Browsing by Subject "Biochemistry"
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Item Discovery-Driven Proteomics to Advance the Evaluation of the Role of Lipids and Lipid Peroxidation-Related Carbonyl Stress in Biological Model Systems(2013-05-01) Talamantes, Tatjana D.; Laszlo ProkaiResearch has come a long way with the advancement of sequenced genomes in regards to a variety of organisms. In addition, the technology used to explore biological information encoded by these genome sequences has also been enhanced. However, the knowledge of the role that simple components play in biological functions and cellular processes of the organism, from a global perspective, remains limited. With the advent of proteomics, exploring pathways and networks influenced by cellular components such as lipids, has become possible in the context of systems biology. This dissertation has been divided into two parts. Both will focus on a discovery-driven approach to elucidate the diverse biological functions of lipids as well as stress and its consequences in two biological model systems using mass spectrometry-based proteomics. The first part investigates the broader role of phospholipids (specifically phosphatidylglycerol), essential components of biological membranes in prokaryotes and eukaryotes, in a genetically modified strain of cyanobacteria. Ultimately, understanding the impact of lipid-regulation in this simple organism is expected to reveal previously unknown lipid-signaling mechanisms, which could be further investigated in higher-order organisms. The second part is dedicated to the investigation of lipid-signaling mechanisms generated by electrophilic products of oxidative stress-induced lipid peroxidation, and its consequences in disturbing homeostasis in zebrafish embryos. Exploring free radical-mediated lipid peroxidation involved in cellular responses promises a comprehensive understanding of the role and/or contribution that electrophilic products of lipid peroxidation play in modulating oxidative stress-related signaling pathways potentially involved in various pathophysiological dysfunctions. Bioinformatics software successfully constructed networks from the proteins identified as being regulated by the induced carbonyl-stress including intra- and intercellular processes involved in eIF2 signaling, glycolysis, and remodeling of epithelial adherens junctions.Item EGCG and Its Role in Prostate Cancer Angiogenesis(2005-05-01) Thomas, Rusha; Porunelloor Mathew; Ming-Chi Wu; Dan DimitrijevichThomas, Rusha, EGCG and its role in prostate cancer angiogenesis. Master of Science (Biochemistry and Molecular Biology), May 2005, 47 pages, 14 illustrations, reference list, 44 titles. Hypoxia inducible factor-1 (HIF-1)-mediated upregulation of vascular endothelial growth factor (VEGF) has been implicated in angiogenesis associated with malignancies. HIF-1 consists of a constitutively expressed HIF-1β subunit, and a hypoxia-inducible HIF-1α subunit. Hypoxic induction of HIF-1α correlates with increased transcriptional activation of its downstream target genes, including VEGF. Epidemiologic and laboratory studies indicate that green tea has cancer preventive activity which has been attributed to its polyphenol components, the major one being epigallocatechin gallate (EGCG). This study investigated the effect of EGCG on normoxic VEGF expression in PC-3ML human prostate cancer cells. In contrast to previous studies where EGCG inhibited VEGF expression in breast and colon cancer cell lines, our results demonstrated that EGCG has the ability to upregulate HIF-1α transcription factor via inhibition of prolyl hydroxylation and subsequent von Hippel-Lindau protein interaction. HIF-1α upregulation by EGCG led to increased VEGF promoter activity and protein expression.Item Forensic Chemistry(Edward Arnold & Co., 1921-01-01) Lucas, A.Item Sexually Dimorphic Anxiety-Like Interoceptive Discriminative Stimuli(1997-12-01) Jung, Marianna E.; Walls, Cleatus; Downey, H. Fred; Forster, MichaelJung, Marianna E., Sexually Dimorphic Anxiety-Like Interoceptive Discriminative Stimuli. Doctor of Philosophy (Biomedical Sciences), December 1997, 150 pp, introduction, 2 chapters, discussion, bibliography, 109 titles. This study compared gender differences in the anxiogenic stimuli induced by either a GABA-A antagonist, pentylenetetrazol (PTZ) or by a 5-HT1b/2 agonist, m-chlorophenylpiperazine (m-CPP) before and during ethanol withdrawal (EW). Rats were trained to discriminate either PTZ (16mg/kg, IP) or m-CPP (1.2 mg/kg, IP) from saline in a two lever choice task for food reward. Male and female rats were gonadectomized or sham-operated, and ovariectomized (OVX) female rats were tested during replacement treatment with 17β estradiol (2.5 mg, 21 day release, sc). The dose-response for the discrimination of the interoceptive stimulus (IDS) produced by PTZ (0-16 mg/kg) or m-CPP (0 to 1.2 mg/kg) was measured under all hormonal conditions. For m-CPP trained rats, latency to first lever-press response was also tested. Results: sham and estradiol-replaced female rats had higher ED50s for discrimination of the PTZ or m-CPP IDS than intact males or OVX rats. There is a dose-related impairment of operant responding after mCPP injection. Sham and estradiol replaced OVX rats showed an increased delay to the initiation of response after m-CPP injection as compared to sham or castrated male rats or OVX rats that showed no effect at the doses tested. Rats then received a chronic ethanol diet (6.5%) for 10 days. At twelve hours of ethanol withdrawl, they were tested for lever selection after saline injection. Fewer sham female and estradiol-replaced female rats responded on the drug lever during acute EW as compared to sham male, castrated or OVX rats. In general, the anxiogenic drug lever selection of OVX rats resembled that of male rats but was restored toward that of sham female rats by estradiol replacement. Castration did not alter the response of male rats to either PTZ or mCPP. Serum β –estradiol concentrations were determined by radioimmunoassay for sham, OVX, and estradiol-replaced female rats. The concentration was significantly higher in hormone-replaced female rats than in OVX. The estradiol concentration in sham female rats showed a cyclic pattern over 4 consecutive days, but this pattern did not correlate with any difference in IDS. Blood ethanol concentration (BEC) was determined using head space gas chromatography. BEC was higher in intact female rats than in intact male rats after ethanol injection (2 g/kg, ip), but did not differ during EW. Conclusions: females produce less anxiogenic IDS in response to either GABA inhibition or 5-HT1b/2 activation, but are more impaired by m-CPP in their ability to initiate operant responses than male rats. In addition, fewer intact females developed a spontaneous IDS during EW than males which is not the result of lower BEC. Estrogen appears to play a trophic role in altering responsiveness to anxiogenic stimuli.Item STAT6 and Its Relationship with PSA and Annexin A2 in Human Prostate Cancer(2008-05-01) Roth, Cherice P.; Singh, Meharvan; Jones, Harlan P.; Sharma, RajendraRoth, Cherice, STAT6 and its relationship with PSA and Annexin A2 in Human Prostate Cancer. Master of Science (Biochemistry and Molecular Biology), May 2008, 49 pages, 13 illustrations, reference list, 54 titles. The increase of signal transducer and activator of transcription (STAT6) has been correlated with increased prostate tumor size as well as Gleason score. This molecule’s exact role in prostate cancer is still unknown. This research focused on the relationships of STAT6 in prostate specific antigen (PSA) expression as well as its novel interaction with annexin A2. These data show that STAT6 is involved in an alternate PSA expression pathway. It is also concluded that the interaction of STAT6 and annexin A2 increased the activated STAT6 (p-STAT) but not total STAT6. Chromatin immunoprecipitation also confirmed the novel protein-protein interaction between STAT6 and annexin A2 is nuclear.Item Study of Cross Bridge Kinetics in Hypertrophic Ventricular Muscle(2009-05-01) Muthu, Priya; Borejdo, JulianCardiovascular diseases are the leading cause of mortality worldwide; with heart failure being highly prevalent in most affluent parts of the world. There is a need for a better understanding of the mechanism underlying these diseases. Familial hypertrophic cardiomyopathy (FHC), one such disease, is a genetic disorder of the heart characterized by increased growth or hypertrophy in the thickness of the wall of the left ventricle, the largest of the four chambers of the heart. This research project is focused on one kind of FHC, the D166V mutation in the regulatory light chain in myosin, which is associated with a particularly malignant form of the disease. The overall goal of this project was to study cross bridge kinetics (contraction and ATP utilization) in cardiac muscle from transgenic mice and to develop assays to apply this to human samples. The real time orientation changes of myosin and actin during a single cross bridge cycle beginning in a state of rigor was studied by Fluorescence anisotropy. Rabbit psoas fibers were isolated and used to achieve imaging of a few fluorophores or cross bridges. This technique was then applied to study cardiac myofibrils from transgenic mice, carrying the mutation causing the disease (FHC). Methods to achieve single molecule detection to aid studying human samples suffering from this disease were developed using silver island films, monolayers of nanoparticles and surface plasmon coupled emission. The conclusions of this dissertation were that a mutation in a light chain in myosin cause changes in the cross bridge kinetics. Myofibrils from the mutated mice displayed a significant slower rate of detachment during contraction as well as increased ATPase activity, which if severe enough could cause the heart to compensate by increasing wall thickness (hypertrophy). Despite significant clinical advances in the treatment of various cardiovascular diseases, mortality rates remain high. No therapy currently exists to treat or delay progression from hypertrophy to heart failure. This proposal help answer an important question regarding the molecular basis of FHC-mediated pathology in the heart. Also, achieving imaging of a single fluorophore has numerous implications in the biological field, like studying ligand-receptor interactions in live cells, involvement of protein molecules in internalization of bacteria by cells, monitoring the conformational fluctuations of DNA, diagnosis of prion diseases and also in detection of viruses at an early phase of infection.Item The Effect of Late-Life Antioxidant Supplementaion on Brain Function(2007-10-01) Shetty, Ritu A.; Forster, Michael J.; Sumien, Nathalie; Singh, MeharvanShetty, Ritu A., The effect of late-life antioxidant supplementation on brain function. Doctor of Philolosophy (Biomedical Sciences), October, 2007, 229 pp., 5 tables, 18 figures, bibliography, 284 titles. Purpose: Aging is associated with mild to moderate loss in brain function over time. These functional losses are thought to involve reversible changes disrupting important cellular signaling processes. One of the theories that proposes to explain the reversible losses of function is the ‘oxidative stress’ hypothesis of aging. According to the oxidative stress hypothesis, there is an inherent cellular imbalance between production of oxidants and antioxidative defenses that increases with age and that leads to an increase in oxidative damage to macromolecules that are involved in crucial cell functions. Previous studies have established a link between these cellular changes associated with aging and the impairments in cognitive and psychomotor function. Further it has also been suggested that dietary interventions can modulate the level of oxidative stress, reducing oxidative damage and perhaps even ameliorate age-related dysfunction. Most interventions have been implemented relatively early in life and maintained until old age. However, the current studies were based on the rationale that interventions initiated in late-life could potentially lower oxidative damage and thereby alter cellular components responsible for functional impairments. Methods: In study I, separate groups of young (4 months) and old mice male C57BL/6 (18 months) were fed a control diet or a diet supplemented with low (105 mg/kg/day) or high (368 mg/kg/day) concentrations of CoQ10 for a period of 15 weeks. After 6 weeks on the diets, the mice were subjected to a battery of age-sensitive behavioral tests. In study II, separate groups of male C57BL/6 young mice aged 3-4 months and old mice 17-18 months (total of n=124) were fed ad libitum either a control diet (cyclodextrin in base diet), or the same diet supplemented with D- α-tocopheryl acetate (Toc) (200 mg/kg body wt/day), or with CoQ10 (148 mg/kg body wt/day) or a diet containing a combination of CoQ and Toc (200 mg/kg body wt/day + 148 mg/kg body wt/day) for a period of 13-14 weeks. In both studies mice were subjected to a battery of behavioral tests that required utilization of various component of memory and learning and sensorimotor reflexes. Results: In study I, low CoQ10 failed to improve cognitive and psychomotor function in old mice. However, the high CoQ10 marginally helped the old mice to navigate in the swim maze task with greater efficiency than control mice but did not affect their performance in probe trials. Conversely, the high CoQ10 diet selectively impaired the spatial performance in young mice in probe trials. The results from study I indicated that intake of CoQ10 initiated in late-life had minimal beneficial effects on behavior function. In study II, an age-associated decline of behavioral functioning was observed; however CoQ10 treatment failed to improve the performance of mice in any of the age-sensitive tests. Moreover, young mice supplemented with a high CoQ diet performed poorly in the probe trial in a swim maze task, suggesting a possible deleterious effect. The results from study II indicated that there was a significant improvement in performance of old mice in the coordinated running and the learning ability in discriminated avoidance task when supplemented with Toc or with a combination of CoQ10 and Toc. Conclusions: In conclusion, these studies suggest that benefits of single antioxidant supplementation when initiated late in life are limited; however dietary supplementation with a combination of antioxidants has a greater impact in reversing age-related decline in behavioral function.Item The Effects of a 14-3-3 inhibitor peptide on cardiomyocyte hypertrophic gene expression(2006-05-01) Ellis, Joel James; Dillon, Glenn; Mallet, Robert T.; Shepard, AllanJoel James Ellis, The Effects of a 14-3-3 inhibitor peptide on cardiomyocyte hypertrophic gene expression. Doctor of Philosophy (Biomedical Sciences), May 2006, 164 pp, 2 tables, 29 illustrations, references, 117 titles. The myocyte enhancer factor-2 (MEF2) family of transcription factors regulates transcription of muscle-dependent genes in skeletal, smooth and cardiac muscle types. MEF2 is activated by calcium/calmodulin (CaM)-dependent protein kinases I and IV and silenced by CaM kinase IlδC. MEF2 is held inactive in the nucleus by class I histone deacetylases (HDAC4&5) until phosphorylated by either CaM kinase I or IV. This phosphorylation results in HDAC transport out of the nucleus via a 14-3-3-dependent mechanism, thereby freeing MEF2 to drive transcription. 14-3-3 proteins exist as homodimers, which are modulated by the phosphorylation of serines 60 and 65 in the dimerization region. In this study, a HIV TAT protein transduction domain (PTD) fused 14-3-3 peptide inhibitor was generated that is designed to prevent the dimerization of 14-3-3 proteins. The data presented demonstrates that the 14-3-3 inhibitor peptide freely enters cardiomyocytes and is not cytotoxic under culture conditions. The presence of this 14-3-3 inhibitor promotes nuclear localization of class II HDACs in the presence of hypertrophic stimuli. Moreover, the 14-3-3 inhibitor prevented dimerization of wild type 14-3-3β in ventricular cardiomyocytes. Finally, increased MEF2-dependent transcriptional activity, due to CaMKI, CaMKIV and PE, was effectively silenced by this 14-3-3 inhibitor in cardiomyocytes. Atrial natriuretic peptide (ANP) transcriptional activity was also pressed in the presence of the 14-3-3 inhibitor under these same conditions. Taken together, these data suggest that the 14-3-3 inhibitor peptide is able to affect dimerization of 14-3-3, revealing a key regulatory point in the signaling of cardiac hypertrophy. Information from these results may provide a promising point of therapeutic intervention in the progression of heart disease due to cardiomyocyte hypertrophy.