<p> The cellular stress response (CSR) is one of the most highly conserved mechanisms among all organisms. Cellular stress can be defined as damage or the threat of damage to proteins, macromolecules and/or DNA. The response to damage can involve cell cycle regulation, protein chaperoning, DNA repair or, if macromolecular damage is too severe, apoptotic mechanisms can be initiated. This thesis details experiments that were designed to examine the cellular response to non-lethal environmental stressors at the protein level, using two fish species as study models. Two proteins that can cause cell cycle arrest and apoptosis mechanisms were examined. Expression of the CCAAT enhancer binding protein-delta (C/EBP-&delta;) was examined in the zebrafish, <i>Danio rerio,</i> exposed to acute, non-lethal hypoxic conditions. While C/EBP-&delta; was expressed constitutively in control individuals during all time points, exposure to hypoxic conditions did not have a consistent significant effect on C/EBP-&delta; expression (two-way ANOVA, P>0.05) in zebrafish white muscle tissue. In a second study, the expression of the growth arrest and DNA damage 45-alpha protein (gadd45-&alpha;), a mediator of cell cycle arrest and perhaps apoptosis was examined in heat-stressed liver tissue of an extremely cold-adapted Antarctic fish, <i>Trematomus bernacchii.</i> Gadd45-&alpha; levels were higher in fish exposure to 2&deg;C across all time points (one-way ANOVA; P&lt;0.05). The findings in these two studies expand our understanding of the CSR and how two genes that are involved in cell cycle regulation respond to acute, non-lethal environmental stress.</p>

<p> Coordinated movement of cardiac valves controls unidirectional flow of the blood with every heart beat. Cardiac valves are composed of thin, pliable leaflets that withstand compressive tension, fluid shear stress, and bending stress as blood flows through them. The structure and the mechanical properties of the valves render them durable during the lifetime of human beings. However, changes in hemodynamic environment, inflammatory responses, and congenital valvular defects can all cause valves to undergo irreversible structural changes, one of which is calcific aortic stenosis (CAS). CAS affects 2-3% of the population over 65 years old in the western world, and the only effective treatment is valve replacement surgery. CAS is characterized by tissue stiffening and the formation of calcified nodules, the development of which is associated with abnormal differentiation of resident fibroblasts known as valvular interstitial cells (VICs). Upon tissue injury, VICs are activated to myofibroblasts which deposit excessive collagen and stiffen the matrix. Understanding how the pathogenic phenotype of VICs is regulated by cues from the matrix may lead to new therapeutic treatments for CAS. In this thesis, I examined how matrix elasticity and TGF-&beta;1 regulate VIC phenotypes. First, I characterized the VIC population from porcine aortic valves and showed that this population is relatively homogeneous. When I cultured these primary cells on different substrates, I found that poly(ethylene glycol) hydrogels mimicked the native valve matrix better than tissue culture polystyrene plates with respect to preserving the quiescent fibroblast phenotype. At the level of signaling, I demonstrated that this is mediated through an elasticity-regulated PI3K/AKT pathway. Additionally, I showed that reduced matrix rigidity redirected activated valvular myofibroblasts into dormant fibroblasts without inducing significant apoptosis. Finally, I examined the effect of TGF-&beta;1 on VIC gene expression over time with microarray-based gene expression profiling and found that TGF-&beta;1 up-regulated cell-cell contact proteins (e.g., OB-cadherin, N-cadherin) in order to regulate valvular myofibroblast activation. Collectively, my thesis work revealed novel mechanosensing mechanisms employed by VICs to respond to matrix elasticity and explored the complex interactions among multiple extracellular cues, including matrix elasticity, TGF-&beta;1 and cell-cell adhesion, to direct the cellular fate of VICs.</p>

<p>This work investigates the functional heterogeneity of influenza virus quasispecies through quantitative analysis of cellular responses to the entry of noninfectious biologically active particles, the effect of reassortment of gene segments on the generation and function of these particle subpopulations, and the potential of these subpopulations as <i>in vitro</i> correlates of <i>in vivo</i> effectiveness of live-attenuated influenza vaccines (LAIVs). </p><p> For the first time, the clonogenic assay was used to show that populations of most influenza A viruses contained cell-killing particles in excess of infectious particles when tested in the same host cell. Thus, a new class of influenza virus particles was revealed &ndash; noninfectious cell-killing particles which required the synthesis of a specific viral polymerase subunit to kill cells and the expression of NS1 protein to temporally delay apoptosis/cell-killing. </p><p> The noninfectious cell-killing particles were clearly distinguished from the well known defective-interfering particles by differences in their numbers in standard influenza virus populations, their temporal appearance and quantity during serial high multiplicity propagation in mammalian and chicken cells, an inability of defective-interfering particles to kill cells or interfere with the cell-killing capacity of noninfectious cell-killing particles, genetic requirements (a small DI RNA &sim;350 nt and a large RNA &sim;2,300 nt for defective-interfering and noninfectious cell-killing particle activities, respectively), and the extracellular T&half; at 40.5 &deg;C (&sim;40h and &sim;85h for noninfectious cell-killing particles and defective-interfering particles, respectively). </p><p> Specific exchange of the <i>NS</i> gene segment from lethal A/HK/156/97 (H5N1) (NS1: E92, or E92D) virus for the cognate <i>NS</i> gene segment of A/PR/834 (H1N1) (NS1: D92) virus caused <i>de novo</i> generation of large defective-interfering particle subpopulations and >10-fold enhancement of interferon-inducing particle efficiency. These changes were attributed to dysfunction of the H5N1 virus <i>NS1</i> gene. </p><p> Populations of two effective LAIVs (Vac⁺) in chickens were characterized by high defective-interfering to interferon-inducing particle ratios and induction of large amounts of interferon in chicken cells. Interferon is an antiviral cytokine that acts as a potent natural adjuvant of adaptive immune responses in chickens. Populations of two ineffective LAIVs (Vac^-) in chickens had lower defective-interfering to interferon-inducing particle ratios and induced less interferon. Unexpectedly, these phenotypes were reversed in mammalian cells. Populations of Vac^- (in chickens) LAIV candidates were excellent interferon inducers with high defective-interfering to interferon-inducing particle ratios in mammalian cells. In contrast, populations of Vac⁺ (in chickens) LAIV candidates were poor interferon inducers with low defective-interfering to interferon-inducing particle ratios in mammalian cells. As predicted by the <i>in vitro</i> screen, the Vac phenotypes were reversed <i>in vivo</i> (in mice) relative to chickens. </p><p> Overall, this study shows that the majority of noninfectious particles of influenza virus are biologically active, reassortment can change the subpopulation make of influenza virus, and a high defective-interfering to interferon-inducing particle ratio is a strong <i>in vitro</i> correlate of the effectiveness of self-adjuvanting LAIVs. Taken together, these attributes of an influenza virus population represent a novel ensemble of <i>in vitro</i> parameters that may be used to distinguish between Vac⁺ and Vac^- LAIV candidates. </p>

<p> Amphidromous species migrate between fresh water and the sea for larval development. Many caridean shrimps, especially <i>Macrobrachium </i> spp., are amphidromous, and some populations are found far-upstream within continental river systems. This project tested the hypothesis that populations of <i>Macrobrachium ohione</i> from the Atchafalaya and Mississippi Rivers are amphidromous. </p><p> In the laboratory, I tested the hypothesis that upstream populations of <i>M. ohione</i> have freshwater larval development. My results indicated that saline habitats are essential for <i>M. ohione</i> development, and larval mortality increased after 3-4 days of freshwater drifting. This provides indirect evidence that upstream populations have extended marine larval development. </p><p> Due to their limited freshwater survival, <i>M. ohione</i> must deliver larvae to the sea. Spatial-temporal analysis in the Atchafalaya and Mississippi Rivers reported an influx of reproductive-sized shrimps and females with near-hatching broods into coastal sites. This suggests that females are migrating downstream to hatch larvae in downstream habitats. </p><p> Stable isotope analysis indicated that the upstream juvenile migration originates from saline habitats. Video surveillance revealed that juveniles migrate throughout the night at an average speed of &sim; 0.56 km hr^-1, and swimming speeds were related to the water velocity they swam against. From these results, I estimated that juveniles are capable of migrating to far-upstream habitats within their life span (1-2 years). </p><p> Lastly, I investigated how dams affect the juvenile migration, and tested juvenile migrant climbing abilities. This study reported greater densities of juveniles downstream of dams than upstream of dams, indicating the dams impede the juvenile migration downstream of Old River Control. Shrimp climbing studies revealed that at various inclinations and water velocities, ~ 52% of the shrimps were climbing the shrimp ramp and ~ 12% completed the climb. These results demonstrated juveniles can climb bypass structures with detectable water flows. </p><p> My findings suggest that <i>M. ohione</i> populations within the Mississippi River System are amphidromous, because they require marine larval development and long-distance migrations are conducted to and from the sea. This study may serve as a general model for migrations of amphidromous shrimps in comparable large rivers, and potentially contribute to freshwater shrimp conservation.</p>

<p> Islet amyloid polypeptide or amylin is a recently discovered 37 amino acid residue signaling protein (hormone) that is produced and co-secreted along with insulin by pancreatic beta-cells. In late onset of diabetes, amylin readily aggregates forming protein deposits or plaques that are toxic to beta-cells, resulting in beta-cell apoptosis. Given the well-known role of cholesterol and lipids in etiology of diabetes, I explored whether these two essential PM components regulate amylin assembly and aggregation on artificial (synthetic) membranes. Using high resolution imaging and spectroscopic approaches, I demonstrated that amylin undergoes facilitated aggregation and conformational changes in the presence of membranes composed of anionic lipids such as phosphatidylserine (PS). The presence of cholesterol on the other hand inhibited lipid-induced aggregation of amylin in solution and on model planar membranes. However, the patho-physiological consequence of cholesterol-regulated amylin polymerization on membranes, and biochemical mechanisms that protect beta-cells from amylin toxicity are poorly understood. Hence, in my subsequent study, I reported that PM cholesterol plays a key role in molecular recognition, sorting and internalization of toxic amylin oligomers but not monomers in pancreatic rat insulinoma and human islet cells. Depletion of PM cholesterol or the disruption of the cytoskeleton network inhibited internalization of amylin oligomers, which in turn enhanced extracellular oligomer accumulation and potentiated amylin toxicity. In contrast to oligomers, amylin monomers followed clathrin-dependent endocytosis, which was not sensitive to cholesterol depletion. Our studies identified an actin-mediated and cholesterol-dependent mechanism for selective uptake and clearance of amylin oligomers, impairment of which greatly potentiated amylin toxicity. </p><p> However, the exact uptake mechanism and trafficking routes of these molecular forms and their significance for amylin toxicity are yet to be determined. Hence, in my further study, I observed that pancreatic cells employed different strategies to eliminate amylin's toxic and non-toxic molecular forms. My study also revealed that macropinocytosis serves a major cyto-protective role in these cells, by clearing of amylin molecular forms. The overreaching goal was to fully elucidate the internalization and trafficking pathways of human amylin monomers and toxic oligomers in pancreatic cells.</p>

<p> Alkali bees, <i>Nomia melanderi</i>, are native, solitary, soil&ndash;nesting bees commercially managed in southeastern Washington State. They nest in dense aggregations and are important pollinators of alfalfa produced for seed. The Washington State Department of Transportation (WSDOT) proposed safety improvements to US Highway 12 through the Touchet&ndash; Lowden&ndash;Gardena alfalfa seed growing district, an area critical to alfalfa seed production. This includes northern realignment to accommodate a wider roadway and avoid impacting any towns. Relocation of the highway will bisect several <i> N. melanderi</i> nesting aggregations and alfalfa fields. The study has three objectives: 1) survey the population abundance of <i>N. melanderi </i> across the region by comparing two sampling techniques; 2) determine bee flight heights across roads; and 3) determine <i>N. melanderi</i> foraging range using transgenic pollen. </p><p> Regression was significant between the two population sampling methods. Mean emergence hole counts, mean prepupal counts, and the surface area of the nesting aggregations, were used to estimate the abundance of<i> N. melanderi</i> in each bee bed. We constructed a &ldquo;vehicular bee sweeper&rdquo; designed to capture insects at specific heights over the roadway. The majority of <i>N. melanderi</i> flew below 2.1 m when no other factors were considered, but environmental conditions like temperature and wind speed affected number and flight height of <i>N. melanderi</i>. To determine <i>N. melanderi</i> foraging distance, adults were collected from their nest sites, and pollen on their hind tibia was tested for the presence or absence of Roundup&ndash;^&reg;Ready alfalfa (RRA). The minimum foraging distance was 0.04 km and the maximum was 4.62 km. These distances suggest that <i>N. melanderi</i> will cross the highway for floral resources, increasing potential mortality. </p><p> Studying <i>N. melanderi</i> population abundance and flight characteristics allows us to understand the potential impacts of the proposed highway on bee populations and on alfalfa seed producers. The non&ndash;destructive quadrat method of sampling <i>N. melanderi</i> populations is robust compared to the destructive, labor-intensive, soil core method. Due to the low-flying nature and foraging distance of <i>N. melanderi</i>, vehicle strikes can be expected to cause mortality in bisected populations. Ultimately, recommendations will be made to highway designers to minimize and mitigate these effects. &#8195;</p>

<p> Acidic aqueous-phase pretreatment is a promising approach that has been directed at maximizing intermediates yields (e.g. sugars, sugar degradation products, and lignin) from biomass for fuel and chemical production. This dissertation explores the kinetic fundamentals of biomass hydrolysis in acidic aqueous-phase with different catalysts (e.g. sulfuric acid, metal chlorides), operating conditions (e.g. temperature, time pressure), and equipment configurations (e.g. batch, flowthough). </p><p> The kinetic analysis revealed that crystalline cellulose is insusceptible to hydrolysis compared with agarose at low temperature (e.g.140 &deg;C), while it decomposed rapidly at elevated temperature (e.g. 220 &deg;C). Higher temperature with reduced time was desirable for glucose production whereas lower temperature with prolonged time was preferred for xylose generation. In acidic conditions, furfural and levulinic acid were stable whereas 5-hydroxymethylfurfural was susceptible to decomposition with high rate constant. MgCl₂ can promote the cleavage of C-O-C bond in polysaccharides (e.g. agarose) and enhance the subsequent dehydration reaction to 5-hydroxymethylfurfural. Unlike transition metal chlorides and H2SO₄, MgCl₂ has little ability to induce retro aldol and rehydration reactions to generate byproducts like lactic acid and levulinic acid. Mg²⁺ possessing hgiher activity than other alkali and alkaline earth metal chlorides (Na⁺ and Ca2⁺) resulted in 40.7% yield and 49.1% selectivity of 5-hydroxymethylfurfural. </p><p> Dissolution of biomass was significantly enhance using acidic hot water flowthrough pretreatment at 200&mdash;280&deg;C. Significant cellulose removal accompanied with the transformation of cellulose I to cellulose II and amorphous cellulose were observed when temperature was above 240 &deg;C for water-only and 220 &deg;C for dilute acid. Approximately100% of the xylan and &sim;90% of the cellulose were solubilized and recovered. Up to 15% of the lignin was solubilized, while the remaining lignin was insoluble. Over 90% sugar yields were obtained from pretreated whole slurries using less than 10 FPU/g cellulase plus hemicellulase enzyme. </p><p> A kinetic model was developed to depict the biomass degradation in flowthrough system. This model predicted the sugar generation more precisely than the conventional homogeneous first-order reaction models. Mass transfer limitations were minimized using 4mm biomass particle sizes with 4g biomass loading at 25mL/min flow rate, produced hydrolyzate slurries with 13g/L potential sugar concentrations.</p>

<p> The abstract is not available for copy and paste.</p>

<p> Several methods of enumerating Enterococci in water are suggested in the literature, notably membrane filtration and mEA plating. To establish optimal growth conditions, including incubation time, (24 and 48 hr) and temperature (35&deg;C and 41&deg;C), samples of 0.1 mL, 1 mL and 10 mL filtered water collected from Lake Artemisia, MD, USA were amended with known concentrations of Enterococcus faecalis (ATCC 29212), filtered using 0.45 &micro;m membrane filters, and incubated on mEA agar under different conditions: 35&deg;C/24h, 35&deg;C/48h, and 41&deg;C/48h, following U. S. Environmental Protection Agency guidelines. Results demonstrated no significant difference among the volume and time of incubations used but a significant difference in the temperatures employed. Being the etiological agent of cholera, V. cholerae is a major public health problem in several developing countries. The prevalence of &beta;-lactamase-producing strains and their isolation from life-threatening infections as well as the environment is alarming and presents a major therapeutic challenge for clinicians. The extended-spectrum &beta;-lactamase profile of a collection of 210 V. cholerae O1 strains isolated from clinical and water samples was investigated. The strains were collected during ongoing epidemiological and ecological cholera surveillance in the provinces of Chhatak and Mathbaria in Bangladesh, between March 2009 and April 2012. Resistance to penicillins, monobactams, carbapenems, second-, third- and fourth- generation cephalosporins were tested by disk diffusion. Genotypic analysis of the resistance determinants was performed by PCR to detect ESBL (blaCTX, blaTEM, blaSHV), carbapenemases (blaIMP, blaSPM, blaVIM, blaBIC, blaNDM, blaKPC, blaAIM, blaSIM, blaDIM, and blaGIM). All strains were sensitive to the 4th-generation beta-lactam cefepime. This is the first report documenting such extensive resistance to monobactams and third-generation cephalosporin in V. cholerae.</p>

<p> Nordihydroguaiaretic acid (NDGA) is the principal compound in the resinous leaf coating of <i>Larrea tridentata</i> (creosote bush), the dominant shrub of North American deserts. <i>L. tridentata</i> exists as three polyploid races: diploid (2X = 26), tetraploid (4X = 52), and hexaploid (6X = 78). The distributions of these ploidy levels are strongly associated with the three major deserts of the region where diploids primarily reside in the cooler, wetter Chihuahuan desert, tetraploids in the Sonoran desert, and hexaploids in the hot, dry Mojave desert. NDGA is a secondary metabolite of creosote bush that functions to protect plants from biotic and abiotic stressors such as extreme drought, harmful UV radiation, and herbivory. Here, I investigated the role of polyploidy and environmental variables on the production of NDGA by quantifying concentrations from field and greenhouse-grown polyploids. Citizen scientists were utilized to facilitate simultaneous sampling across the entire distributional range of this species, for one full year. Under natural conditions, shrubs produced significantly higher NDGA concentrations than when removed from the harsh desert environment. In field and greenhouse treatments, hexaploids exhibited higher NDGA concentrations than diploids or tetraploids. Within the diploid cytotype, I documented environmental influences on NDGA concentration based on comparisons between a field site experiencing severe drought, a watered field site, and greenhouse-grown diploids. Principal components analysis revealed that NDGA response to environmental variables successfully predicts the current ploidy distribution of this species. These observations highlight the complexity of plant-environment-genotype interactions and suggest that evolution in production of secondary metabolites may be driven by long-term changes in environmental conditions, and potentially influence species distribution regimes.</p>