaired. We discovered that faster-growing cells arise spontaneously in ?efp cultures, suggesting that translation defects could be circumvented by mutating other genes. This thesis presents a genetic and biochemical analysis of a mechanism ?efp cells employ to overcome translation stress. Using a dual luciferase reporter system, we found that transpeptidation remained hindered in the faster growing ?efp cells. Whole genome sequencing of several fast-growing strains revealed mutations in a poorly characterized RNA helicase called HrpA. We determined that deletion of hrpA, or mutations at several conserved residues critical for HrpA's function, was sufficient to improve the fitness of ?efp cells. HrpA is a DEAH-box RNA helicase and represents a large class of enigmatic proteins that use ATP to restructure cellular RNAs; however, it's direct function in cellular physiology has yet to be clearly demonstrated. Several HrpA mutants were engineered to interrogate the molecular mechanism of HrpA and how its function impairs ?efp cells. Complementation in ?efp ?hrpA cells showed that a number of these mutants were unable to restore sickness, suggesting they were defective in key aspects of RNA processing. It was discovered that wild-type HrpA is associated with actively translating ribosomes and several of the inactive HrpA mutants impose substantial deleterious effects on translation and ribosome production. In sum, the work presented here describes a mechanism by which cells overcome translation stress involving a novel genetic and biochemical relationship between EF-P and HrpA.
Show less - Date Issued
- 2018
- Identifier
- CFE0007267, ucf:52178
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007267
- Title
- SPLIT DEOXYRIBOZYME PROBE FOR EFFICIENT DETECTION OF HIGHLY STRUCTURED RNA TARGETS.
- Creator
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Solarez, Sheila Raquel, Gerasimova, Yulia, De Bekker, Charissa, University of Central Florida
- Abstract / Description
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Transfer RNAs (tRNAs) are known for their role as adaptors during translation of the genetic information and as regulators for gene expression; uncharged tRNAs regulate global gene expression in response to changes in amino acid pools in the cell. Aminoacylated tRNAs play a role in non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosynthesis.[1] tRNAs have a highly stable structure that can present a...
Show moreTransfer RNAs (tRNAs) are known for their role as adaptors during translation of the genetic information and as regulators for gene expression; uncharged tRNAs regulate global gene expression in response to changes in amino acid pools in the cell. Aminoacylated tRNAs play a role in non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosynthesis.[1] tRNAs have a highly stable structure that can present a challenge for their detection using conventional techniques.[2] To enable signal amplification and lower detection limits, a split probe - split deoxyribozyme (sDz or BiDz) probe, which uses a double-labeled fluorogenic substrate as a reporter - has been introduced. In this project we developed an assay based on sDz probe to detect yeast tRNA[Phe] as a proof-of-principle highly structured target. An sDz probe was designed specific to tRNA[phe] that could efficiently unwind stable secondary and tertiary structure of the target RNA thereby providing an efficient tool for tRNA detection.[3] The efficiency of the developed sDz probe was compared with a currently used state-of-the-art hybridization probe - molecular beacon probe. The results obtained in the project further demonstrate the power of sDz probes for the detection of highly structured RNA analytes. The split probes show signal amplification capabilities in detection of structured analytes, which will benefit diagnostics, fundamental molecular biology research and therapeutic fields.
Show less - Date Issued
- 2018
- Identifier
- CFH2000311, ucf:45728
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000311
- Title
- Computational Methods for Comparative Non-coding RNA Analysis: from Secondary Structures to Tertiary Structures.
- Creator
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Ge, Ping, Zhang, Shaojie, Guha, Ratan, Stanley, Kenneth, Jha, Sumit, Song, Hojun, University of Central Florida
- Abstract / Description
-
Unlike message RNAs (mRNAs) whose information is encoded in the primary sequences, the cellular roles of non-coding RNAs (ncRNAs) originate from the structures. Therefore studying the structural conservation in ncRNAs is important to yield an in-depth understanding of their functionalities. In the past years, many computational methods have been proposed to analyze the common structural patterns in ncRNAs using comparative methods. However, the RNA structural comparison is not a trivial task,...
Show moreUnlike message RNAs (mRNAs) whose information is encoded in the primary sequences, the cellular roles of non-coding RNAs (ncRNAs) originate from the structures. Therefore studying the structural conservation in ncRNAs is important to yield an in-depth understanding of their functionalities. In the past years, many computational methods have been proposed to analyze the common structural patterns in ncRNAs using comparative methods. However, the RNA structural comparison is not a trivial task, and the existing approaches still have numerous issues in efficiency and accuracy. In this dissertation, we will introduce a suite ofnovel computational tools that extend the classic models for ncRNA secondary and tertiary structure comparisons.For RNA secondary structure analysis, we first developed a computational tool, named PhyloRNAalifold, to integrate the phylogenetic information into the consensus structural folding. The underlying idea of this algorithm is that the importance of a co-varying mutation should be determined by its position on the phylogenetic tree. By assigning high scores to the critical covariances, the prediction of RNA secondary structure can be more accurate. Besides structure prediction, we also developed a computational tool, named ProbeAlign, to improvethe efficiency of genome-wide ncRNA screening by using high-throughput RNA structural probing data. It treats the chemical reactivities embedded in the probing information as pairing attributes of the searching targets. This approach can avoid the time-consuming base pair matching in the secondary structure alignment. The application of ProbeAlign to the FragSeq datasets shows its capability of genome-wide ncRNAs analysis.For RNA tertiary structure analysis, we first developed a computational tool, named STAR3D, to find the global conservation in RNA 3D structures. STAR3D aims at finding the consensus of stacks by using 2D topology and 3D geometry together. Then, the loop regions can be ordered and aligned according to their relative positions in the consensus. This stack-guided alignment method adopts the divide-and-conquer strategy into RNA 3D structural alignment, which has improved its efficiency dramatically. Furthermore, we also have clustered all loop regions in non-redundant RNA 3D structures to de novo detect plausible RNA structural motifs. The computational pipeline, named RNAMSC, was extended to handle large-scale PDB datasets, and solid downstream analysis was performed to ensure the clustering results are valid and easily to be applied to further research. The final results contain many interesting variations of known motifs, such as GNAA tetraloop, kink-turn, sarcin-ricin and t-loops. We also discovered novel functional motifs that conserved in a wide range of ncRNAs, including ribosomal RNA, sgRNA, SRP RNA, GlmS riboswitch and twister ribozyme.
Show less - Date Issued
- 2016
- Identifier
- CFE0006104, ucf:51212
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006104
- Title
- Preparation, Characterization, and Delivery of Antibodies Binding to a Model Oncogenic RNA, Human Initiator tRNA.
- Creator
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Archer, Jennifer, Santra, Swadeshmukul, Ye, Jingdong, Ye, Jingdong, Self, William, Khaled, Annette, University of Central Florida
- Abstract / Description
-
Non-coding RNAs (ncRNAs) account for a higher percent of the genome than coding mRNAs, and are implicated in human disease such as cancer, neurological, cardiac and many others. While the majority of ncRNAs involved in disease were originally attributed to a class of RNAs called micro RNAs (miRNAs) with a small size of only about 19 -24 base pairs, emerging research has now demonstrated a class of long non-coding RNAs (lncRNAs) that have a size of over 200 base pairs to be responsible for...
Show moreNon-coding RNAs (ncRNAs) account for a higher percent of the genome than coding mRNAs, and are implicated in human disease such as cancer, neurological, cardiac and many others. While the majority of ncRNAs involved in disease were originally attributed to a class of RNAs called micro RNAs (miRNAs) with a small size of only about 19 -24 base pairs, emerging research has now demonstrated a class of long non-coding RNAs (lncRNAs) that have a size of over 200 base pairs to be responsible for gene regulation and other functional roles and have also found to contribute to pathogenesis in humans. The increased size and structural complexity require novel tools to study their interactions beyond RNA interference. Synthetic antibodies are classic tools and therapeutics utilized to study and treat proteins involved in human disease. Likewise we hypothesize that structured RNAs can also take advantage of synthetic antibodies to probe their functions and be utilized as therapeutics.Currently, antibodies have been raised against microbial riboswitches and other structured RNAs of single-celled organisms, and only one human structured RNA to the best of our knowledge. However, no one has yet to create a synthetic antibody capable of behaving as a therapeutic against a structured RNA. We therefore sought to raise an antibody Fab against a structured RNA, human initiator tRNA, a model oncogenic non-coding RNA and demonstrate its efficacy in vitro. We then characterized the antibody and explored delivery options in cancer cells including the use of nanoparticle delivery systems. With the emerging transcriptome revealing new ncRNAs implicated in human disease, our research has begun to address a new therapeutic strategy, laying down the foundation for the future of structured RNA-targeted therapies.
Show less - Date Issued
- 2014
- Identifier
- CFE0005756, ucf:50072
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005756
- Title
- MICRORNA REGULATION OF PROSTATE CANCER DESENSITIZATION TO ANDROGEN RECEPTOR ANTAGONIST DRUGS DURING ANDROGEN DEPRIVATION THERAPY.
- Creator
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Lorch, Robert, Chakrabarti, Ratna, University of Central Florida
- Abstract / Description
-
The current standard treatment of prostate cancer by androgen deprivation therapy involves using drugs such as bicalutamide (Casodex) to antagonistically block androgen receptors that are normally present within prostate cells. Usually, the therapy is successful in the short run at limiting the growth of prostate cancer. However, in virtually all cases tumors begin to grow aggressively again after several months of treatment and new therapies must be started. The mechanism by which these...
Show moreThe current standard treatment of prostate cancer by androgen deprivation therapy involves using drugs such as bicalutamide (Casodex) to antagonistically block androgen receptors that are normally present within prostate cells. Usually, the therapy is successful in the short run at limiting the growth of prostate cancer. However, in virtually all cases tumors begin to grow aggressively again after several months of treatment and new therapies must be started. The mechanism by which these prostate cells transform from androgen sensitive to androgen independent and anti-androgen resistant is unclear. In this study, we investigated the role of microRNAs, small 15 to 18 nucleotide regulatory RNAs, in regulating the desensitization of prostate cancer cells to the androgen receptor antagonist drug bicalutamide. In order to identify significant microRNAs, quantitative PCR was used to obtain genome-wide microRNA expression levels of 885 human microRNAs at different timepoints for androgen sensitive LNCaP cancer cells treated with bicalutamide and for untreated control cells in tissue culture. Analysis of microRNA expression by clustering analysis and by statistical comparisons of treatment groups resulted in identification of 28 microRNAs that have altered expression in the progression process. In silico target prediction analysis was performed with the microRNAs shown to have altered expression, and a group of genes predicted to be under microRNA regulatory control during cancer progression to resistance was identified. A microRNA expression profile can be useful in developing more effective prognostic and therapeutic tools for prostate cancer.
Show less - Date Issued
- 2011
- Identifier
- CFH0003826, ucf:44740
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0003826
- Title
- THE ROLE OF FRABIN (FGD4) IN AGGRESSIVE PROSTATE CANCER.
- Creator
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Bossan, Alexia M, Chakrabarti, Ratna, University of Central Florida
- Abstract / Description
-
A major problem in prostate cancer (PCa) management is the development of drug resistance. It is known that there are changes in PCa biology upon prolonged treatment with drugs, including anti-androgen drugs that alter cellular signaling processes leading to the development of castration resistant PCa. MicroRNAs (miRNAs) are regulatory molecules that modulate gene expression through inhibition of protein translation and modulate cellular functions. Altered expression of miRNAs is often noted...
Show moreA major problem in prostate cancer (PCa) management is the development of drug resistance. It is known that there are changes in PCa biology upon prolonged treatment with drugs, including anti-androgen drugs that alter cellular signaling processes leading to the development of castration resistant PCa. MicroRNAs (miRNAs) are regulatory molecules that modulate gene expression through inhibition of protein translation and modulate cellular functions. Altered expression of miRNAs is often noted in drug resistant cancer including PCa. Studies from our laboratory have identified a number of down-regulated miRNAs in PCa, including miR-l 7-92a miRNAs. Frabin (FGD4) is a target of the miR-l 7-92a cluster that was found to be up-regulated in PCa cells. For this paper's investigation, an FGD4 knockdown approach was used to identify the effects on cell viability, cell cycle progression, cell migration and drug sensitivity. Two PCa cells lines, LNCaP-104S (androgen sensitive) and PC-3 (androgen independent), were used for our studies. MTS assays for both cell lines showed significant reduction in cell viability following knockdown of FGD4 compared to transfection with control siRNAs. Cell cycle analysis revealed an arrest in the G2/M phase of the cells that were transfected with FGD4 siRNAs. Cell migration assays revealed a decrease in migration rate of PC-3 cells after knockdown, which supports the involvement of FGD4 in actin- cytoskeleton rearrangement. Treatments with anti-mitotic drug Docetaxel (PC-3) or androgen receptor antagonist bicalutamide/Casodex (LNCaP-104S) showed improved sensitivity of the FGD4 siRNA treated cells to these drugs. Our results suggest the potential for FGD4 knockdown to be used in combination with currently used drugs, increasing the effectiveness of frontline chemotherapeutics.
Show less - Date Issued
- 2017
- Identifier
- CFH2000162, ucf:45937
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000162
- Title
- Computational Methods for Analyzing RNA Folding Landscapes and its Applications.
- Creator
-
Li, Yuan, Zhang, Shaojie, Hua, Kien, Jha, Sumit, Hu, Haiyan, Li, Xiaoman, University of Central Florida
- Abstract / Description
-
Non-protein-coding RNAs play critical regulatory roles in cellular life. Many ncRNAs fold into specific structures in order to perform their biological functions. Some of the RNAs, such as riboswitches, can even fold into alternative structural conformations in order to participate in different biological processes. In addition, these RNAs can transit dynamically between different functional structures along folding pathways on their energy landscapes. These alternative functional structures...
Show moreNon-protein-coding RNAs play critical regulatory roles in cellular life. Many ncRNAs fold into specific structures in order to perform their biological functions. Some of the RNAs, such as riboswitches, can even fold into alternative structural conformations in order to participate in different biological processes. In addition, these RNAs can transit dynamically between different functional structures along folding pathways on their energy landscapes. These alternative functional structures are usually energetically favored and are stable in their local energy landscapes. Moreover, conformational transitions between any pair of alternate structures usually involve high energy barriers, such that RNAs can become kinetically trapped by these stable and local optimal structures.We have proposed a suite of computational approaches for analyzing and discovering regulatory RNAs through studying folding pathways, alternative structures and energy landscapes associated with conformational transitions of regulatory RNAs. First, we developed an approach, RNAEAPath, which can predict low-barrier folding pathways between two conformational structures of a single RNA molecule. Using RNAEAPath, we can analyze folding pathways between two functional RNA structures, and therefore study the mechanism behind RNA functional transitions from a thermodynamic perspective. Second, we introduced an approach, RNASLOpt, for finding all the stable and local optimal structures on the energy landscape of a single RNA molecule. We can use the generated stable and local optimal structures to represent the RNA energy landscape in a compact manner. In addition, we applied RNASLOpt to several known riboswitches and predicted their alternate functional structures accurately. Third, we integrated a comparative approach with RNASLOpt, and developed RNAConSLOpt, which can find all the consensus stable and local optimal structuresthat are conserved among a set of homologous regulatory RNAs. We can use RNAConSLOpt to predict alternate functional structures for regulatory RNA families. Finally, we have proposed a pipeline making use of RNAConSLOpt to computationally discover novel riboswitches in bacterial genomes. An application of the proposed pipeline to a set of bacteria in Bacillus genus results in the re-discovery of many known riboswitches, and the detection of several novel putative riboswitch elements.
Show less - Date Issued
- 2012
- Identifier
- CFE0004400, ucf:49365
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004400
- Title
- Finding Consensus Energy Folding Landscapes Between RNA Sequences.
- Creator
-
Burbridge, Joshua, Zhang, Shaojie, Hu, Haiyan, Jha, Sumit, University of Central Florida
- Abstract / Description
-
In molecular biology, the secondary structure of a ribonucleic acid (RNA) molecule is closely related to its biological function. One problem in structural bioinformatics is to determine the two- and three-dimensional structure of RNA using only sequencing information, which can be obtained at low cost. This entails designing sophisticated algorithms to simulate the process of RNA folding using detailed sets of thermodynamic parameters. The set of all chemically feasible structures an RNA...
Show moreIn molecular biology, the secondary structure of a ribonucleic acid (RNA) molecule is closely related to its biological function. One problem in structural bioinformatics is to determine the two- and three-dimensional structure of RNA using only sequencing information, which can be obtained at low cost. This entails designing sophisticated algorithms to simulate the process of RNA folding using detailed sets of thermodynamic parameters. The set of all chemically feasible structures an RNA molecule can assume, as well as the energy associated with each structure, is called its energy folding landscape. This research focuses on defining and solving the problem of finding the consensus landscape between multiple RNA molecules. Specifically, we discuss how this problem is equivalent to the problem of Balanced Global Network Alignment, and what effect a solution to this problem would have on our understanding of RNA.Because this problem is known to be NP-hard, we instead define an approximate consensus on a landscape of reduced size, which dramatically reduces the searching space associated with the problem. We use the program RNASLOpt to enumerate all stable local optimal secondary structures in multiple landscapes within a certain energy and stability range of the minimum free energy (MFE) structure. We then encode these using an extended structural alphabet and perform sequence alignment using a structural substitution matrix to find and rank the best matches between the sets based on stability, energy, and structural distance. We apply this method to twenty landscapes from four sets of riboswitches from Bacillus subtillis in order to predict their native (")on(") and (")off(") structures. We find that this method significantly reduces the size of the list of candidate structures, as well as increasing the ranking of previously obscure secondary structures, resulting in more accurate predictions overall. Advances in the field of structural bioinformatics can help elucidate the underlying mechanisms of many genetic diseases.
Show less - Date Issued
- 2015
- Identifier
- CFE0006210, ucf:51109
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006210
- Title
- DEVELOPMENT OF METHODS TO MODULATE NATURAL KILLER CELLS.
- Creator
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Shaver, Kari A, Copik, Alicja, University of Central Florida
- Abstract / Description
-
Natural Killer (NK) cell based immunotherapies have demonstrated success against malignancies and hematological cancers. However, tumors have developed mechanisms to evade detection by and suppress the immune system, commonly through altering the expression of cell-surface proteins. Overexpression of human leukocyte antigen-E (HLA-E), which binds to the inhibitory NKG2A on NK cells, protects malignant cells from lysis. Downregulating the NKG2A receptor on NK cells should release NK cell...
Show moreNatural Killer (NK) cell based immunotherapies have demonstrated success against malignancies and hematological cancers. However, tumors have developed mechanisms to evade detection by and suppress the immune system, commonly through altering the expression of cell-surface proteins. Overexpression of human leukocyte antigen-E (HLA-E), which binds to the inhibitory NKG2A on NK cells, protects malignant cells from lysis. Downregulating the NKG2A receptor on NK cells should release NK cell inhibition, but proves challenging as NK cells are difficult to transfect and no good methods currently exist. This project is designed to investigate the use of exosomes - small vesicles and natural carriers of regulatory microRNAs (miRNAs) and proteins that are shed from cells - as delivery vehicles for small RNAs (sRNAs) to immune cells. Exosomes are biologically compatible, immunologically inert, and interact with target cells through receptor-ligand interactions, allowing for targeted delivery of cargo. Exosomes loaded with shRNA against NKG2A were cultured in vitro with NK cells. Delivery success was assessed by monitoring NKG2A receptor expression on NK cells through flow cytometry. This research will provide valuable information that will likely impact the delivery of RNA therapeutics and unlock the full cytotoxic potential of NK immunotherapy.
Show less - Date Issued
- 2018
- Identifier
- CFH2000455, ucf:45720
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000455
- Title
- A COMPARISON OF PAPER-PENCIL VERSUS VIDEO-CONFERENCING ADMINISTRATION OF A NEUROBEHAVIORAL SCREENING TEST.
- Creator
-
Duffield, Tyler, Fouty, Homer, University of Central Florida
- Abstract / Description
-
Regardless of the reason, many patients/clients do not have access to face-to-face medical, neuropsychological, or mental health consultation, assessment, or treatment (Cowain, 2001). The term Remote Neuropsychological Assessment (RNA) has been proposed by Browndyke to denote the general use of telecommunication and Internet-based technologies in neuropsychological assessment and practice (as cited in Schatz & Browndyke, 2002). RNA (Telemedicine) offers a plausible, potentially cost-effective...
Show moreRegardless of the reason, many patients/clients do not have access to face-to-face medical, neuropsychological, or mental health consultation, assessment, or treatment (Cowain, 2001). The term Remote Neuropsychological Assessment (RNA) has been proposed by Browndyke to denote the general use of telecommunication and Internet-based technologies in neuropsychological assessment and practice (as cited in Schatz & Browndyke, 2002). RNA (Telemedicine) offers a plausible, potentially cost-effective solution to individuals in need of medical, neuropsychological, or mental health consultation, assessment, or treatment that are located in geographical areas away from the specialist (Armstrong, 2006; Berman, 2005; Cowain, 2001; Jacobsen, Sprenger, Andersson, & Krogstad, 2003). The purpose of this study was to examine if test performance for RNA administration of the Cognistat is comparable to test performance for the pencil-paper administration. A one-way repeated measures multivariate analysis of variance (MANOVA) was used to analyze the data. The main effect for administration modality was not significant, F(9, 126) = .375, p = .945. The present study demonstrated the utility of a widely used neurobehavioral screening test that provides a differentiated profile of cognitive status can now reliably be used through a video-conferencing administration. The importance of this finding is that a more comprehensive detection of deficits in multiple domains of cognitive functioning for screening purposes is now possible remotely.
Show less - Date Issued
- 2011
- Identifier
- CFE0003943, ucf:48687
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0003943
- Title
- Computational Methods for Comparative Non-coding RNA Analysis: From Structural Motif Identification to Genome-wide Functional Classification.
- Creator
-
Zhong, Cuncong, Zhang, Shaojie, Hu, Haiyan, Hua, Kien, Li, Xiaoman, University of Central Florida
- Abstract / Description
-
Non-coding RNA (ncRNA) plays critical functional roles such as regulation, catalysis, and modification etc. in the biological system. Non-coding RNAs exert their functions based on their specific structures, which makes the thorough understanding of their structures a key step towards their complete functional annotation. In this dissertation, we will cover a suite of computational methods for the comparison of ncRNA secondary and 3D structures, and their applications to ncRNA molecular...
Show moreNon-coding RNA (ncRNA) plays critical functional roles such as regulation, catalysis, and modification etc. in the biological system. Non-coding RNAs exert their functions based on their specific structures, which makes the thorough understanding of their structures a key step towards their complete functional annotation. In this dissertation, we will cover a suite of computational methods for the comparison of ncRNA secondary and 3D structures, and their applications to ncRNA molecular structural annotation and their genome-wide functional survey.Specifically, we have contributed the following five computational methods. First, we have developed an alignment algorithm to compare RNA structural motifs, which are recurrent RNA 3D structural fragments. Second, we have improved upon the previous alignment algorithm by incorporating base-stacking information and devise a new branch-and-bond algorithm. Third, we have developed a clustering pipeline for RNA structural motif classification using the above alignment methods. Fourth, we have generalized the clustering pipeline to a genome-wide analysis of RNA secondary structures. Finally, we have devised an ultra-fast alignment algorithm for RNA secondary structure by using the sparse dynamic programming technique.A large number of novel RNA structural motif instances and ncRNA elements have been discovered throughout these studies. We anticipate that these computational methods will significantly facilitate the analysis of ncRNA structures in the future.
Show less - Date Issued
- 2013
- Identifier
- CFE0004966, ucf:49580
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004966
- Title
- Methodological Improvements in the mRNA Profiling Assays for Incorporation into DNA Casework Workflows.
- Creator
-
Volk, Paris, Ballantyne, John, Gerasimova, Yulia, Baudelet, Matthieu, University of Central Florida
- Abstract / Description
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Currently, DNA profiling is the gold standard to identify an individual. However, determining body fluid origin is important in criminal investigations, offering additional information surrounding the circumstances of a crime. However, crime labs can only definitively identify blood and semen and presumptively saliva using techniques that consume time and sample and do not simultaneously identify all forensically relevant body fluids. This causes many crime labs to want to bypass body fluid...
Show moreCurrently, DNA profiling is the gold standard to identify an individual. However, determining body fluid origin is important in criminal investigations, offering additional information surrounding the circumstances of a crime. However, crime labs can only definitively identify blood and semen and presumptively saliva using techniques that consume time and sample and do not simultaneously identify all forensically relevant body fluids. This causes many crime labs to want to bypass body fluid identification altogether. Therefore, advances into more definitive molecular-based body fluid methods are necessary. One such technique is mRNA profiling because it provides a highly sensitive and specific approach to definitively identifying all relevant body fluids in parallel. Although advancements have been made, improvements to mRNA profiling methodologies still need to be researched such as 1) possible mRNA recovery from established DNA workflows and 2) possible integration of mRNA profiling into an upfront male DNA screening assay for triaging sexual-assault evidence likely to contain male DNA and reduce/eliminate a significant bottleneck in the standard DNA workflow of microscopic sperm identification. This study was designed to address these two issues by evaluating a novel way to recover RNA, for body fluid identification, from the waste fractions of a PrepFiler(TM) DNA extraction, and from the DNA extracts directly. Next, this study aimed to provide a relatively quick molecular-based approach for screening sexual-assault evidence. It involves extraction of RNA using the Dynabeads(TM) mRNA DIRECT(TM) Kit, while saving the extraction waste fractions for downstream male-DNA quantitation and STR profiling. The RNA is then used in a rapid and sensitive 1-step combined reverse transcription-HRM assay to positively detect the presence of sperm. Both non-conventional co-extraction methods successfully addressed current body fluid identification challenges and allowed for easy integration into existing workflows when single sourced, mixture and mock casework samples were analyzed.
Show less - Date Issued
- 2019
- Identifier
- CFE0007551, ucf:52627
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007551
- Title
- Multi-target high-throughput screening assays for antimicrobial drug discovery.
- Creator
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Grube, Christopher, Roy, Herve, Chakrabarti, Debopam, Moore, Sean, Koculi, Eda, University of Central Florida
- Abstract / Description
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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.
Show less - Date Issued
- 2019
- Identifier
- CFE0007642, ucf:52469
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007642
- Title
- MESSENGER RNA PROFILING: A PROTOTYPE METHOD FOR BODY FLUIDAND TISSUE IDENTIFICATION.
- Creator
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Juusola, Jane, Ballantyne, Jack, University of Central Florida
- Abstract / Description
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Conventional methods of body fluid identification use labor-intensive, technologically diverse techniques that are performed in a series, not parallel, manner and are costly in terms of time and sample. Furthermore, for some frequently encountered body fluids, such as saliva or vaginal secretions, no confirmatory technique exists. Terminally differentiated cells, such as blood lymphocytes or epithelial cells lining the oral cavity, have a unique pattern of gene expression, which is evinced by...
Show moreConventional methods of body fluid identification use labor-intensive, technologically diverse techniques that are performed in a series, not parallel, manner and are costly in terms of time and sample. Furthermore, for some frequently encountered body fluids, such as saliva or vaginal secretions, no confirmatory technique exists. Terminally differentiated cells, such as blood lymphocytes or epithelial cells lining the oral cavity, have a unique pattern of gene expression, which is evinced by the presence and relative abundance of specific mRNA species. If the type and abundance of mRNAs can be determined in a stain or tissue sample recovered at the crime scene, it would be possible to definitively identify the tissue or body fluid in question. Advantages of an mRNA-based approach, compared to conventional biochemical analysis, include greater specificity, simultaneous and semi-automated analysis though a common assay format, improved timeliness, decreased sample consumption and compatibility with DNA extraction methodologies. In this report, we demonstrate that RNA is stable in biological stains and can be recovered in sufficient quantity and quality for analysis using reverse transcriptasepolymerase chain reaction assay (RT-PCR). We have identified sets of candidate tissuespecific genes for body fluids and tissues of forensic interest, namely blood, saliva, semen, vaginal secretions, menstrual blood, urine, skin, muscle, adipose, and brain. We also report the identification of a new housekeeping gene for use in mRNA based assays. Select body fluid-specific genes have been incorporated into multiplex PCR and real-time PCR assays. These assays allow for the positive identification of blood, saliva, semen,vaginal secretions, and/or menstrual blood in a stain. The final task of this work was the molecular characterization of mRNA degradation patterns in biological stains, which not only has fundamental importance in possibly revealing mRNA degradation pathways in dried biological stains, but may ultimately lead to better assay design strategies for mRNA markers for forensic use. An mRNA-based approach described in this report could allow the facile identification of the tissue components present in a body fluid stain and could conceivably supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory.
Show less - Date Issued
- 2005
- Identifier
- CFE0000862, ucf:46668
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000862
- Title
- Structural and Functional Studies of Glycine Riboswitches and Development of Fab Chaperone Assisted RNA Crystallography.
- Creator
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Sherman, Eileen, Ye, Jingdong, Kolpashchikov, Dmitry, Koculi, Eda, Harper, James, Self, William, University of Central Florida
- Abstract / Description
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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.
Show less - Date Issued
- 2014
- Identifier
- CFE0005549, ucf:50285
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005549
- Title
- THE EFFECT OF MISMATCH PRIMERS ON THE EFFICIENCY OF AMPLIFICATION IN QUANTITATIVE POLYMERASE CHAIN REACTIONS.
- Creator
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Dawkins, Molly C, Moore, Sean, University of Central Florida
- Abstract / Description
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Polymerase chain reaction (PCR) is a method used in many research protocols to amplify a small amount of a short segment of DNA to millions of copies. PCR is used for many taxonomic studies, as well as for some medical diagnostic procedures. Through PCR, short DNA primers bind to the template DNA to allow the thermostable DNA polymerase to copy the DNA. Often, researchers create universal primers to target a conserved region of DNA in multiple species, for example, the 16S rRNA gene in...
Show morePolymerase chain reaction (PCR) is a method used in many research protocols to amplify a small amount of a short segment of DNA to millions of copies. PCR is used for many taxonomic studies, as well as for some medical diagnostic procedures. Through PCR, short DNA primers bind to the template DNA to allow the thermostable DNA polymerase to copy the DNA. Often, researchers create universal primers to target a conserved region of DNA in multiple species, for example, the 16S rRNA gene in bacteria. The problem with these universal primers is that they do not always perfectly match the target DNA. The mismatch primers can still bind to the template, but could affect the efficiency of the PCR amplification. The effect of mismatch primers on the efficiency of the amplification in PCR is the focus of this thesis. Four forward primers with various mismatch overhangs were generated and incorporated into a DNA template through an initial PCR. These primers contained the binding region complementary to the V3/V4 region of the 16S rRNA bacterial gene. Further quantitative PCR (qPCR) reactions were run on these newly-made templates using two sets of primers complementary to the 16S rRNA gene region – one with ambiguous base pairs, one with unambiguous base pairs. The qPCR amplification curves, the Cq values, and the initial concentrations of DNA products (seed values) were analyzed and compared. The results showed differences in the Cq values and seed values between the reactions containing mismatches and those not containing mismatches. Other variables including annealing temperature, addition of Illumina sequencing tails to the primers, and initial primer concentration were also tested to determine if these variables had an effect on the amplification. The results from these reactions using different variables were inconclusive.
Show less - Date Issued
- 2018
- Identifier
- CFH2000361, ucf:45761
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000361
- Title
- THE RELATIVE RECOVERABILITY OF DNA AND RNA PROFILES FROM FORENSICALLY RELEVANT BODY FLUID STAINS.
- Creator
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Parker, Charly, Ballantyne, Jack, University of Central Florida
- Abstract / Description
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Biological material (fluids or tissues) whether from the victim or suspect is often collected as forensic evidence, and methods to obtain and analyze the DNA found in that material have been well established. The type of body fluid (i.e. blood, saliva, semen, vaginal secretions, and menstrual blood) from which the DNA originated is also of interest, and messenger RNA typing provides a specific and sensitive means of body fluid identification. In order for mRNA profiling to be utilized in...
Show moreBiological material (fluids or tissues) whether from the victim or suspect is often collected as forensic evidence, and methods to obtain and analyze the DNA found in that material have been well established. The type of body fluid (i.e. blood, saliva, semen, vaginal secretions, and menstrual blood) from which the DNA originated is also of interest, and messenger RNA typing provides a specific and sensitive means of body fluid identification. In order for mRNA profiling to be utilized in routine forensic casework, RNA of sufficient quantity and quality must be obtained from biological fluid stains and the methods used for RNA analysis must be fully compatible with current DNA analysis methodologies. Several DNA/RNA co-extraction methods were evaluated based on the quantity and quality of DNA and RNA recovered and were also compared to standard non-co-extraction methods. The two most promising methods, the in-house developed NCFS co-extraction and the commercially available AllPrep DNA/RNA Mini kit, were then optimized by improving nucleic acid recovery and consistency of CE (capillary electrophoresis) detection results. The sensitivity of the two methods was also evaluated, and DNA and RNA profiles could be obtained for the lowest amount of blood (0.2 µL) and saliva and semen (1 µL) tested. Both extraction methods were found to be acceptable for use with forensic samples, and the ability to obtain full DNA profiles was not hindered by the co-extraction of RNA. It is generally believed that RNA is less stable than DNA which may prevent its use in forensic casework. However, the degradation rates of DNA and RNA in the same biological fluid stain have not been directly compared. To determine the relative stability of DNA and RNA, the optimized NCFS co-extraction protocol was used to isolate DNA and RNA from environmentally compromised stains. Dried blood, saliva, and semen stains and vaginal secretions swabs were incubated at set temperatures and outside for up to 1 year. Even at 56°C, DNA and RNA were both stable out to 1 year in the blood and semen stains, out to 3 months (DNA) and 1 year (RNA) in the saliva stains, and out to 6 months (DNA) and 3 months (RNA) in the vaginal secretions swabs. The recoverability of both nucleic acids was reduced when the samples were exposed to increased humidity, sunlight, and rain. In general, DNA and RNA stability was found to be similar with a loss in ability to obtain a DNA or RNA profile occurring at the same time point; however, there were instances where RNA body fluid markers were detected when a poor/no DNA profile was obtained, indicating that RNA in dried stains is sufficiently stable for mRNA body fluid typing to be used in forensic casework.
Show less - Date Issued
- 2011
- Identifier
- CFE0003596, ucf:48849
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0003596
- Title
- NEW COMPUTATIONAL APPROACHES FOR MULTIPLE RNA ALIGNMENT AND RNA SEARCH.
- Creator
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DeBlasio, Daniel, Zhang, Shaojie, University of Central Florida
- Abstract / Description
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In this thesis we explore the the theory and history behind RNA alignment. Normal sequence alignments as studied by computer scientists can be completed in $O(n^2)$ time in the naive case. The process involves taking two input sequences and finding the list of edits that can transform one sequence into the other. This process is applied to biology in many forms, such as the creation of multiple alignments and the search of genomic sequences. When you take into account the RNA sequence...
Show moreIn this thesis we explore the the theory and history behind RNA alignment. Normal sequence alignments as studied by computer scientists can be completed in $O(n^2)$ time in the naive case. The process involves taking two input sequences and finding the list of edits that can transform one sequence into the other. This process is applied to biology in many forms, such as the creation of multiple alignments and the search of genomic sequences. When you take into account the RNA sequence structure the problem becomes even harder. Multiple RNA structure alignment is particularly challenging because covarying mutations make sequence information alone insufficient. Existing tools for multiple RNA alignments first generate pair-wise RNA structure alignments and then build the multiple alignment using only the sequence information. Here we present PMFastR, an algorithm which iteratively uses a sequence-structure alignment procedure to build a multiple RNA structure alignment. PMFastR also has low memory consumption allowing for the alignment of large sequences such as 16S and 23S rRNA. Specifically, we reduce the memory consumption to $\sim O(band^2*m)$ where $band$ is the banding size. Other solutions are $\sim O(n^2*m)$ where $n$ and $m$ are the lengths of the target and query respectively. The algorithm also provides a method to utilize a multi-core environment. We present results on benchmark data sets from BRAliBase, which shows PMFastR outperforms other state-of-the-art programs. Furthermore, we regenerate 607 Rfam seed alignments and show that our automated process creates similar multiple alignments to the manually-curated Rfam seed alignments. While these methods can also be applied directly to genome sequence search, the abundance of new multiple species genome alignments presents a new area for exploration. Many multiple alignments of whole genomes are available and these alignments keep growing in size. These alignments can provide more information to the searcher than just a single sequence. Using the methodology from sequence-structure alignment we developed AlnAlign, which searches an entire genome alignment using RNA sequence structure. While programs have been readily available to align alignments, this is the first to our knowledge that is specifically designed for RNA sequences. This algorithm is presented only in theory and is yet to be tested.
Show less - Date Issued
- 2009
- Identifier
- CFE0002736, ucf:48166
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0002736