Browsing by Subject "molecular biology"
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Item Characterization of marine exopolymeric substance (EPS) responsible for binding of thorium (IV) isotopes(Texas A&M University, 2005-08-29) Alvarado Quiroz, Nicolas GabrielThe functional group composition of acid polysaccharides was determined after isolation using cross-flow ultrafiltration, radiolabeling with 234Th(IV) and other isotopes, and separation using isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). Phosphate and sulphate concentrations were determined from cultured bacterial and phytoplankton colloid, particulate and colloidal samples collected from the Gulf of M??xico (GOM). Characterization of the 234Th(IV)-binding biomolecule was performed using ion chromatography (IC), and gas chromatography-mass spectrometry (GC-MS). Radiotracer experiments and culture experiments were conducted in determining the binding environment of the 234Th(IV)-binding ligand (i.e., sorption onto suspended particles), as well as the origin of the ligand in seawater systems. In all samples, 234Th(IV) isoelectric focusing profiles indicated that 49% to 65% of the 234Th(IV) labeled EPS from Roseobacter gallaeciensis, Sagittula stellata, Emiliania huxleyi, Synechococcus elongatus and GOM Station 4-72m was found at a pHIEF of 2 in the IEF spectrum. The carboxylic acid group appeared at the same pHIEF as 234Th(IV) for EPS from Roseobacter gallaeciensis, Emiliania huxleyi, Synechococcus elongatus and GOM colloidal organic matter sample. The phosphate group appeared at the same pHIEF as 234Th(IV) for EPS from Roseobacter gallaeciensis, and Synechococcus elongatus sample. The sulphate group was found at the same pHIEF as 234Th(IV) for EPS from S. elongatus and GOM colloidal organic matter sample. The total polysaccharide content was only 14% and 8%, uronic acids were approximately 5.4% and 87.1%, and total protein content was 2.6% and 6.2% of total carbon content of Sagittula stellata and Synechococcus elongatus, respectively. Monosaccharides identified in both Sagittula stellata and Synechococcus elongatus were galactose, glucose, and xylose in common. In addition, Sagittula stellata contained mannose and Synechococcus elongatus had galactoglucuronic acid. Thus, depending on the species, the size, structural composition, and functional groups of the 234Th(IV)-binding, acidic polysaccharides will vary. From these observations, it is concluded that the steric environment and not necessarily the exact functional group might actually be responsible for thorium-234 complexation to macromolecular organic matter. This research helped to improve our understanding of the observed variability in POC/234Th ratios in the ocean and provided insights into factors that regulate organic carbon export fluxes.Item Circuits attenuating seizures under well-fed and food-deprived conditions in C. elegans male sex muscles(2010-07-14) Leboeuf, Brigitte L.The circuits that allow organisms to control behavioral timing need to be tightly regulated to ensure execution of appropriate environmental responses. Disrupting such regulation results in individuals unable to perform tasks necessary for survival and propagation. Identifying the molecular components regulating behaviors will enable compensation where behavioral impediments to survival exist. To identify circuits of behavioral regulation, I studied male mating behavior in the nematode Caenorhabditis elegans. Specifically, I focused on the step wherein the male inserts his copulatory spicules into the hermaphrodite vulva, as vulva penetration is required for successful sperm transfer. This step must be tightly regulated; if the spicules protract too soon or not at all, vulva penetration and thus successful mating will not occur. In this dissertation, I elucidate the circuits regulating sex-muscle excitability under standard conditions and describe how these pathways are augmented to further reduce excitability under food deprivation conditions. I employ a variety of assays to identify and analyze these circuits, including genetic manipulation, biochemical techniques, and behavioral assays. Under standard conditions the calcium/calmodulin-dependent protein kinase II (CaMKII) encoded by unc-43 is required to inhibit C. elegans male sex-muscle seizures; under conditions where food is scarce, I propose that CaMKII is further up-regulated to activate the EAG K+ channel EGL-2 through a direct interaction. The CaMKII/EGL-2 interaction functions to attenuate calcium influx from L-type voltage-sensitive calcium channels (L-VGCCs), while CaMKII also downregulates calcium influx from ryanodine receptors. Additionally, another K+ channel, the voltage- and calcium-sensitive big current channel SLO-1, attenuates sex-muscle excitability by inhibiting L-VGCCs under food deprivation conditions. In conclusion, CaMKII and EGL-2?s paralog, UNC-103/ERG-like K+ channel, are required when food is plentiful to prevent premature sex-muscle contractions, while food deprivation reduces cell excitability and thereby inhibits inappropriate seizures through CaMKII, EGL-2, and SLO-1.Item Comparison of the Prevalence and Genotypic Characteristics of Clostridium difficile in a Closed and Integrated Human and Swine Population in Texas(2011-10-21) Norman, Keri NoelleClostridium difficile has been recognized as one of the leading causes of nosocomial diarrhea and pseudomembranous colitis in human hospitals and nursing homes since the 1970s; however, recent occurrences of community-acquired cases have led researchers to search for additional sources of these infections. Some of the possible sources being investigated include food animals and retail meat. The objective of this study was to compare the prevalence and genotypic characteristics of C. difficile isolated from a closed population in Texas consisting of both humans and swine. Implicit in this objective, we seek to investigate the possible food safety and occupational risks associated with swine and C. difficile. Isolation of C. difficile was performed utilizing an enrichment technique and restrictive media. Polymerase chain reaction (PCR) was used to test for the presence of the toxin A and B genes, the tcdC gene deletion, and the binary toxin gene. Genotypic characteristics were compared using PCR toxinotyping and pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility was tested using commercially available tests (ETest?) for 11 different antibiotics. Statistical comparisons (both parametric and non-parametric, and appropriate to the data) were performed both between and among host species. We tested 2,292 aggregated human wastewater samples and 2,936 swine fecal samples from 2004 to 2006 and found 271 (11.8 percent) and 252 (8.6 percent) to be positive for C. difficile, respectively. The prevalence of C. difficile among swine production groups differed significantly (p<0.05); however, prevalence in the human occupational group cohorts (swine workers and non-workers) did not differ (p=0.81). The majority of the human and swine isolates were a PFGE NAP7 (a variant pattern with 90.5 percent similarity) toxinotype V strain. Antimicrobial resistance levels and multi-resistance patterns were generally similar between host species; however, there was decreased susceptibility (p<0.05) to ampicillin, clindamycin, and imipenem observed in swine isolates, whereas there was decreased susceptibility (p<0.05) to ciprofloxacin in the human isolates. The similarity in C. difficile prevalence between swine workers and non-workers suggests a low occupational hazard of working with swine as it relates to C. difficile source. We also found that there is a decreased prevalence of C. difficile in late production groups in swine suggesting a lowered risk of food-borne exposure. However, the majority of the isolates derived from the human wastewater and swine appeared to be of very similar strain types, suggesting that a common environmental point source predominates for both hosts.