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CELLULAR IMMUNE RESPONSE AND GENE EXPRESSION PROFILING IN CROHN'S DISEASE PATIENTS ASSOCIATED WITH MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS
Title
CELLULAR IMMUNE RESPONSE AND GENE EXPRESSION PROFILING IN CROHN'S DISEASE PATIENTS ASSOCIATED WITH MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS.
Creator
Romero, Claudia, Naser, Saleh A., University of Central Florida
Abstract / Description
Despite the chronic debate in the etiology of crohn's disease (cd), a debilitating inflammatory bowel disease (ibd) closely related to ulcerative colitis (uc), an emerging interest in a possible mycobacterial role has been marked. Granuloma and pathologic manifestations in cd resemble aspects found in tuberculosis, leprosy and paratuberculosis. The latter, a chronic enteritis in cattle, goat, sheep and primates, which is similar to human enteritis, also known as cd, is caused by a fastidious,...
Show moreDespite the chronic debate in the etiology of crohn's disease (cd), a debilitating inflammatory bowel disease (ibd) closely related to ulcerative colitis (uc), an emerging interest in a possible mycobacterial role has been marked. Granuloma and pathologic manifestations in cd resemble aspects found in tuberculosis, leprosy and paratuberculosis. The latter, a chronic enteritis in cattle, goat, sheep and primates, which is similar to human enteritis, also known as cd, is caused by a fastidious, slow growing mycobacterium avium subspecies paratuberculosis (map). Due to the similarities between cd and paratuberculosis, a mycobacterial cause in cd has been proposed. Recent discovery of a possible association between nod2/card15 mutations and risk of cd added support to microorganism-host interactions. In this study, a possible mycobacterial role in cd etiology has been evaluated by investigating the presence of map dna, the state of the cellular immune response and microarray gene expression profiling in peripheral blood and surgical tissue from cd, uc and healthy control subjects. Nested pcr detected map dna in tissue from 10/12(83%) cd patients compared to 1/6(17%) non-ibd subjects. Fluorescence in situ hybridization (fish) with the aid of confocal scanning laser microscopy (cslm) detected map dna in 8/12(67%) cd subjects compared to 0/6(0%) in non-ibd subjects. The detection of map dna by either technique in tissue from cd subjects is significant compared to non-ibd subjects (p < 0.05). Map dna was also detected in both inflamed and non-inflamed tissue from patients with cd suggesting map infiltration in human tissue. Correlation of possible map presence and the function of polymorphonuclear leukocytes (pmn) and peripheral blood mononuclear cells (pbmc) in 19 cd patients and 12 controls have been evaluated. Pmn phagocytosis of viable fitc-map was suppressed in 13/19(68%) cd patients compared to 0/12(0%) in healthy controls (p<0.05). Pbmc phagocytosis of viable fitc-map was suppressed in 5/19(26%) of cd patients compared to 0/12(0%) of healthy controls (p<0.05). The proliferative response of pbmc with t-cell majority from cd and controls subjects was evaluated against pha, candida albicans, pwm and map ppd. Dysfunctional proliferative response against pha was found in 8/19(42%) cd patients compared to 1/12(8.3%) in controls suggesting possible t-cell anergy. Pbmc from 11 cd subjects reacted normally to pha, 7/11(64%) reacted strongly to map ppd suggesting previous exposure to mycobacteria, and 3/11(27%) did not react with map ppd suggesting lack of pre-exposure to mycobacteria. From the seven mycobacterial pre-exposed samples, 6/7(86%) showed a normal ability to recall antigens by activated macrophages when exposed to c. Albicans, and all 7 samples had a normal pwm response. Finally, microarray-chip technology was employed to identify the expression profile of genes that have a role in the immune response of cd patients. Rna was isolated from fresh buffy coats from 8 healthy controls, 2 cd, and 1 uc patients. Chips with an estimated of 30,000 human genes were hybridized to cdna from these samples. We found that 17% of the total number of genes was differentially expressed. Over 200 genes were involved in the immune response, 7 genes where common to both forms of ibd (uc and cd), and 8 genes were found to be either downregulated in cd and upregulated in uc or viceversa. The ifngr1 gene, which encodes the ligand-binding chain of the ifn-gamma receptor, was found to be downregulated in 2/2(100%) of cd patients, but not in uc patients. It is known that defects in ifngr1 are a cause of atypical mycobacterial infection and bcg infection. Patients suffering from this deficiency have an immunologic defect predisposing them to infection with mycobacteria. This correlates with the proposed theory as map being the causative agent of cd. Furthermore, the results indicate a host susceptibility requirement for the establishment of mycobacterial infection in cd patients. Further characterization of ifngr1 using real-time pcr is underway. Collectively, detection of map dna in the majority of cd tissue and the alteration in pmn and pbmc to respond efficiently to map may be related to the fact that mycobacterial pathogens infect phagocytic cells of susceptible hosts and consequently the immune response is dysregulated. Furthermore, the fact that a gene linked to mycobacterial susceptibility was found to be downregulated in cd patients only, strengthens the mycobacterial etiology of cd. In general, the data suggest a possible role for a bacterial pathogen in cd pathogenesis.
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Date Issued
2004
Identifier
CFE0000170, ucf:46170
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0000170
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
Delineating key genetic components on linear plasmid 36 that contribute to its essential role in Borrelia burgdorferi mammalian infectivity.
Title
Delineating key genetic components on linear plasmid 36 that contribute to its essential role in Borrelia burgdorferi mammalian infectivity.
Creator
Choudhury, Tisha, Jewett, Mollie, Khaled, Annette, Self, William, Cole, Alexander, University of Central Florida
Abstract / Description
The spirochete Borrelia burgdorferi is the etiologic agent of Lyme disease. This pathogen has a complex enzootic life cycle that involves passage between the tick vector (Ixodes scapularis) and various vertebrate hosts with humans being inadvertent hosts. There is a pressing need to study the genetic aspects of the B. burgdorferi infectious cycle and particularly spirochete genes involved in mammalian infectivity so as to develop novel therapeutic and diagnostic strategies to combat Lyme...
Show moreThe spirochete Borrelia burgdorferi is the etiologic agent of Lyme disease. This pathogen has a complex enzootic life cycle that involves passage between the tick vector (Ixodes scapularis) and various vertebrate hosts with humans being inadvertent hosts. There is a pressing need to study the genetic aspects of the B. burgdorferi infectious cycle and particularly spirochete genes involved in mammalian infectivity so as to develop novel therapeutic and diagnostic strategies to combat Lyme disease. The B. burgdorferi genome is fragmented and comprised of a single 900 kb linear chromosome and multiple linear and circular plasmids. It has been observed that plasmids are lost during serial passage and manipulation in vitro and the loss of some of the plasmids has been shown to be related to the loss of infectivity and persistence in the host. One such plasmid is linear plasmid 36 (lp36). lp36 is approximately 36kb in size and carries 56 putative open reading frames a majority of which have no predicted function. B. burgdorferi lacking lp36 show no deficiency in survival in ticks; however, these mutant spirochetes are highly attenuated for mammalian infectivity. The genetic components of this plasmid that contribute to its function in mammalian infectivity have yet to be clearly defined. Using an in vivo expression technology (IVET) based genetic screen the lp36-encoded gene bbk46 was identified as a candidate B. burgdorferi gene that is expressed during mammalian infection. Herein we present evidence that bbk46 is required for B. burgdorferi persistent infection of immunocompetent mice. Our data support a molecular model of immune evasion by which bbk46 functions as an RNA to regulate expression of the antigenic variation protein VlsE. These data represent the first demonstration of a regulatory mechanism critical for controlling vlsE gene expression. Moreover these findings further define the critical role of linear plasmid 36 in Borrelia burgdorferi pathogenesis.
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Date Issued
2013
Identifier
CFE0004982, ucf:49566
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004982
GENETIC AND PHYSIOLOGICAL CONTRIBUTION OF ADRENERGIC CELLS IN HEART DEVELOPMENT
Title
GENETIC AND PHYSIOLOGICAL CONTRIBUTION OF ADRENERGIC CELLS IN HEART DEVELOPMENT.
Creator
Osuala, Kingsley, Ebert, Steven, University of Central Florida
Abstract / Description
The adrenergic hormones norepinephrine (NE) and epinephrine (EPI) are essential for cardiovascular development as embryos lacking NE/EPI begin to die abruptly between embryonic days 10.5 and 11.5 due to apparent cardiac failure. The objective of this research aimed to elucidate the mechanism of embryonic fatality observed in the NE/EPI deficient mouse model. We utilized the dopamine [two]-hydroxylase knockout (Dbh-/-) mouse model, which lacks the gene and subsequent enzyme necessary for...
Show moreThe adrenergic hormones norepinephrine (NE) and epinephrine (EPI) are essential for cardiovascular development as embryos lacking NE/EPI begin to die abruptly between embryonic days 10.5 and 11.5 due to apparent cardiac failure. The objective of this research aimed to elucidate the mechanism of embryonic fatality observed in the NE/EPI deficient mouse model. We utilized the dopamine [two]-hydroxylase knockout (Dbh-/-) mouse model, which lacks the gene and subsequent enzyme necessary for conversion of dopamine to NE. We utilized embryonic mouse hearts at E10.5 from Dbh+/+ (control) and Dbh-/- (experimental model) mice for mRNA transcript expression profiling. Gene expression data suggests a novel connection between the ability of the heart to synthesize adrenergic hormones and the gene expression of enzymes involved in the production of retinoic acid. We found a statistically significant change in transcriptional expression of the retinol binding protein-1 (Rbp-1) [+ 1.4 fold], retinol dehydrogenase 12 (Rdh-12) [+ 2.1 fold], and the beta carotene monooxygenase-1(Bcmo1) [- 2.7 fold] genes in the hearts of E10.5 Dbh-/- embryos. These genes encode enzymes responsible for the transport and enzymatic conversion of retinoic acid precursor molecules. Since alterations in retinoic acid concentration have been shown to cause mid-gestational embryonic teratogenesis and lethality, we chose to quantify retinoic acid present in the Dbh-/-embryo at E10.5. Our results showed a significantly higher concentration of retinoic acid in E10.5 Dbh-/- embryos as compared to wild-type controls. This finding indicates that altered expression of genes involved in retinoic acid synthesis lead to a physiological change in retinoic acid concentration which may contribute to the mid-gestational lethality of the Dbh-/- embryos. Previous studies have shown that adrenergic hormones are produced within the heart itself beginning early in embryonic development, but little is known about the fate and disposition of adrenergic cells within the heart at later stages and into adulthood. To investigate this, we utilized a genetic mouse model that expresses [two]-galactosidase ([two]-Gal) in cells capable of synthesizing EPI in order to identify the spatial and temporal distribution of adrenergic-derived cells in the developing heart. The model was designed so that cells capable of expressing the gene phenylethanolamine N-methyltransferase (Pnmt), which is responsible for the synthesis of epinephrine, also produce the enzyme [two]-Gal as a reporter. Evaluation of the location of these cells in the embryonic heart showed a preferential distribution at the sinoatrial node and atrioventricular sulcus at E10.5, and later at E18.5 a more widely distributed ventricular pattern was observed. In the adult heart, the right myocardium showed two small cclusters of XGAL positive cells, one near the apex and another region of the sinoatrial node. However the left heart myocardium showed XGAL positive cells throughout the left atrium and scattered through the LV where the staining appeared localized to myocytes. Interestingly, the left-sided distribution in the LV appeared to be non-random and non-uniform, since specific regions near the base, mid-section, and apex were consistently XGAL-positive. These findings suggest that adrenergic cells differentiate into cardiac muscle tissue that is predominantly found on the left side of the heart by adult stages of development. Taken collectively, this study has shown a novel connection between adrenergic hormones and RA synthesis, two crucial developmental signaling pathways in the embryonic heart. Remarkably, adrenergic derived cells were also found to persist in the adult heart where they constitute relatively large sections of the left ventricle and atrium. These findings provide important new insights into the mechanism of adrenergic actions in the developing heart and a previously unanticipated structural role for cells descending from an adrenergic lineage in the formation of left myocardial tissue.
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Date Issued
2011
Identifier
CFE0003987, ucf:48653
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003987
PHYSICAL CHARACTERISTICS OF AN INDIVIDUAL: THE IDENTIFICATION OF BIOMARKERS FOR BIOLOGICAL AGE DETERMINATION
Title
PHYSICAL CHARACTERISTICS OF AN INDIVIDUAL: THE IDENTIFICATION OF BIOMARKERS FOR BIOLOGICAL AGE DETERMINATION.
Creator
Alvarez, Michelle, Ballantyne, Jack, University of Central Florida
Abstract / Description
It is now a matter of routine for the forensic scientist to obtain the genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. Potential contributors of the stain must either be known to investigators (i.e. a developed suspect) or the questioned profile must be searched against a database of DNA profiles such as those maintained in the CODIS National DNA database. However, in those instances where there is no developed suspect and no match is...
Show moreIt is now a matter of routine for the forensic scientist to obtain the genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. Potential contributors of the stain must either be known to investigators (i.e. a developed suspect) or the questioned profile must be searched against a database of DNA profiles such as those maintained in the CODIS National DNA database. However, in those instances where there is no developed suspect and no match is obtained after interrogation of appropriate DNA databases, the DNA profile per se presently provides no meaningful information to investigators, with the notable exception of gender determination. In these situations it would be advantageous to the investigation, if additional probative information could be obtained from the biological stain. A useful biometric that could provide important probative information, and one that may be amenable to molecular genetic analysis, is the biological age of an individual. The ability to provide investigators with information as to whether a DNA donor is a newborn, infant, toddler, child, adolescent, adult, middle-aged or elderly individual could be useful in certain cases, particularly those involving young children such as kidnappings or in providing additional intelligence during terrorist investigations. Currently no validated molecular assays exist for age determination. Biological human ageing can be defined by two distinct processes, degenerative and developmental ageing. The degenerative process of ageing is based on theories which identify an increase or decrease in physiological conditions with increasing age. In contrast, the developmental process of ageing is based on the theory that as individuals increase in chronological age, there will be subtle corresponding molecular based biological changes, each requiring genes to be expressed or silenced, indicative of that particular stage of life. We investigated the degenerative process of chromosomal telomere shortening, as well as the developmental process of gene expression profiling analysis, in an attempt to identify biomarkers of biological age in a self-renewing tissue such as blood. While telomere length analysis was an ineffective method for age determination; gene expression analysis revealed three gene transcripts expressed in an age-dependent physiological manner. These species namely- COL1A2, HBE1 and IGFBP3, were found to be expressed at elevated levels in younger individuals, newborns, or post-pubertal individuals, respectively. The biological process of hemoglobin switching was also investigated for the possibility of determining human age. While experimenting with the potential of using the gamma-hemoglobin chains, as newborn specific gene candidates, we serendipitously discovered four novel truncated transcripts, which we have termed HBG1n1, HBG1n2, HBG2n2 and HBG2n3; whose expression was restricted to whole-blood newborn samples and specific fetal tissues. The molecular origin of these transcripts appears to be at the RNA level, being produced by specific rearrangement events occurring in the standard gamma hemoglobin transcripts (HBG1 and HBG2), which yield these new isoforms that are expressed in a highly regulated tissue specific manner.
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Date Issued
2007
Identifier
CFE0001737, ucf:47297
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0001737