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DEVELOPMENT OF LUMINESCENT TOOLS FOR USE IN THE STUDY OF MYCOBACTERIUM TUBERCULOSIS
Title
DEVELOPMENT OF LUMINESCENT TOOLS FOR USE IN THE STUDY OF MYCOBACTERIUM TUBERCULOSIS.
Creator
Moore, Krista A, Rohde, Kyle, University of Central Florida
Abstract / Description
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a growing problem worldwide due to the emergence of multi-drug resistant and extensively-drug resistant strains of the bacteria. A key to combatting the spread of these strains lies in the understanding of gene expression occurring in Mtb. This study focuses on the development and optimization of a luciferase-based bioluminescent transcriptional reporter that can be used to monitor gene expression in Mtb. The...
Show moreMycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a growing problem worldwide due to the emergence of multi-drug resistant and extensively-drug resistant strains of the bacteria. A key to combatting the spread of these strains lies in the understanding of gene expression occurring in Mtb. This study focuses on the development and optimization of a luciferase-based bioluminescent transcriptional reporter that can be used to monitor gene expression in Mtb. The luminescent signal emitted from the reporter can be measured and correlated with the level of transcription of certain genes. This study focuses specifically on a gene called whiB7 which encodes a transcription factor known to contribute to the drug resistance of Mtb. The drug-inducible whiB7 promoter was cloned into various locations in the luciferase plasmid in order to determine the ideal configuration of the reporter for maximum luminescence. The optimized luciferase reporter was then compared with a fluorescent transcriptional reporter, mCherry, also under control of the whiB7 promoter. Fluorescent reporters present some disadvantages including delayed kinetics and inability to accurately reflect gene downregulation due to long half-life of reporter proteins. It was hypothesized that the luciferase reporter would solve these problems by offering a more sensitive and dynamic tool to monitor gene expression. Quantitative real-time PCR was used to measure whiB7 mRNA present in cultures containing either the luciferase or mCherry reporters. The luminescent and fluorescent signal given from these reporters was then compared to actual mRNA expression. It was observed that the signal from the luciferase reporter more closely matched mRNA expression at each timepoint, indicating that the luciferase reporter is a better gauge of actual gene expression levels than the mCherry reporter.
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Date Issued
2019
Identifier
CFH2000478, ucf:45912
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH2000478
Transcriptional and Post-transcriptional Regulation of Gene Expression
Title
Transcriptional and Post-transcriptional Regulation of Gene Expression.
Creator
Ding, Jun, Hu, Haiyan, Li, Xiaoman, Zhang, Shaojie, Jin, Yier, University of Central Florida
Abstract / Description
Regulation of gene expression includes a variety of mechanisms to increase or decrease specific gene products. Gene expression can be regulated at any stage from transcription to post-transcription and it's essential to almost all living organisms, as it increases the versatility and adaptability by allowing the cell to express the needed proteins. In this dissertation, we comprehensively studied the gene regulation from both transcriptional and post-transcriptional points of view....
Show moreRegulation of gene expression includes a variety of mechanisms to increase or decrease specific gene products. Gene expression can be regulated at any stage from transcription to post-transcription and it's essential to almost all living organisms, as it increases the versatility and adaptability by allowing the cell to express the needed proteins. In this dissertation, we comprehensively studied the gene regulation from both transcriptional and post-transcriptional points of view. Transcriptional regulation is by which cells regulate the transcription from DNA to RNA, thereby directing gene activity. Transcriptional factors (TFs) play a very important role in transcriptional regulation and they are proteins that bind to specific DNA sequences (regulatory elements) to regulate the gene expression. Current studies on TF binding are still very limited and thus, it leaves much to be improved on understanding the TF binding mechanism. To fill this gap, we proposed a variety of computational methods for predicting TF binding elements, which have been proved to be more efficient and accurate compared with other existing tools such as DREME and RSAT peaks-motif. On the other hand, studying only the transcriptional gene regulation is not enough for a comprehensive understanding. Therefore, we also studied the gene regulation at the post-transcriptional level. MicroRNAs (miRNAs) are believed to post-transcriptionally regulate the expression of thousands of target mRNAs, yet the miRNA binding mechanism is still not well understood. In this dissertation, we explored both the traditional and novel features of miRNA-binding and proposed several computational models for miRNA target prediction. The developed tools outperformed the traditional microRNA target prediction methods (.e.g miRanda and TargetScan) in terms of prediction accuracy (precision, recall) and time efficiency.
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Date Issued
2016
Identifier
CFE0006098, ucf:51197
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006098
FATHERS' LANGUAGE INFLUENCE ON THEIR SIX-MONTH-OLD INFANTS' VOCALIZATION DURING FREE-PLAY
Title
FATHERS' LANGUAGE INFLUENCE ON THEIR SIX-MONTH-OLD INFANTS' VOCALIZATION DURING FREE-PLAY.
Creator
Xia, Lu, Culp, Rex, University of Central Florida
Abstract / Description
Data for this study were derived from videotapes of 26 father-infant dyads, specifically from a five minute period of free-play. The first step was the creation of a literal transcription of the father-infant dyads interaction. Subsequently, nine variables of fathers' language characteristics and one infant characteristic were coded employing the literal transcriptions and observing the videotapes. The fathers' language variables were number of : (1) father utterances, (2) father...
Show moreData for this study were derived from videotapes of 26 father-infant dyads, specifically from a five minute period of free-play. The first step was the creation of a literal transcription of the father-infant dyads interaction. Subsequently, nine variables of fathers' language characteristics and one infant characteristic were coded employing the literal transcriptions and observing the videotapes. The fathers' language variables were number of : (1) father utterances, (2) father words, (3) father contingent responses, (4) father teaching utterances, (5) father descriptive teaching utterances, (6) father directive teaching utterances - making commands, (7) father directive teaching utterance – asking questions, (8) percentage of father teaching utterances, and (9) mean length of father utterances (MLU). The infant variable was number of vocalizations. Eight out of the nine variables were positively correlated to infant vocalizations, indicating the importance of fathers input in child language development. The only negative correlation in the present study was between Mean Length of Utterance (MLU) and infant vocalizations and the possible reasons are discussed. The findings support the idea that there are positive relationships between fathers' language characteristics and infant vocalizations. Recommendations are made that fathers should be involved in early intervention programs.
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Date Issued
2010
Identifier
CFE0003229, ucf:48553
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003229
The utility of verbal display redundancy in managing pilot's cognitive load during controller-pilot voice communications
Title
The utility of verbal display redundancy in managing pilot's cognitive load during controller-pilot voice communications.
Creator
Kratchounova, Daniela, Jentsch, Florian, Mouloua, Mustapha, Hancock, Peter, Wise, John, University of Central Florida
Abstract / Description
Miscommunication between controllers and pilots, potentially resulting from a high pilot cognitive load, has been a causal or contributing factor in a large number of aviation accidents. In this context, failure to communicate can be attributed, among other factors, to an inadequate human-system interface design, the related high cognitive load imposed on the pilot, and poor performance reflected by a higher error rate. To date, voice radio remains in service without any means for managing...
Show moreMiscommunication between controllers and pilots, potentially resulting from a high pilot cognitive load, has been a causal or contributing factor in a large number of aviation accidents. In this context, failure to communicate can be attributed, among other factors, to an inadequate human-system interface design, the related high cognitive load imposed on the pilot, and poor performance reflected by a higher error rate. To date, voice radio remains in service without any means for managing pilot cognitive load by design (as opposed to training or procedures). Such an oversight is what prompted this dissertation. The goals of this study were (a) to investigate the utility of a voice-to-text transcription (V-T-T) of ATC clearances in managing pilot's cognitive load during controller-pilot communications within the context of a modern flight deck environment, and (b) to validate whether a model of variable relationships which is generated in the domain of learning and instruction would (")transfer("), and to what extend, to an operational domain. First, within the theoretical framework built for this dissertation, all the pertaining factors were analyzed. Second, by using the process of synthesis, and based on guidelines generated from that theoretical framework, a redundant verbal display of ATC clearances (i.e., a V-T-T) was constructed. Third, the synthesized device was empirically examined. Thirty four pilots participated in the study (-) seventeen pilots with 100-250 total flight hours and seventeen with (>)500 total flight hours. All participants had flown within sixty days prior to attending the study. The experiment was conducted one pilot at a time in 2.5-hour blocks. A 2 Verbal Display Redundancy (no-redundancy and redundancy) X 2 Verbal Input Complexity (low and high) X 2 Level of Expertise (novices and experts) mixed-model design was used for the study with 5 IFR clearances in each Redundancy X Complexity condition. The results showed that the amounts of reduction of cognitive load and improvement of performance, when verbal display redundancy was provided, were in the range of about 20%. These results indicated that V-T-T is a device which has a tremendous potential to serve as (a) a pilot memory aid, (b) a way to verify a clearance has been captured correctly without having to make a (")Say again(") call, and (c) to ultimately improve the margin of safety by reducing the propensity for human error for the majority of pilot populations including those with English as a second language. Fourth, the results from the validation of theoretical models (")transfer(") showed that although cognitive load remained as a significant predictor of performance, both complexity and redundancy also had unique significant effects on performance. Furthermore, these results indicated that the relationship between these variables was not as (")clear-cut(") in the operational domain investigated here as the models from the domain of learning and instruction suggested. Until further research is conducted, (a) to investigate how changes in the operational task settings via adding additional coding (e.g., permanent record of clearances which can serve as both a memory aid and a way to verify a clearance is captured correctly) affect performance through mechanisms other than cognitive load; and (b) unless the theoretical models are modified to reflect how changes in the input variables impact the outcome in a variety of ways; a degree of prudence should be exercised when the results from the model (")transfer(") validation are applied to operational environments similar to the one investigated in this dissertation research.
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Date Issued
2012
Identifier
CFE0004251, ucf:49504
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004251
STUDIES ON A NOVEL HUMAN CARDIOSPECIFIC TRANSCRIPTION FACTOR AND ITS INVOLVEMENT IN OMI/HTRA2 MEDIATED CELL DEATH
Title
STUDIES ON A NOVEL HUMAN CARDIOSPECIFIC TRANSCRIPTION FACTOR AND ITS INVOLVEMENT IN OMI/HTRA2 MEDIATED CELL DEATH.
Creator
Puthucode Balakrishnan, Meenakshi, Zervos, Antonis, University of Central Florida
Abstract / Description
Omi/HtrA2 is a mitochondrial serine protease that is known to translocate to the cytoplasm upon induction of apoptosis and to activate caspase-dependent and caspase-independent cell death. The molecular mechanism of Omi/HtrA2ÂÂ's function is not clear but involves degradation of specific substrates. These substrates include cytoplasmic, mitochondrial, as well as nuclear proteins. We have isolated a new Omi/HtrA2 interactor, the THAP5 protein. THAP5 is a fifth member of...
Show moreOmi/HtrA2 is a mitochondrial serine protease that is known to translocate to the cytoplasm upon induction of apoptosis and to activate caspase-dependent and caspase-independent cell death. The molecular mechanism of Omi/HtrA2ÂÂ's function is not clear but involves degradation of specific substrates. These substrates include cytoplasmic, mitochondrial, as well as nuclear proteins. We have isolated a new Omi/HtrA2 interactor, the THAP5 protein. THAP5 is a fifth member of a large family of transcription factors that are involved in cell proliferation, apoptosis, cell cycle control, chromosome segregation, chromatin modification and transcriptional regulation. THAP5 is an approximately 50kDa nuclear protein, with a restricted pattern of expression. Furthermore, there is no mouse or rat homolog for this protein. THAP5 mRNA is highly expressed in the human heart but some expression is also seen in the brain and skeletal muscle. The normal function of THAP5 in the heart or heart disease is unknown. THAP5 protein level is significantly reduced in the myocardial infarction (MI) area in the heart of patients with coronary artery disease (CAD). This part of the heart sustains most of the cellular damage and apoptosis. Our data clearly show that THAP5 is a specific substrate of the proapoptotic Omi/HtrA2 protease and is cleaved and removed during cell death. The molecular mechanism of THAP5ÂÂ's function is unclear. THAP5 can bind to a specific DNA sequence and repress transcription of a reporter gene. Our work suggests that THAP5 is a tissue specific transcriptional repressor that plays an important role in the normal function of the human heart as well as in the development of heart disease.
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Date Issued
2010
Identifier
CFE0003412, ucf:48409
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003412
Implication of alpha-synuclein transcriptional regulation and mutagenesis in the pathogenesis of sporadic Parkinson's disease
Title
Implication of alpha-synuclein transcriptional regulation and mutagenesis in the pathogenesis of sporadic Parkinson's disease.
Creator
Basu, Sambuddha, Kim, Yoon-Seong, King, Stephen, Estevez, Alvaro, Altomare, Deborah, University of Central Florida
Abstract / Description
Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by selective loss of dopaminergic neurons (DA neurons) from the substantia nigra (SN) of the mid-brain. PD is classically associated with cytoplasmic inclusion of aggregated proteins called Lewy bodies. alpha-synuclein (?-SYN) coded by the gene SNCA, is one of the major components of Lewy body and neurite along with several other proteins like ubiquitin, neurofilament to name a few. PD is broadly categorized...
Show moreParkinson's disease (PD) is an age-related neurodegenerative disorder characterized by selective loss of dopaminergic neurons (DA neurons) from the substantia nigra (SN) of the mid-brain. PD is classically associated with cytoplasmic inclusion of aggregated proteins called Lewy bodies. alpha-synuclein (?-SYN) coded by the gene SNCA, is one of the major components of Lewy body and neurite along with several other proteins like ubiquitin, neurofilament to name a few. PD is broadly categorized into two groups based on their incidence of occurrence. First is the familial form that occurs due to known genetic aberrations like mutation, gene duplication/triplication in important PD associated gene like SNCA which in turn leads to early-onset PD (EOPD). Second is the late-onset idiopathic or sporadic form, whose origin of occurrence is often unknown. Interestingly, more than 90%-95% of reported PD cases belong to the latter category. Although, the familial and the idiopathic form of PD are different in their respective cause of occurrence, aggregation of ?-SYN into Lewy body is a common pathologic hallmark seen in both. Aggregation of ?-SYN in turn is strongly implicated by the transcriptional upregulation of the gene as seen in both familial forms as well as idiopathic forms. In this thesis, we first describe the designing and functioning of a novel tool to monitor real-time SNCA transcription in Human Embryonic Kidney (HEK) 293T cells. In the next part, we shed light into a novel transcriptional deregulation phenomenon called transcriptional mutagenesis, which leads to accelerated aggregation of ?-SYN as seen in sporadic PD. In brief, the focus of this work is to highlight the importance of transcriptional regulation of SNCA gene, through development of a tool and a mechanism affecting the fidelity of transcription under pathologic condition. In the first study, we developed a stable cell line in HEK293T cells in which ?-SYN was tagged with Nanoluc luciferase reporter using CRISPR/Cas9-mediated genome editing. Nanoluc is a small stable reporter of 19KDa size, which is 150 fold brighter compared to firefly and Renilla luciferase, thus making it a very good candidate for endogenous monitoring of gene regulations. We successfully integrated the Nanoluc at the 3'end of the SNCA before the stop codon. Successful integration of the Nanoluc was demonstrated by the fusion ?-SYN protein containing the Nanoluc. This allowed efficient monitoring of ?-SYN transcription keeping its native epigenetic landscape unperturbed which was otherwise difficult using exogenous luciferase reporter assays. The Nanoluc activity monitored by a simple two-step assay faithfully reflected the endogenous deregulation of SNCA following treatment with different drugs including epigenetic modulators and dopamine which were already known to up-regulate SNCA transcription. Interestingly, use of exogenous promoter-reporter assays (firefly luciferase assays) failed to reproduce the similar outcomes. In fact, exogenous system showed contradictory results in terms of the ?-SYN regulation which aroused from spurious effects of the drug on the reporter system. To our knowledge, this is the first report showing endogenous monitoring of ?-SYN transcription, thus making it an efficient drug screening tool that can be very effectively used for therapeutic intervention in PD. In the next study, we investigated the effect of oxidative DNA damage in the form of 8-hydroxy-2-deoxyguanosine (8-oxodG, oxidized guanine) on aggregation of ?-SYN through a novel phenomenon called transcriptional mutagenesis. It is already known that 8-oxodG is repaired by a specific component of the base excision repair machinery of the cell called 8-oxodG-DNA glycosylase 1 (OGG1). If left unrepaired, 8-oxodG can lead to misincorporation of adenine instead of cytosine (C?A transversion) in the synthesized mRNA during transcription for post-mitotic cells like neurons. This phenomenon is called transcriptional mutagenesis (TM) and can generate novel mutant variants of any functional protein. ?-SYN, which is implicated very strongly in the pathogenesis of PD, has been shown to become aggregation prone by specific point mutation. Previous studies have shown that certain point mutations can make ?-SYN more prone to aggregation and can affect the aggregation of the parental protein as a template directed misfolding mechanism. We used SNCA as a model gene and predicted the generation of forty-three different positions that can be mutated by the TM event. We investigated the generation of three out of the forty-three possible TM mutants from the SN of post-mortem PD and age-matched control brain cohorts based on their potential to aggregate as predicted by aggregation prediction software TANGO. The three mutants were Serine42Tyrosine (S42Y), Alanine53Glutamate (A53E) and Serine129Tyrosine (S129Y). We confirmed the presence of all the three mutant ?-SYN (S42Y, A53E and S129Y) in SNCA mRNA from the SN of human post-mortem PD brain using a PCR-based detection technique. As expected, analysis of the overall distribution of the three mutants showed a higher rate of occurance in the PD cohort compared to the age-matched controls. Sequencing genomic DNA of the same PD sample from the same region of ?-SYN revealed no mutations at the genomic DNA level, thus implying its generation during transcription. Although we could detect the presence of S42Y, A53E and S129Y ?-SYN in the cohort of PD patients, we focused to analyse the contribution of S42Y towards the aggregation of wild-type (WT) ?-SYN parental protein based on its higher potential to aggregate. By using cell-based biochemical and recombinant protein assays, we saw that S42Y-?-SYN can accelerate the aggregation process involving the WT protein even when present in significantly lower proportion (100 times less compared to the WT). Importantly, we developed antibody to specifically detect the S42Y ?-SYN in human PD cohort. Immunohistochemical analysis of serial post-mortem PD brain sections with Hematoxylin and Eosin staining (H(&)E), anti-ubiquitin staining and anti-S42Y ?-SYN staining, showed Lewy bodies that stained positively with S42Y ? -SYN. To our knowledge, this is the first report about TM related mutations of ?-SYN in Parkinson's disease and their role in the pathogenesis.
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Date Issued
2017
Identifier
CFE0006719, ucf:51882
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006719