Theses/Dissertations - Biology
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Browsing Theses/Dissertations - Biology by Author "Baylor University. Institute of Biomedical Studies."
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Item Anti-tumor properties of CD40 ligand when delivered as a transgene by the conditional replicative oncolytic adenovirus AdEH to breast cancer cells.(2007-02-14T21:54:39Z) Gomes, Erica Manuela.; Tong, Alex W.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.Cancer-selective biotherapy and gene therapy have been considered to be the next horizon towards developing a cure for breast cancer. CD40 ligand (CD40L), a member of the tumor necrosis factor superfamily, relays critical growth signals in various hematological malignancies and carcinomas. We previously demonstrated that recombinant CD40L can directly inhibit breast cancer cell growth. However a potential limitation of CD40L therapy is systemic toxicity. To improve efficacy of gene delivery and limit CD40L expression to the tumor micro-environment, we have generated a conditionally replicative virus (AdEHCD40L) that delivers CD40L selectively to breast carcinomas. Tumor/tissue-specific promoters (hypoxic/HIF-1α response and estrogen response elements) were incorporated to limit CD40L expression to the tumor microenvironment. Viral E1A and CD40L transgene expression was examined in breast cancer lines with low constitutive (T47D) or no (BT-20) HIF-1α expression. Both cell lines displayed significantly increased CD40L expression under viral- permissive conditions (T47D: 65.5 ± 3.9% with increased HIF-1α vs. 38.5 ± 2.8% under uninduced condition, p = 0.01; BT20: 43.2 ± 14.9% vs. 10.6 ± 0.2%, p = 0.03). AdEHCD40L produced markedly stronger inhibition compared to the parental construct (T47D: 53.7 ± 15.2% vs. 32.1 ± 11.7%, p = 0.02; BT20: 25.8 ± 10.2% vs. 15.2 ± 6.8%, p = 0.03), suggesting that growth inhibition encompassed CD40L-mediated and viral oncolytic events. Replicative activity of AdEHCD40L was comparable to the wild type adenovirus in breast cancer cells and attenuated in normal lung fibroblast cells, with reduced growth inhibitory impact. Preliminary findings on mechanisms of AdEHCD40L cytotoxicity indicated increased apoptotic (Annexin V+) and necrotic (propidium iodide incorporation) activities that were accompanied by reduced IBα, phosphorylation, G2M/S cell cycle arrest, culminating in an increased sub-G0 fraction, and altered chemokine/cytokine expression. AdEHCD40L biodistribution and its maximum tolerated dose (2x108 pfu) evaluated in mice were comparable to that of other conditional-replicative adenoviruses. Anti-tumor efficacy of AdEHCD40L showed a reduction in mean tumor diameter of MDA-MB-231 (44-58%) and T47D (49%) human breast cancer xenografts in SCID mice. These findings illustrate the applicability of CD40L gene transfer approach for experimental treatment of human breast cancer.Item Gamma Hydroxybutyrate (GHB): mechanisms of central nervous system toxicity.(2006-07-31T01:14:52Z) Lyng, Eric E.; Bottiglieri, Teodoro, 1958-; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.Gamma Hydroxybutyrate (GHB) is an endogenous metabolite of gamma-aminobutyric acid (GABA) and a putative neurotransmitter found in mammalian brain. The illicit use of GHB is a growing health care concern in the U.S. Low doses have euphoric and stimulatory effects while high doses act as a CNS depressant and can cause respiratory failure. In addition to fatalities and drug facilitated rape, in 2004 over 7000 GHB overdoses were reported in the U.S. Gamma Butyrolactone (GBL) and 1,4-Butanediol (1,4-BD), precursors of GHB, can cause similar effects after being converted to GHB in the body. While GHB is a Schedule I compound, recently it was given a Schedule III classification as a drug for treatment of cataplexy associated with narcolepsy. Individuals affected by the inherited disorder succinic semialdehyde dehydrogenase (SSADH) deficiency have significant elevation of GABA and GHB in body tissues, and a range of neurological complications. Currently there is no treatment option for a GHB overdose situation or for SSADH patients. GHB has been shown to inhibit striatal dopamine release leading to sedation and loss of locomotor activity in rodents. However, GHB’s mechanism of action is poorly understood. This dissertation investigated acute and chronic effects of GBL, a precursor to GHB, on locomotor function and monoamine neurotransmitter metabolism in mice. Dose response studies were performed to characterize the effects of GBL. Compounds aimed at increasing central dopaminergic activity or antagonism of GABAergic activity were tested for their ability to antagonize the locomotor effects induced by GBL. In total, eight different compounds were studied of which pergolide and nomifensine were successful at antagonizing the loss of locomotor activity when administered either prior to or after GBL. Chronic administration of GBL over the course of 14 days was evaluated in mice using a rotarod system. These studies did not reveal any long term detrimental effect of GBL on locomotor function. This dissertation is the first report showing the ability of pergolide and nomifensine to antagonize the loss of locomotor activity induced by GBL. These studies provide insight into treatment options for GHB toxicity or overdose as well as for patients with SSADH deficiency.Item The recombinant expression of two pollen allergens using plant-viral and yeast expression systems.(2006-05-09T19:12:08Z) Moehnke, Marcie H.; Kearney, Christopher Michel, 1958-; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.Allergic disease causes much distress within industrialized areas of the world, affecting approximately a quarter of the population in such areas. Current immunotherapy involves the administration of increasing concentrations of crude allergen extracts over a period of time, in an attempt to switch the individual's allergic response to that of a non-allergic individual. Such therapy is largely ineffective, especially for cedar hypersensitivity where only 30% of individuals respond after two years of weekly injections, and unwanted and sometimes life-threatening side effects can accompany specific immunotherapy. In an effort to increase its efficacy, as well as circumvent these negative side effects, recombinant DNA technology is being used to produce recombinant allergens that will take the place of crude allergen extracts found in immunotherapy injections. A main cause of allergic disease within south central Texas is pollen produced by mountain cedars, Juniperus ashei. In one study, I cloned a particular mountain cedar allergen, Jun a 3, into a tobacco mosaic virus vector under the regulation of a subgenomic promoter. Infectious viral transcripts were inoculated onto Nicotiana benthamiana plants, and recombinant Jun a 3 protein was detected within these plants at 21 days post-inoculation. The recombinant protein was able to bind anti-Jun a 3 IgG antibodies as well as IgE from mountain cedar allergic patient sera. A separate study also demonstrated the successful expression of recombinant Jun a 3, but in a yeast expression system. The Jun a 3 cDNA was cloned into a yeast expression vector and transformed into Pichia pastoris cells for expression. Western blotting and ELISA experiments confirmed the recombinant Jun a 3 produced by the yeast bound anti-Jun a 3 IgG antibodies and IgE from allergic patient sera. A third study utilized the tobacco mosaic virus-based plant expression system to produce the main Italian cypress allergen, Cup s 1, from Cupressus sempervirens. This recombinant allergen behaved very similarly to a native cross-reactive allergen in its binding to IgG antibodies and allergic patient sera.