Identification of therapeutically relevant molecules is necessary for the advancement of non-viral reprogramming of human cells for regenerative medicine. We have developed a novel non-viral model system that transforms primary human dermal fibroblasts into cells with induced regeneration competence (iRC). Low oxygen-mediated effects of fibroblast growth factor FGF2 lead to an increased cellular lifespan with a two fold increase in population doublings before senescence, remaining non-tumorigenic when injected into SCID mice while maintaining regeneration competence. This system allows us to study molecules that participate in increased cellular lifespan in a non-tumorigenic system. Analysis of chromatin modification enzymes by hybridization array, RT-PCR, and Western blots revealed upregulation of the arginine methyltransferase PRMT8 in iRC cells, challenging the paradigm that PRMT8 is solely expressed in brain tissue at the plasma membrane. Possibly leading to the erroneous conclusions that PRMT8 is brain specific at the plasma membrane is the fact that PRMT8 has several mRNA variants and protein isoforms. Here, I report expression of a novel PRMT8 variant in human dermal fibroblasts. Essential participation of PRMT8 in cellular proliferation was identified as a novel function for this enzyme through siRNA-mediated knockdown in both non-tumorigenic and tumorigenic cell lines. While other members of the PRMT family have known roles in cell cycle progression, I show for the first time that PRMT8 expression is reduced in both natural senescence and by premature induction of replicative senescence using sub-cytotoxic levels of hydrogen peroxide, implicating a correlation between PRMT8 expression and cell cycle progression. However, PRMT8 overexpression causes no significant change in the number of population doublings or the amount of time spent in culture prior to senescence, and does not alter the expression of key cell cycle regulatory genes. These results suggest that maintenance of PRMT8 expression is critical for cellular proliferation, but overexpression of PRMT8 alone is not sufficient to increase cellular lifespan. I determined that oxygen is the primary mediator of PRMT8 upregulation in the iRC system and therefore investigate histone occupancy of the PRMT8 promoter at hypoxia response elements. Through this analysis, I found bivalent occupancy regardless of culture conditions, indicating that PRMT8 maintains a state of poised readiness for transcriptional accessibility. The mechanism by which PRMT8 participates in cellular proliferation was investigated through binding partner identification. A binding partner of endogenous PRMT8 is identified here for the first time as FGF2 using co-IP and mass spectrometry. As iRC cells demonstrate a unique phenotype that uncouples the mechanisms of increased lifespan from tumorigenesis, I investigated the feasibility of PRMT8 as a cancer biomarker by mining publically available data in light of our own. I showed that PRMT8 is not only expressed in a variety of cancers, but that its expression is amplified. Moreover, PRMT8 expression significantly correlates to patient survival in specific cancers, strengthening the feasibility of this molecule as a biomarker. Aberrant expression of most PRMT family members has been described in various cancers, and specific PRMT variants are currently being used as prognostic markers. As such, I analyzed variant-specific PRMT8 expression in primary cancer cell lines and show that tumorigenic glioblastomas express PRMT8 mRNA variant 2. These data suggest that PRMT8 is a viable candidate for further study as a prognostic cancer biomarker, specifically for brain cancer.

Thesis (Ph.D.)--Ohio University, March, 2006. / Includes bibliographical references (leaves 152-160)

The work reported in this thesis is an investigation of certain aspects of both the blood pressure (BP) elevating properties and BP lowering properties of arginine vasopressin (AVP). The hypothesis that endothelin (ET) contributes to the exaggerated pressor responsiveness of the spontaneously hypertensive rat (SHR) to AVP was tested by comparing the changes of BP, cardiac output (CO), and total peripheral conductance (TPC) to AVP in SHR to those in Wistar-Kyoto rats (WKY) both in the presence and absence of bosentan, a non-selective ET antagonist. Bosentan antagonized the BP responses to exogenous ET-1 in a competitive fashion. The pressor effects of AVP and Ang II were exaggerated in the SHR compared to WKY. Except for the highest dose of AVP, pre-treatment with bosentan blunted the increases in BP and the decreases in total peripheral conductance (TPC) evoked by AVP in the SHR, but not in the WKY. In contrast to AVP, bosentan blunted the increases in BP evoked by lower doses of Ang II in both strains, although the effect was more pronounced in the SHR. These results suggest that ET contributes to the hemodynamic effects of AVP in the SHR and to the effects of Ang II in both strains. The findings support the hypothesis that ET contributes to the exaggerated pressor responsiveness of SHR to AVP. Cessation of a 3 hour infusion of AVP (20 ng/kg/min) results in a dramatic and prolonged decrease in BP below pre-infusion basal levels in hypertensive rats, but not in normotensive control rats. This phenomenon has been termed the "withdrawal-induced antihypertensive phenomenon" (WAP). In order to determine the time course of the WAP, and the role of CO and TPC in the WAP, BP was recorded by radiotelemetry and CO was recorded from aonic flowprobes in conscious unrestrained rats before, during, and after a 3 hr i.v. infusion of 20 ng/kg/min of AVP. Baseline mean arterial BP values were lower, and the magnitude of the WAP was less in SHR when BP was recorded with radiotelemetric implants than in another group in which BP was recorded with conventional externalized femoral arterial catheters. Strikingly, absolute BP values recorded both during and after the AVP infusion were similar in the two groups. BP remained decreased for several days in SHR infused with AVP with complete recovery requiring 6-7 days. In rats instrumented with aortic flow probes, the fall in pressure following cessation of the AVP infusion was associated with a large decrease in CO below control levels in the SHR. The time-course of the CO responses approximated the time-course of the pressure responses. These results lead to the following conclusions: firstly, telemetry is a superior method for recording BP in hypertensive animals, and the lower magnitude of the WAP was probably related to the lower basal BPs recorded by this method; secondly, the mechanism accounting for the WAP must be of a long duration; thirdly, the WAP is mediated by a fall in CO and not by an increase in TPC. In conclusion, the results of the thesis support the hypothesis that ET contributes to the BP elevating properties of AVP and, consequently, the exaggerated pressor responsiveness of SHR to the peptide, and that the BP lowering properties of AVP are mediated by a fall in CO.

Reproduction de : Thèse de doctorat : Médecine. Nutrition périnatale : Nantes : 2008. / Bibliogr.

Thesis (Ph.D.), Memorial University of Newfoundland, 2000. / Bibliography: leaves 140-154.

Thesis (M.S.)--California Polytechnic State University, 2009. / Title from PDF title page; viewed on January 6, 2010. Major professor: Brooke D. Humphrey, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Agriculture, with Specialization in Animal Science." "October 2009." Includes bibliographical references (p. 102).

The twin-arginine translocase allows the translocation of folded protein substrates across the cytoplasmic membrane of bacteria and archaea or the thylakoid membrane of plants. In Escherichia coli, its protein components TatA, TatB and TatC assemble dynamically upon interaction with protein substrates. Prior to export, the machinery performs a quality control check so that only correctly folded proteins are translocated. The first objective of this work was to derive and apply new methodologies based on the inherent qualities of the pathway. We developed a new bacterial two-hybrid system that capitalizes on the folding quality control mechanism of the Tat pathway. One protein (prey) is fused to Tat-specific signal peptide. A second (bait) protein is produced as a fusion to a reporter that produces a "signal" (growth or enzymatic activity) only when the bait-reporter fusion binds to the prey and the resulting complex is exported into the periplasm via the Tat pathway. As a second biotechnological application of the Tat pathway, we developed a phage display system that allows the protein of interest to fold within the cytoplasm prior export and display onto phage particles. This is in contrast to the conventional phage display system, in which displayed protein folds in the periplasm. We took advantage of this new system to screen a library of 2 x 10⁶ of fluorescent GFP variants containing a hexameric peptide insertion for ligand binding. Despite the diversity of the hexamer, we were not able to isolate single GFP variants that would bind with specificity to various ligands. This highlights the difficulty in engineering GFP variants that can bind to other proteins while retaining the ability to fluoresce. The second aspect of this research was to examine mechanistic aspects of the Tat pathway. TatB and TatC are responsible for the recognition of Tat signal peptides. Here, we established the importance of TatC as the crucial component of the Tat pathway for the interaction with the hallmark twin-arginine motif within Tat signal peptide. Substitution of the RR dipeptide with a KK sequence completely abolishes export. In a genetic screen using a ssTorA(KK)-GFP-SsrA as a reporter. We identified several amino acid substitutions within TatC that allowed the alteration of the substrate specificity of the pathway as indicated by the impairment of indigenous Tat substrates. Finally, we analyzed the conformational dynamics of TatA using GFP fusions and by incorporation of the chemically reactive, non-canonical amino acid azidohomoalanine.

No description available.

Rapid expansion of the aquaculture industry in recent decades has resulted in infectious diseases emerging as a major constraint to fish production, causing large economical losses worldwide. Therefore, prevention practices are indispensable for maintaining the industry's profitability and sustainability. Vaccination is a proven effective strategy for disease control in aquaculture; however, improvements in vaccine efficacy are still needed. Because amino acid supplementation not only enhances fish growth but also immune responses, a series of experiments were conducted to test the hypothesis that dietary supplementation of arginine and glutamine, two amino acids with immunomodulatory roles, may promote growth and increase the efficacy of vaccination against Edwardsiella ictaluri in channel catfish. An initial experiment demonstrated that dietary arginine supplementation at 2 and 4% of diet enhanced growth and feed efficiency of channel catfish. Dietary arginine deficiency diminished plasma levels of arginine, citrulline, ornithine, glutamine and glutamate, and impaired innate performance of macrophages and neutrophils. In a separate experiment, dietary glutamine supplementation failed to enhance growth responses; however, supplementation at 2% of diet had strong positive effects on intestinal histology and enterocyte migration rate. In addition, serine, asparagine, glycine and threonine were increased in plasma of fish fed the diet with glutamine at 2%. A third experiment revealed that activated macrophages utilized large quantities of glutamine in media and to a lesser extent arginine. These two amino acids also were the most utilized by proliferating lymphocytes. Supplementing media with these amino acids positively modulated phagocytosis and bactericidal capacity of macrophages, as well as increased the proliferation rate of lymphocytes. A final experiment indicated that dietary supplementation of arginine (4%) and glutamine (2%) optimized the nutritional and immunological status of channel catfish, and enhanced responses to E. ictaluri vaccination. At the same time, this supplementation ameliorated some short-term adverse effects of vaccination on growth. Higher specific antibody titers, better lymphocyte responsiveness and survival to the bacterium were seen in vaccinated fish fed arginine- and glutamine-supplemented diets. These results support an expanded role of dietary arginine and glutamine manipulation as a tool to improve growth and vaccine efficacy of channel catfish.

This study was conducted to test the hypothesis that dietary supplementation with L-arginine during early pregnancy will ameliorate embryonic loss in pigs. Gilts were bred at the second estrus, and housed individually in pens and fed twice daily 1 kg of a corn- and soybean meal-based diet supplemented with 0.0%, 0.4%, or 0.8% L-arginine (w/w) between d 0 and 25 of gestation (Experiment 1) or between d 14 and 25 of gestation (Experiments 2 and 3). At d 25 (Experiment 1 and 2) or d 60 (Experiment 3) of gestation, gilts were hysterectomized to obtain uteri and conceptuses. Total RNA and protein were extracted from the frozen tissues. Quantitative RT-PCR, western blotting, and microarray analyses were performed to determine the changes of gene expression at mRNA and protein levels. Dietary supplementation with 0.8% L-arginine between d 0 and 25 of gestation decreased uterine weight, total number of fetuses, number of corpora lutea (CL), total fetal weight, total volume of allantoic and amniotic fluids, concentrations of progesterone in maternal plasma and allantoic fluid, compared to the control group. However, dietary supplementation with 0.4% or 0.8% L-arginine between d 14 and 25 of gestation increased total volume of amniotic fluid, total amounts of arginine in allantoic and amniotic fluids, total amounts of fructose and most amino acids in amniotic fluid, placental growth, and the number of viable fetuses per litter by 2. Dietary supplementation with 0.4% or 0.8% L-arginine between d 14 and 25 of gestation increased the total number of fetuses and number of live fetuses, rate of embryonic survival, and volumes of allantoic and amniotic fluids in gilts with 15 to 18 CL on d 60 of gestation compared with the control group. The abundance of placental protein and expression of mRNA related to the genes for arginine transport and metabolism, including cationic amino acid transporter 1, endothelial nitric oxide synthase (NOS3), phosphorylated-NOS3, ornithine decarboxylase, and guanosine triphosphate cyclohydrolase-I was increased by dietary supplementation with 0.8% L-arginine between d 0 and 25 of gestation. The abundance of total and phosphorylated mechanistic target of rapamycin was also enhanced by dietary 0.8% L-arginine supplementation between d 0 and 25 of gestation. Microarray analysis revealed that supplementation with 0.8% arginine between d 14 and 25 of gestation affected placental expression of 575 genes. Findings from the current study not only advance basic knowledge of mammalian reproductive biology, but also have important implications for developing practical means to enhance fertility in female pigs.