Species introductions have been linked to the decline of native taxa, and in many cases have resulted in the elimination of native species in both terrestrial and aquatic systems throughout the United States. In aquatic systems, a particular threat is the introduction of large piscivorous fish that may alter the native fish community structure. For example, introductions of large ictalurids such as blue catfish, (lctalurus furcatus), and flathead catfish, (Pylodictus olivaris), into coastal Virginia rivers, including the James River twenty years ago have resulted in the establishment of these large, predatory fishes. This study described the trophic ecology of four ictalurid catfishes in the tidal James River, Virginia including the native white catfish (Ameiurus catus), the possibly introduced channel catfish (Ictalurus punctatus), and the recently introduced blue catfish and flathead catfish. The objectives of this study were to determine the trophic ecology of these four catfishes in a coastal Virginia river, and to assess the potential predatory effects of large, recently introduced piscivorous ictalurids on the native fish assemblage, and especially anadromous clupeid fishes. A stratified sample of 4, 164 catfish was taken throughout the tidal freshwater reach of the James River during the summer and fall, 1996 and spring, 1997. Stomach content analysis revealed that blue catfish and flathead catfish are highly piscivorous, feeding on several families of native fishes. Flathead catfish consumed over 90% (frequency of occurrence) fish prey in most predator size classes and began consuming more fish prey at smaller sizes than blue catfish. Blue catfish shifted to a mostly piscivorous diet at predator lengths > 500 mm. Both blue catfish and flathead catfish consumed adult anadromous clupeids. The greatest numeric proportion (0.41) of anadromous clupeids consumed were juvenile Alosa spp. (<100 mm) taken by small blue catfish (<500 mm) during the fall sampling season. Piscivory was much less extensive in channel catfish and white catfish (less than 10% frequency of occurrence for all predator size classes). There is evidence of negative consequences to native fishes associated with the introductions of blue catfish and flathead catfish into Atlantic slope rivers. These consequences may conflict with current restoration efforts for native fishes such as the anadromous clupeids in these rivers.

Mast cells have long been appreciated as the primary effector cells in allergy and asthma, and recently have been implicated in other inflammatory diseases. Using high density oligonucleotide probe arrays, we assessed genome-wide transcriptional profiles after FcεRI aggregation for 90 minutes, 5 hours and 24 hours. We describe novel gene regulation in response to FcεRI signaling, including altered expression of CD44, Pari, osteopontin, Nur77, E4BP4, and NDRG 1. In addition, the gene encoding FcεRI β was downregulated 5 hours after mast cell activation according to both microarray analysis and RPA, and western blotting confirmed the downregulation of the beta subunit protein. Moreover, this downregulation of beta mRNA correlated with the decreased FcεRI surface expression after mast cell activation. Very little is known about the transcriptional regulation of the beta subunit of FcεRI. These transcriptome profiling experiments are revealing novel and clinically relevant insight into how FcεRI signaling may be controlled. Continuing with our focus on how mast cell activation is regulated, we examined the effects of Th2 cytokines on expression of important surface receptors. Murine mast cells co-express the activation receptor FcγRIII and the inhibitory receptor FcγRIIb and can be activated by lgG immune complexes. Using mouse bone marrow-derived mast cells, we report that IL-4 selectively increases FcγRIII expression without altering FcγRIIb. This enhanced expression could be induced by Stat6 activation alone, and appeared to be mediated in part by increased FcγRIIIα protein synthesis without significant changes in transcription. The increase in FcγRIII expression was functionally significant, as it was matched by enhanced FcγR-mediated degranulation and cytokine production. Selective regulation of mast cell FcγR by IL-4 could alter inflammatory lgG responses and subsequently disease severity and progression. Collectively, our studies have used genome-wide screening and reductionist studies to demonstrate mechanisms by which mast cell function is regulated.

The results of this chapter lead to a modification of the standard stopping rule which breathes new life into the method of steepest ascent, but which is unfortunately not sufficient to effect a total resuscitation. As expected, there is no uniformly optimum stopping rule, nor even a uniformly best test statistic; both the optimum statistic and the optimum test level depend on the model parameters y1 and y2. One can, however, adopt a compromise rule which works reasonably well for all parameter combinations by testing at the 25% level with the statistic zn = 1/2(yn -yn-2). In many cases the loss incurred by the compromise rule is less than half that incurred by the existing rule; in the few cases where the compromise rule is less efficient, the difference in losses is small. Nevertheless, the compromise rule is nearly always inferior to the parameter-dependent optimum rule, and the optimum itself is not always desirable.

The presence of an adenyl cyclase system within the haemocyte of Periplaneta americana has been confirmed by previous investigations, with corresponding experiments using dibutyryl cyclic AMP to show enhancement of 14c-tyrosine uptake into the cuticle linked to stimulation of the sclerotization process. In the present studies, American cockroaches held at depressed temperatures were treated with bursicon, revealing an increase in haemocyte cyclic AMP concentrations. The levels declined with progression of time, probably resulting from enzymatic hydrolysis. When animals which had been held at room temperature were treated with bursicon, the levels of haemocyte cyclic AMP were lowered, with a declination to sub-control levels with time progression. These results were' duplicated using dihydroxyphenylethylamine (dopamine) and isobutylmethylxanthine in the treatment medium. Analysis of haemocyte protein binding by cyclic AMP revealed large amounts bound by proteins greater than sixty.thousand daltons. Any remaining cyclic AMP was either unbound or converted to one of several metabolites. Inhibition of phosphodiesterase by removal of a sulfhydryl protecting agent and addition of EDTA caused a net decrease in protein bound label and an increase in intact cyclic AMP recovered from the reaction. Bursicon added to the reaction caused a slight depression of the binding reaction, presumably due to competition by native cyclic AMP. The addition of dibutyryl cyclic AMP to haemocyte preparations resulted in net increases in uptake of tyrosine and leucine into the cells as a function of the dibutyryl cyclic AMP concentration. The levels of uptake were enhanced corresponding to each concentration level of dibutyryl cyclic AMP, but did not maintain their rapid uptake rates over an extended time course. Results presented in this work indicated that the mode of action of bursicon is via adenyl cyclase activation and resultant cyclic AMP production. Rapid binding reactions to enzymes such as phosphodiesterase and protein kinase appear to regulate the actions of cyclic AMP in the haemocyte.

Homologous recombination has been studied in many plant and animal systems. In maize, most recombination occurs intragenically. The current study assessed the frequency and location of meiotic recombination at the maize red color (r1) gene. Three independent mutant (colorless seed) alleles derived from the colored seed allele, R-sc:124, were made heterozygous with the colored plant allele, r-r:n142. Each mutant resulted from the insertion of a Dissociation (Ds) transposable element into the 3' end of R-sc:124. In the presence of Activator (Ac), each Ds insertion allele (r-sc:mutables) produced spotted seeds and germinal reversion to fully colored seeds due to the vi excision of Ds. In the absence of Ac, each insertion allele stable and produces colorless or very faintly pigmented seeds. The r-sc:mutable/r-r:n142 heterozygotes were pollinated with R-g:8pale, an r1 allele that produces pale brown seeds, in the absence of Ac, to recover revertant progeny likely resulting from recombination rather than Ds excision. Revertant progeny were self pollinated to verify paternity. Molecular analysis using polymerase chain reaction, Southern blot and DNA sequence analyses showed that 92 out of 94 of the revertant alleles arose via a crossover event between the r-sc:mutable and r-r:n142 chromosomes. The remaining two revertant alleles likely arose via gene conversion, double cross over or cryptic Ac activity. The ratio of genetic to physical distance (1/ρ) calculated for the 3' end of r1 is approximately 0.07 cM/kb, which is comparable to 1/ρ values calculated for a1, b1, bz1 and wx1 loci. Given the average value for 1/ρ throughout the genome is 0.00021 cM/kb, this analysis supports the hypothesis that recombination in maize primarily occurs intragenically and that r1 serves as a recombination hotspot in the maize genome. Molecular analysis also revealed that the majority of exchanges occurred at the 3' end of r1 as has been previously observed. The pattern of recombination observed at r1 is different from those observed at other maize loci. The recovery of substantially more cross over events (92/94) relative to non-crossover events (2/94) or the nature of the recombination event is consistent with previous observations at a1, b1, and bz1. The frequency of potential gene conversion is estimated at 3.97 x 1 o-s and the gene conversion tract length is maximally 2.5 kb. The possibility of the influence of insertional mutations, amount and structure of DNA sequence homology, cis- and trans- factors and preference for cross over versus non-crossover events could explain the observed pattern at r1.

The incorporation of phosphoproteins and phenol-bound proteins into the cuticle of Periplaneta americana was analyzed. Radioactive monosodium phosphate was injected into newly ecdysed cockroaches. Incorporation into haemolymph proteins as well as tanned cuticle was observed. Phosphoproteins were observed to be combined with the cuticle to a greater extent than monosodium phosphate. This suggests that phosphate is being incorporated into the cuticle more readily than monosodium phosphate. The relevance of these observations is discussed in light of phosphate being a possible crosslinking agent between chitin and cuticle protein. Binding of dopamine metabolites to cuticle proteins was investigated. Specific metabolites studied were N-acetyldopamine-3-0-sulphate and dopamine-3-0-sulphate. In an in vitro experiment, these metabolites were readily bound to cuticular, structural proteins while they remained unchanged when incubated with soluble cuticular proteins. The presence of sulphatase activity was also evidenced in the structural proteins but not in the soluble proteins. The binding of N-acetyldopamine to lysyl-lysine was accomplished in vitro. Binding was observed with and without newly ecdysed cuticle homogenate. The products of the binding were separated wia P-2 polyacrylamide gel chromatography, paper chromatography and high voltage electrophoresis. Absorbancy at 280 nm, as well as the reaction with ninhydrin were monitored for each fraction. A metabolite (or metabolites) was separated which was both UV positive and ninhydrin positive. This metabolite was analyzed by NMR and no aromatic protons were observed thus indicating complete substitution on the ring. These data support the existing theory that suggests protein binding to the sclerotization agent occurs on the ring.

<p> Low back pain (LBP) is the leading cause of disability worldwide, and intervertebral disc (IVD) degeneration is the most common diagnosis for non-specific LBP. A need exists to develop and understand an animal model of LBP with similarities to the human condition that can be used to understand underlying pathophysiology and as a screening tool in therapeutic studies. Although a wealth of previous research exists regarding the causes of IVD degeneration, no studies to date have directly related molecular measures to a functional pain behavior in an induced lumbar IVD injury model. Therefore, the purpose of this project was to determine the predictive causes of pain in a lumbar IVD puncture rat model. Pain behavior, IVD degeneration, IVD height, intradiscal pro-inflammatory cytokine expression, and dorsal root ganglia (DRG) Substance P (SubP) expression were measured and their relationships analyzed with single and multi-variate regressions. Using a multi-variate step-wise linear regression model, IVD height was the only predictive factor for pain behavior in this rat model. This suggests the induced annular injury utilized in this model produces pain via DRG impingement by foraminal stenosis or by increased motion segment instability. TNF-&alpha; injection also increased pain, suggesting an additional inflammatory pain component in this model. These neuropathic and nociceptive pain contributions of the model share many characteristics with the human IVD degeneration condition and therefore this model has much potential to identify mechanisms of IVD degeneration-related pain and therapeutic screening. </p>

<p>It is increasingly evident that evolutionary processes play a role in how ecological communities are assembled. However the extend to which evolution influences how plants respond to spatial and environmental gradients and interact with each other is less clear. In this dissertation I leverage evolutionary tools and thinking to understand how space and environment affect community composition and patterns of gene flow in a unique system of Atlantic rainforest and restinga (sandy coastal plains) habitats in Southeastern Brazil.</p><p>In chapter one I investigate how space and environment affect the population genetic structure and gene flow of Aechmea nudicaulis, a bromeliad species that co-occurs in forest and restinga habitats. I genotyped seven microsatellite loci and sequenced one chloroplast DNA region for individuals collected in 7 pairs of forest / restinga sites. Bayesian genetic clustering analyses show that populations of A. nudicaulis are geographically structured in northern and southern populations, a pattern consistent with broader scale phylogeographic dynamics of the Atlantic rainforest. On the other hand, explicit migration models based on the coalescent estimate that inter-habitat gene flow is less common than gene flow between populations in the same habitat type, despite their geographic discontinuity. I conclude that there is evidence for repeated colonization of the restingas from forest populations even though the steep environmental gradient between habitats is a stronger barrier to gene flow than geographic distance.</p><p>In chapter two I use data on 2800 individual plants finely mapped in a restinga plot and on first-year survival of 500 seedlings to understand the roles of phylogeny, functional traits and abiotic conditions in the spatial structuring of that community. I demonstrate that phylogeny is a poor predictor of functional traits in and that convergence in these traits is pervasive. In general, the community is not phylogenetically structured, with at best 14% of the plots deviating significantly from the null model. The functional traits SLA, leaf dry matter content (LDMC), and maximum height also showed no clear pattern of spatial structuring. On the other hand, leaf area is strongly overdispersed across all spatial scales. Although leaf area overdispersion would be generally taken as evidence of competition, I argue that interpretation is probably misleading. Finally, I show that seedling survival is dramatically increased when they grow shaded by an adult individual, suggesting that seedlings are being facilitated. Phylogenetic distance to their adult neighbor has no influence on rates of survival though. Taken together, these results indicate that phylogeny has very limited influence on the fine scale assembly of restinga communities.</p> / Dissertation

A cytochemical study of the nucleus of Sarcoma 180 cancer cells from Crocker albino mice, grown ^n vitro, was made. The cancerous cells were treated with various reagents in an attempt to elucidate the nature of the nucleus. The Feulgen nuclear reaction revealed the presence of large quantities of DKA (deoxyribonucleic acid) within the nucleus of these malignant cells, which was not only evenly distributed throughout the structure, but also concentrated near one end of the nuclear membrane in some cells. Cells were treated with 0.2% and 1.0% DNAase (adjusted to a pH of 6.0) and incubated at 37° C. for a one hour period. Treatment with the enzyme caused rapid breakdown of nuclear material leaving an apparently empty structure except for the presence of very conspicuous nucleoli. The enzyme revealed an apparent thickened nuclear membrane. The thickening was probably a result of the penetration of the membrane by the enzyme, or due to a breakdown of the chromatin material that has long been established as being in association with the nuclear membrane. Some of the cultures were treated with varying concentrations of a soap-like detergent, digitonin. When cells were treated with strong concentrations of the substance (0.5%, 1.0%, and 5.0%) and incubated for a 12 hr. period, both the nuclei and cytoplasm became solubilized. Whereas, when weak concentrations (0.1% and 0.2%) were utilized, only the cytoplasm was solubilized leaving the nuclei intact. This property of digitonin, wherein nuclei were left intact after treatment, may be due to the inability of weak concentrations of the soap-like detergent to breakdown the disulfide bonds of the proteins of which the nuclear membrane is composed, thus allowing the nuclei to maintain their integrity.

The ability of Porphyromonas gingivalis to acquire iron in the iron-limited environment of the host is crucial to the colonization of this organism. Here, the isolation and characterization of a transpositional insertion mutant of P. gingivalis A7436 (designated MSM-3) which is defective in the utilization and transport of hemin, is reported. P. gingivalis MSM-3 was initially selected on the basis of its non-pigmented colony phenotype on anaerobic blood agar plates following mutagenesis with the Bacteroides fragilis transposon Tn4351. P. gingivalis MSM-3 grew poorly when supplies with hemin as a sole source of iron; however growth was observed with hemoglobin or inorganic iron. P. gingivalis MSM-3 grown under hemin-replete or hemin-deplete conditions bound and transported less [14C] hemin and [59Fe] hemin than did the parental strain A7436. At 4 hr, P. gingivalis MSM-3 grown under hemin-replete conditions transported only 10,000 pmol of hemin per mg total cellular protein, or 14% of the amount transported by P. gingivalis A7436 grown under similar conditions. Unlike P. gingivalis A7436, hemin binding and transport by P. gingivalis MSM-3 were not tightly regulated by hemin or iron. Examination of P. gingivalis MSM-3 cultures by electron microscopy revealed an overproduction of membrane vesicles, and determination of the dry weight of purified vesicles indicated that P. gingivalis MSM-3 produced twice the amount of membrane vesicles as did strain A7436. Extracellular vesicles isolated from P. gingivalis MSM-3 were found to express higher hemagglutination titers, as well as enhanced hemolytic and arginine-X-specific protease activities. In vivo studies revealed that P. gingivalis MSM-3 was more infectious and invasive than the parent strain, as indicated by secondary lesion formation and death in experimental mice. Genetic analysis has revealed that while Tn4351 has inserted into a non-coding region (60 bp downstream from a known P. gingivalis insertion sequence element, IS1126), its insertion into the host chromosome resulted in the mobility of IS 1126. We have determined that P. gingivalis MSM-3 contains two additional copies of IS1126 as compared to the parent strain. The insertion site of one of the additional IS1126 elements has been sequenced. Genetic analysis reveals that it contains numerous open reading frames and does not provide evidence of an open reading frame which may encode for a gene involved in iron acquisition. However, approximately 1 kb downstream of the insertion site, a 1.014 kb open reading frame (designated hemB) has been located. Analysis has revealed that this open reading frame shares homology with several genes that encode for hemin/iron receptors or iron-regulated outer membrane proteins. Taken together, the results presented in this study provide evidence for the coordinate regulation of P. gingivalis virulence factors by hemin. Additionally, the capability of mobilization of an insertion sequence element, a phenomenon not previously reported in P. gingivalis, has been demonstrated. Further characterization of hemB and the second additional IS1126 element's insertion site may provide evidence for the role of specific proteins involved in hemin/iron acquisition by P. gingivalis.