Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

Riboswitches are naturally occurring RNA aptamers that form a precise three-­dimensional structure and selectively bind to cellular target molecules. Binding of the target molecule initiates an allosteric structural change in the riboswitch that in turn regulates expression of a relevant target gene. Most riboswitches specifically recognize the metabolic product of the gene that is being regulated. Expression may be regulated at either transcription or translation stage of gene expression. Most riboswitches are off switches meaning they turn off expression of metabolite producing gene when metabolite concentration is high enough. The ykkCD putative riboswitch appears to increases production of an efflux pump that expels toxic drugs from the cell by binding to the antibiotic tetracycline. Based on previous data collected the ykkCD putative riboswitch seems to regulate the efflux pump at the transcriptional level. To confirm this hypothesis we want to map the structural change that takes place upon binding of the antibiotic tetracycline to the mRNA. Nucleic acid footprinting studies will be used to map the binding site of tetracycline and the allosteric change that takes place upon tetracycline binding. / Department of Chemistry

Ultraviolet radiation causes an alteration in DNA by modifying neighboring thymine bases resulting in the formation of a dimer. These dimers block the processes of transcription and translation and ultimately no protein is synthesized and the cell dies. However, DNA repair mechanisms correct this damage by excising the dimer from the DNA strand and inserting replacement bases which are joined to the original strand by DNA ligase. This allows transcription to resume and ultimately protein synthesis to take place.This research focused on determining the DNA damage and subsequent repair levels in a connective tissue disorder, namely Marfan syndrome. This information is important in understanding the clinical expression and management of life threatening conditions in Marfan syndrome individuals.Preliminary results indicate that at 20-25J/m2 UV dose (254nm) Marfan syndrome skin cells show a mean reduced survival value of 12% compared to normal human skin cells. Gel electrophoresis indicates a reduced DNA repair level 24h post UV irradiation for Marfan syndrome skin cells compared to normal human skin cells. These results suggest Marfan syndrome skin cells have reduced survival and DNA repair levels compared to normal human skin cells. / Department of Biology

Mouse models (that develop or can be stimulated to develop lymphomas) are used to examine cancer-related processes. Mouse models can be effective tools used to identify new, early, and pre-malignant markers of lymphomas. Signal Transducer and Activator of Transcription (STAT) 6 is a transcription factor activated through the Jak-Stat pathway. Transgenic mice expressing a constantly activated Stat6 (Stat6VT) were previously generated and characterized to have altered lymphocyte homeostasis. Some of these Stat6VT mice developed a lymphoproliferative disorder (LPD). LPD, including lymphomas, develops when lymphocytes are overproduced or act abnormally. These Stat6VT mice may serve as a model for examining lymphoma development. In order to characterize the altered lymphocytes and determine if LPD observed in the Stat6VT mice is characteristic of lymphoma, RT-PCR analysis and Western analysis were done to examine if the presence of Stat6VT alters the expression of the cell cycle genes Gfi-1 and Gfi-1b and if these genes differ in LPD Stat6VT verses control mice. / Department of Biology

Due to the rapid destruction of native orchid habitats it has become necessary to house many endangered orchid species in greenhouse environments where enhanced spread of viral disease occurs due to the close contact between plants. This research was concerned with the construction of a library of genes whose expression is induced in response to viral challenge. In uncovering the genes that are activated during plant-pathogen interactions, it may be possible to manipulate these pathways to develop virus resistant orchids. Furthermore, this research will contribute additional information for the existing framework of plant-pathogen interactions of all plant species.In order to construct a library of genes expressed in response to viral infection, suppression subtractive hybridization was performed using the PCR-Select cDNA Subtraction Kit (CLONTECH, Palo Alto, CA) on Sophrolaeliacattleya Ginny Champion 'Riverbend' clones. RNA was isolated from plants that had been inoculated with the Odontoglossum ringspot virus (ORSV) and from control plants that had not been inoculated with ORSV. Following reverse transcription-PCR (RT-PCR) to obtain cDNA, cDNAs of the tester population (those cDNAs containing differentially expressed messages in response to ORSV) and the driver population (reference cDNAs from uninfected plants) were obtained. The two different cDNA populations are mixed together and hybridized. The sequences common to both populations were subtracted, leaving only the differentially expressed sequences available for PCR amplification.A library containing these genes was constructed, and one clone, chosen at random, was sequenced. Based on homology comparisons to known genes, we have cloned a gene that may contain a nucleotide binding site similar to that of the tobacco N gene, important for plant resistance to pathogens. In the near future, this clone will be used to construct probes for use in northern analysis to determine the timing and localization of the products of this gene. This information will aid in characterizing the function of the orchid N-gene and identifying other members of this signal cascade. In addition, many other clones await sequencing and similar characterization. / Department of Biology

Bacillus cereus causes emesis and/or diarrhea following ingestion of contaminated food due to the production of emetic toxins and enterotoxins. SYBR Green I is used as an intercalating dye and its florescence increases as a result of DNA amplification during real-time PCR. A second-derivative plot is obtained at the end of the PCR run, where amplicons are differentiated based on their DNA melting temperature (Tm). DNA was extracted from Tryptic Soy Broth (TSB) and 2.5% Nonfat Dry Milk (NFDM)-grown B. cereus at cell densities of 10',106,105,104,0,102, and 101 cfu/ml. In order to detect the multiple virulence determinants in pathogenic B. cereus, specific primers were used to target three enterotoxigenic genes (hblC, nheA, and hblA), followed by melt-curve analysis to confirm identity. Conditions used for this experiment allowed for the reproducible distinction of melt curves (characteristic Tm) for each amplicon (hblC = 74.5°C in TSB and 75°C in NFDM; nheA = 78°C; and hblA = 85.5°C in TSB and 84°C in NFDM) with an assay sensitivity of 106 CFU/ml in TSB and 10' CFU/ml in NFDM. B. cereus, nheA expression was examined in cells grown in TSB using transcript-specific, real-time nucleic acid sequence-based amplification (NASBA) with SYBR Green II. NASBA was applied to ascertain relative levels of nheA expression, when cells were subjected to subinhibitory levels of chloramphenicol as a stressor. B. cereus demonstrated consistently high levels of nheA expression at 15 hours when grown in TSB containing subinhibitory concentration (SIC) chloramphenicol (15.625 mg/ml). Relative levels of nheA expression differed in stressed B. cereus cells grown during the 30 hours incubation. / Department of Biology

Childhood acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Several proto-oncogenes that encode nuclear proteins are activated by various chromosomal translocations in ALL including TALI, TAL2, and LMO1 and LMO2. Ectopic TALI expression is observed in about 50 % of T-ALL and is the most common genetic anomaly associated with this pathology. Of interest to the present work is the characterization of various multiprotein complexes and protein protein interactions that drive T-ALL progression (as it relates to TALI and LMO1) and over expression of TALI and LMO1 has been shown to have inhibitory effects on apoptosis. Recent data suggests possible interactions between these two oncoproteins and the protein product of the lipoprotein lipase (LPL) gene. Lipoprotein lipase has a complex pattern of regulation and can be regulated in different ways including down-regulation upon induction of TNF-a in 3T3-L1 cells. Thus, this study was undertaken to determine if LPL is expressed in cells over expressing TAL1 and LMO1. Results from this study demonstrated an increase in LPL expression at both transcriptional and translational level in cells engineered to express TAL1 alone and TAL1 and LMO1 together. This finding is a step forward to understanding mechanisms that result in apoptosis prevention in T-ALL. Therefore, the apoptosis preventive role seen in cells that over express TAL and LMO1 and the presence of LPL in the same cell line, theorizes an apoptosis preventive role for lipoprotein lipase as well. / Department of Physiology and Health Science

Stat6 is a protein that activates the transcription of IL-4-stimulated genes. Amino acids critical for Stat6 function were examined in a mutational analysis of the Src homology (SH2) domain of the Stat6 protein. One mutation, substitution of two Alanines for Valine and Threonine in the N-terminal portion of the SH2 domain, produced a constitutively active form of the molecule that did not require IL-4 for activation. This mutant was named Stat6VT. Mice expressing Stat6VT in lymphocytes were generated, and it was found that approximately 10% of the population of Stat6VT mice, a lympho-proliferative disorder (LPD) occurred. In this study, we are examining genes that have a possible role in the development of this proliferative condition. Specifically, we examined the expression levels of Tiam1, Tacstdl, and Gfi-1 and Gfi-1B (genes known to regulate cellular proliferation and survival) in wildtype, normal Stat6VT and Stat6VT/LPD splenocytes by RT-PCR. Tiam1 results were inconclusive, and Tacstdl was not expressed at levels different from those seen in controls. Interestingly, Gfi-1 B, the homolog of Gfi-1, was expressed at increased levels in a specific subpopulation of cells from Stat6VT/LPD mice. Taken together, these data suggest that in cells expressing a constitutively active Stat6, increased expression of Gfi-1B may play a role in the mechanism of lymphoma development. / Department of Biology

Autoimmune diabetes is a disorder in which the (3-cells in the pancreatic islets of Langerhans are specifically destroyed resulting in absolute insulin deficiency; typically this is a childhood-onset disease, Type 1 Diabetes (T1D). Type 2 Diabetes (T2D) is a metabolic disorder usually developing in adults resulting from defects in insulin secretion and action. Latent Autoimmune Diabetes in Adults (LADA) is a form of diabetes that shares autoimmune disease pathology with T1D but a clinical presentation similar to T2D; LADA patients develop diabetes as adults (>25 years) and do not immediately require insulin treatment for survival. They are therefore often misdiagnosed with T2D. The aims of this work were to characterise immunological and genetic aspects of LADA using a large cohort collected from various patient repositories the United Kingdom to determine if it is a separate disease entity or an age-related extension of T1D. Both T1D and LADA are characterised by autoantibodies to the islet cell protein glutamic acid decarboxylase 65 (GADA) at diagnosis. The persistence and titre of GADA post-diagnosis in LADA was examined at 0.5, 3 and 6 years. GADA persisted in 93% of patients for 6 years; GADA litres decreased between 0.5 and 3 years post-diagnosis and either stabilised or increased again between 3 and 6 years. GADA titre was not associated with age at diagnosis, glycaemic control, β-cell function or other clinical features. GADA titre at 0.5 years was associated with a greater likelihood of requiring more intensive antihyperglycaemic therapy but did not predict therapy or insulin requirement at 3 and 6 years. Autoantibodies against IA-2 plus GADA compared to GADA alone at diagnosis predicted increased therapy requirement by 3 and 6 years and insulin requirement by 3 years postdiagnosis. Variants of the Human Leukocyte Antigen (HLA) genes DRB1 and DQB1, are associated with susceptibility for T1 D. An analysis of these variants in LADA (n = 378) revealed that the predisposing and protective variants in LADA are similar to those reported in T1D; DR3 (in linkage disequilibrium, LD with DQ2) and DR4 (in LD with DQ8) were the main predisposing variants whereas DR2 (in LD with DQ6) was most the protective against LADA. 85% of LADA patients possessed the DR3 and DR4 specificities, compared with 95% seen in T1D, suggesting a reduced predisposition in LADA compared with T1D. Synergistic effects of the DR3 and DR4 specificities occurred in LADA and the DRB1*0401 allele within the DR4 specificity was predisposing to disease, as seen in T1D. No other predisposing variants were identified in LADA. As reported for T1D, DR11, DR13, DQ5, DQ7 and DQ9 were protective against LADA; DQ6 was positively correlated with age at diagnosis. Association analysis of the insulin gene region in LADA (n = 400) showed that the variable number of tandem repeats (VNTR) locus primarily confers susceptibility to disease. Overall, the short Class I alleles predisposed to disease whereas longer Class III alleles conferred dominant protection, as reported in T1D. Fine-structure analysis showed that the Class I haplotypes 'IC+/ID+' and 'ID-' both conferred susceptibility for LADA - unlike in T1D, where the ID- haplotype has been reported to have protective effects. The Class III 'Protective' and Very Protective' haplotypes, conferred equal protection in LADA, as reported forTID. In conclusion; GADA persist post-diagnosis but are not markers for disease progression of LADA. Patterns of susceptibility at the HLA and insulin gene regions in LADA are similar to that reported for T1 D. LADA is likely to represent an age-related extension of T1D rather than a separate disease entity.