A comparison of the structure of isolated and in situ chromatin fibers was performed using starfish (Patiria miniata) sperm nuclei. The simple protein content consisting of five major histones was found. A DNA repeat length of 222bp was calculated. Compact chromatin fibers solubilized from detergent isolated nuclei showed diameters of 38 to 44nm by electron microscopy. Chromatin fibers observed in whole cells and in mildly treated nuclear preparations revealed fiber diameters of approximately 24nm when embedded in epoxy resin. Investigation of the chromatin isolation process uncovered a mechanism where fiber integrity was lost during nuclear isolation. Following nuclease digestion and chromatin release, fiber-like structures reformed in solution. The relationship of the isolated "fibers" to the native structures is not known. Nuclear permeability has been found to be a factor in both release of cleaved chromatin from nuclei and inward diffusion of nuclease. Permeability to various sized dextrans was consistent with a mechanism where cut chromatin exits nuclei as nucleosomal chains and aggregates in solution to form the isolated chromatin state. Attempts to isolate detergent free nuclei revealed that completely isolated nuclei were susceptible to the loss of chromatin fiber integrity suggesting that the medium was incapable of maintaining fiber structure. Other buffer compositions did not improve stability of in situ fibers underscoring a general lack of understanding of the nuclear environment.

Lymphatic filariasis is a human parasitic disease which causes great physical suffering, while also having a substantial economic impact on the peoples of the tropics and sub-tropics. In order to collect accurate epidemiological data which are crucial for designing effective control programs, reliable diagnostic and taxonomic information is needed. The studies described here were designed to obtain DNA sequence data from specific coding and non-coding loci of the parasite genome, which could augment existing morphological, biochemical, and ecological data, to test hypotheses concerning diagnostic and taxonomic classification. Highly repeated DNA elements, members of the Hha I repeat family, were cloned, sequenced and the data employed in a phylogenetic analysis of species, sub-species and distinct geographic isolates of human filarial parasites from the genus Brugia. Comparative analysis reveals that although the 322 base pair (bp) repeat sequences between Brugia pahangi and Brugia malayi are nearly 90% identical overall, there is a small 70 bp region which contains enough divergence to clearly distinguish between these two major species. Nucleotide differences in this and other regions were exploited to draw distinctions between repeats cloned from B. timori, B. patei and various geographic isolates of B. malayi which differ in biological characteristics such as host range, vector preference and periodicity. In addition to the Hha I repeats, the gene encoding a prominent, stage-specific surface antigen from the animal parasite, Brugia pahangi was also cloned and sequenced. Homologous sequences were obtained from the related human pathogen Brugia malayi and a comparative analysis initiated. The results show that the protein coding, flanking and intervening sequences are highly conserved between the two species. In addition to its utility as a taxonomic and phylogenetic tool, the highly conserved nature of this protein sequence makes it a potential candidate for recombinant vaccine development.

The object of my dissertation research was to evaluate the effects of mitogens on ionic currents in A431 cells. the tools used were fluorescence imaging of a membrane potential sensitive dye, di-8-ANEPPS, and whole cell patch-clamp electrophysiology. Imaging studies revealed that application of 100 $\mu$M ATP caused $>$95% of the cells to hyperpolarize on the order of 30 mV. This hyperpolarization lasted for the duration of agonist presence, and was independent of extracellular calcium. Whole-cell electrophysiology was used to determine the ionic nature of the hyperpolarization. At a holding potential of $-$60 mV, ATP caused an inward current after a 4 second latency. This latency indicated a second messenger accumulation. Removal of extracellular calcium served to reduce the average magnitude of the inward current. By making various substitutions of both the intracellular and extracellular solutions, it was determined that chloride efflux made up the majority of the ATP-evoked inward current. At more depolarized holding potentials, ATP caused a transient inward current followed by a prolonged outward current. The outward current was determined to be carried by potassium, and was sensitive to charybdotoxin, which is known to block Ca$\sp{2+}$ -activated K$\sp{+}$ channels. The source of calcium was determined to be intracellular calcium pools. Inclusion of 5 mM BAPTA in the recording electrode abolished ATP activated currents. The role of G-proteins was demonstrated by the ability of GDP$\beta$S in the dialysis electrode to abolish ATP mediated currents. A dose-response study of the ATP effect revealed that at lower concentrations of ATP, fewer cells responded and that the latency prior to response was increased. The magnitude of the response, however, was independent of ATP concentration. My hypothesis for this "all or none" regenerative phenomenon is that a second messenger cascade causes the release of calcium internal stores. This calcium acts in a positive feedback loop to increase permeability of the plasma membrane to external calcium. The sustained rise in cytosolic calcium allows for the activation of multiple calcium dependent ion channels, including Ca$\sp{2+}$-activated K$\sp{+}$ channels, which cause an overall membrane hyperpolarization in A431 cells.

Background Fetal Alcohol Spectrum Disorders (FASDs) refer to the range of developmental abnormalities that occur in a fetus following prenatal alcohol exposure (PAE). It is unclear how PAE affects the development of the embryonic heart. Recent data indicates that the Wnt-signaling pathway may be implicated in congenital heart defects caused by PAE. In previous RNA-Sequencing (RNA-Seq) studies, Wnt7a, Wnt7b, and Wnt11 showed significantly changed expression in embryonic mouse hearts after a single maternal binge ethanol dose at embryonic day 9.5 (E9.5). Hypothesis We hypothesize that there will be significant change in expression of Wnt7a, Wnt7b, and Wnt11 following maternal ethanol binge at E9.5. We also hypothesize a significant decrease in expression of Wnt7a in C2C12 cells following ethanol exposure. Experimental Methods In-vivo, timed pregnant mice were given a single oral gavage of 0.9% saline or 2.5g/kg ethanol at E9.5. RNA from the embryonic heart was quantified and analyzed after 24 hours. Invitro, C2C12 murine myoblasts were cultured and incubated with ethanol or water for 2-24 hours. Cells at 4 different differentiation stages were also exposed to ethanol or water for 24 hours before expression quantification. Results Out of our 3 genes, only Wnt7a showed sustained depressed expression after 24 hours. We also concluded there is no significant impact of alcohol on Wnt7a expression in DM6 C2C12 cells exposed to different doses of ethanol from 2 to 24 hours following exposure. There was a significant change between Wnt7a expression in DM0 controls vs. UD, DM3, and DM6 controls. Conclusion These results suggest that the stage of differentiation plays a large role in Wnt7a activity and its sensitivity to ethanol. This study creates a greater understanding of the Wnt-signaling pathway's response to alcohol in-vivo and Wnt7a's vulnerability to alcohol at various stages of muscle differentiation.

No description available.

A panel of monoclonal antibodies (MAbs) was generated against bovine oviduct cell surface antigens. Male mice were immunized with intact oviduct cells which had been collected by collagenase dissociation. Mice were boosted twice at three week intervals and then fused with Sp2/0-Ag14 hybridoma cells according to conventional procedures. The resulting antibody-secreting hybrid clones were identified by an indirect enzyme-linked immunoadsorbant assay (ELISA) of cell supernatants on intact oviduct cells. Sixty-seven clones were found to be positive. Of these, thirty-three stable clones were established. Indirect immunofluorescence on live cells was used to visualize the staining patterns of each antibody. This panel of MAbs was tested by ELISA for cross-reactivity to cells from other bovine tissues, namely ovary, uterus, trachea, spleen, liver and lymph node. The majority of these MAbs (20) were specific for antigens on the oviduct cell surface. One MAb (#27) cross-reacted only with uterine cells. Eight MAbs cross-reacted with tracheal cells as well as uterine cells. The remaining four MAbs cross-reacted with a number of different cell types. In addition, this panel cross-reacted with sheep oviduct cells (14/33) but not those of rabbit. Also, eleven MAbs cross-reacted with cow oviduct fluid (ODF) while fourteen cross-reacted with sheep ODF. To determine whether any of the MAbs recognized epitopes which were cycle stage-specific, ODF from both the luteal and estrus phases of the reproductive cycle was examined. ELISA data of ODF from cows did not indicate any obvious cycle stage variations. In contrast, ELISA data of sheep ODF did indicate some variations in antigen appearance between the estrus and luteal phases. Antigens that reacted with the MAbs were characterized by immunoblotting analysis of detergent-solubilized membrane antigens after SDS-PAGE fractionation under reducing conditions. Soluble antigens in cow and sheep ODF were also characterized by similar methods. Several MAbs detected broad diffuse bands which suggests that these antigens may be carbohydrate in nature. In an in vitro cell adhesion assay, MAb #27 prevented apical sperm head attachment to oviduct cells at concentrations as low as 5 ug/ml of purified antibody. None of the other MAbs appeared to have an effect on sperm binding. This effect is not due to nonspecific toxicity since inhibition of attachment is reversible. The oviduct can now be examined in greater detail using these MAbs as probes for the molecular components of the system. Such studies should bring to light details of cell surface composition, which will provide further insights into possible oviduct cell surface functions such as sperm storage.

In Chapter 1 we characterized the frequency, amplitude and duration of (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises in fertilized rabbit eggs loaded with fura-2 dextran. Their amplitude decreased and duration increased as fertilization progressed. Injection of 1,4,5 inositol trisphosphate (InsP3; IICR) or addition of thimerosal elicited (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises which were blocked by heparin, an InsP3 receptor antagonist. Ryanodine did not evoke Ca$\sp{2+}$ release. These results indicate that IICR is likely stimulated during fertilization. Fertilization (Ca$\sp{2+}\rbrack\sb{\rm i}$ changes were examined in in vitro matured bovine eggs (Chapter 2). Fertilized eggs exhibited different intervals between (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises which ranged from 15 min to more than 60 min. Unfertilized eggs were responsive to InsP3 injection and thimerosal exposure, although the frequency of the (Ca$\sp{2+}\rbrack\sb{\rm i}$ responses was shorter than the periodicity observed during fertilization. The mechanisms that generate (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations were examined in fertilized rabbit eggs (Chapter 3). Heparin blocked or delayed (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations. Oscillating eggs exhibited Ca$\sp{2+}$ release in response to CaCl2 injection. In unfertilized eggs, injection of GTP (S) induced (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations and enhanced the response to CaCl2 injection. Injection of InsP3 or CaCl2 elicited full Ca$\sp{2+}$ responses that reset the periodicity of ensuing oscillations. Thus, IICR participates in the generation of (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations and the unloading of the stores does not explain the interval between (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises. The signaling pathways possibly stimulated by the sperm during fertilization were investigated in unfertilized bovine eggs (Chapter 4). Injection of GTP (S) or InsP3 evoked (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations. Preinjection of heparin blocked sperm-mediated egg activation. Thimerosal elicited (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations which were not inhibited by heparin or ryanodine. The data suggest that bovine eggs possess a GTP-linked phosphoinositide pathway which appears to be stimulated by the sperm during fertilization. In Chapter 5 the amplitude and duration of (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises elicited by electrical pulses of different strengths and duration administered in variable extracellular Ca$\sp{2+}$ concentrations was reported. As these parameters increased, so did the peak (Ca$\sp{2+}\rbrack\sb{\rm i}$ elicited by the pulse. Young and aged eggs exhibited similar (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises when stimulated with identical pulses. However, their activation rates were different. Thus, aged eggs are more sensitive to a given (Ca$\sp{2+}\rbrack\sb{\rm i}$ rise.

No description available.

This study investigates the psychometric issues and viability of cellphone-based-testing, a novel test administration modality whereby test-takers use a cellphone to respond to items on a web-based assessment. The study explored mode-dependent differences in scores from a web-based version of the Self-Monitoring Scale (SMS) administered across two modalities: desktop computer and cellphone. The selection of the SMS was based on several pre-established criteria. The instrument was simple and brief. Its text-based items included true/false response categories. Its rights of use fell under public domain and it had been previously validated for online administration. The study includes a comprehensive overview of recent literature related to the topic of psychometric equivalency and incorporates numerous methodological approaches to determine test score equivalence, including: comparisons of central tendency, dispersions, and rank order; the Kolmogorov-Smirnov test of equal score distributions; the Pitman procedure for detecting differences in reliability coefficients; a confirmatory factor analysis of the equality of factor structures using LISREL; and an analysis of differential item functioning based on item response theory using BILOG-MG. The study employed a counterbalanced repeated measures design whereby 234 participants took an 18-item web-based version of the SMS using a desktop computer and/or a cellphone. The psychometric equivalency of scores from the two modes of administration was analyzed. All statistical comparisons provided overwhelming support for one general conclusion: There were no mode-dependent differences in scores on the web-based version of the SMS when administered by desktop computer versus cellphone. The study also explored participants’ attitudes toward using cellphones as a test-taking tool. The participants correctly anticipated that their scores would not be affected by using a cellphone, but they categorically rated the cellphone as less enjoyable, more difficult, and more cumbersome than a desktop computer. However, one cannot ignore the tendency of our modern society for being obsessed with information on demand. As cellphone technology continues to improve and the text-messaging generation begins to influence the field of educational and psychological measurement, cellphone-based-testing will likely become an accepted standard for both academic and clinical practice.

No description available.