Biology

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This is the collection for the University of Waterloo's Department of Biology.

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Gaits and Their Development in the Infraorder Pecora
(University of Waterloo, 1967-02) Dagg, Anne Innis
The gaits of twenty-eight species of the Infraorder Pecora are analyzed from motion picture sequences comprising over 45,000 frames. For each gait the percentage time spent on the various supporting legs during each stride and the order in which the combinations of supporting legs were used are tabulated. When possible the times for the strides are calculated. The walk patterns of each of the four families studied are shown to be statistically distinctive. Within each family, those members that live where the vegetation is so dense that they must rely on hearing to warn them of danger use a more stable walk than those species that inhabit open grasslands. A stable walk is that in which diagonal legs are used in preference to lateral legs and three or four supporting legs are used rather than two. With a more stable walk, a species is able to pause quickly and to flee instantly if danger threatens. The data for these species suggest that the antelopes, giraffids and pronghorns evolved in an open environment while the cervids, including the caribou, evolved in a woodland habitat. The variability in the walking strides of members of these species is assessed for the first time. The walk pattern varies with the nature of the terrain and with the speed at which the walk is executed. It also depends upon the age of an individual of a species and on the weight of its antlers or horns. A trotting gait is shown to be the predominant one of the constantly moving caribou. This species has evolved anatomically in ways that ensure an effective trot. The preferred fast gait of several other species is found to be a trot rather than a gallop, owing to their large size, their wooded environment, the swampy nature of the ground underfoot or their heavy antlers or horns. The type of gallop of the Pecoran species is shown to be closely related to their environment and to their anatomy. Light Pecoran species use both flexed and extended suspensions in their fast gallops. The white-tail and the mule deer alone use the extended suspension primarily, since it is in this phase of their gallop that they leap over the bushes and logs that occur throughout their brush environment. Heavy Pecoran species do not use extended suspensions, nor generally do those animals with backs sloping downward posteriorly to relatively short hind legs. A symmetrical bound is shown to be used by both cervids and antelopes either to navigate steep and rough slopes or to increase the field of vision. The times taken for walking, trotting and galloping strides are shown to be longer in larger animals. The reason for this is discussed in terms of the leg considered as a pendulum.
Effect of Fuel Ethanol on Subsurface Microorganisms and its Influence on Biodegradation of BTEX Compounds.
(University of Waterloo, 2000) Araujo, Daniela
Ethanol is used as fuel in neat form in some countries (Brazil and India) or blended with gasoline (Europe, Canada and the United States). The benefits of ethanol use include octane enhancement, a cleaner environment and a secure renewable energy supply. BTEX compounds (benzene, toluene, ethylbenzene, m-xylene, p-xylene and o-xylene) are aromatic hydrocarbons present in gasoline. The fate of these compounds in the environment is of great health concern due to their carcinogenic (benzene) and toxic properties, and due to their high solubility in water compared to the other gasoline hydrocarbons. Ethanol present in gasoline may affect BTEX degradation, in an event of a spill into the subsurface environment. To address the effects of ethanol on subsurface microorganisms, microbial activity and growth in the presence of ethanol (concentrations ranging 0 to 70% v/v) were assessed. Microcosms studies showed that ethanol at concentration ranging 0. 5 to3% (v/v) enhanced microbial activity and did not interfere inmicrobial growth at 10oC temperature, when another source of carbon was present (glucose). Ethanol at 0. 5% concentration enhanced microbial activity over water soluble gasoline components and R2A medium combined. Both microbialactivity and growth were not detected at ethanol concentrations equal and above 5%. Biodegradation study was conducted, in which subsurface material and ground water were exposed to BTEX and ethanol at 0. 5 and 1. 5% (v/v) concentration. The controls had BTEX alone and ethanol alone, sterile and nutrient-free. Total BTEX degradation was observed whenever ethanol was absent. Ethanol and BTEX were simultaneously degraded, however in microcosms containing 0. 5% ethanol, BTEX degradation was slowed, compared to microcosms without ethanol. Competition for inorganic nutrients was the major problem in slowed BTEX degradation in the presence of ethanol. In microcosms where 1. 5% ethanol was present, BTEX compounds and ethanol degradation were not observed.
Sources and Controls of Sulphur Export in Precambrian Shield Catchments in South-central Ontario
(University of Waterloo, 2002) Eimers, M. Catherine
A series of studies was undertaken at Plastic Lake-1 (PC1) to determine the sources and controls on S cycling in small headwater catchments on the Precambrian Shield in south-central Ontario. Two observations were made about the S cycle in this region: (1) all streams exhibit highly coherent temporal patterns in SO4 concentrations and export, and (2) most catchments exported more SO4 in stream water than is received in bulk deposition during the past 2 decades. Synchronous temporal patterns in annual SO4 concentrations in both upland and wetland-draining streams were related to changes in climate, specifically those factors that determine catchment dryness. The number of days with no stream flow or stream flow below a critical threshold was a good predictor of the average stream SO4 concentration in a particular year. Sulphate chemistry in the PC1 outflow is highly dependent on processes occurring in a conifer Sphagnum swamp located immediately upstream of the chemical sampling station. Hydrologic inputs to the swamp during the summer determine whether S is retained or released from peat on an annual basis. Drying and re-wetting of Sphagnum-derived peat caused a substantial increase in soluble SO4 in laboratory experiments, which was slightly enhanced at higher temperature, but alternating moisture conditions had no immediate effect on Sphagnum. Despite large inter-annual changes in SO4 release, over the long-term (i. e. 20-years) SO4 inputs and exports from the swamp are in approximate balance. In contrast, the upland portion of PC1 (i. e. PC1-08) consistently exports more SO4 than is input in bulk deposition in every year of record. Even when inputs are increased to account for potential underestimates in dry deposition or weathering, the majority of catchments in this region exhibit net export in many years. Two internal sources are suggested to account for negative budgets: desorption and mineralization. Adsorption/desorption reactions respond directly to changes in SO4 input concentration, and lysimeter data indicate the importance of these processes for buffering short-term changes in SO4 concentration in LFH percolate. Desorption may be the primary direct response of upland soil to decreasing SO4 inputs in deposition and may substantially extend the period of net SO4 export in catchments that have large adsorbed SO4 pools such as PC1. However, the adsorbed pool may be sustained by continuous net release from mineralization, and should also be considered in budget calculations. Mineralization was shown to be responsive to drying and re-wetting events and temperature, although results varied among different materials. Sulphate release from mineral soil did not appear to be influenced by changing moisture, temperature or deposition chemistry in laboratory experiments, although adsorption/desorption reactions may have largely masked small changes in SO4 release via mineralization. The magnitude of organic S storage in mineral soil indicates that this pool could be an important source of export over the long-term. While it is unknown why (or if) mineralization is a net source of SO4 to drainage streams, changes in climate and/or deposition could potentially influence SO4 release from organic compounds. Soil moisture and temperature are important controls on microbial processes in soil, and changes in climate that bring about changes in soil moisture or temperature conditions could affect decomposition and mineralization processes. Similarly, historically high inputs of S and N in deposition may have brought about slow shifts in litter quality (i. e. decreased C:N, C:S) which could also potentially influence decomposition and mineralization rates. In order to predict the future response of surface water chemistry to changes in SO4 (and N) deposition, it is important to consider not only the magnitude of S pools in soil, but also the potential for SO4 cycling between pools. Likewise, models that predict changes in stream SO4 by adsorption isotherm data alone will underestimate the importance of desorption unless the potential for continual replenishment of the adsorbed pool through the relatively slower process of mineralization is also considered. In general, predictions of recovery from S deposition can only be made from a complete understanding of S pools, transformations, and the effects of climate, which are superimposed upon the long-term trend in deposition.
Characterization of Eukaryotic Translation Initiation Factor 5A-2 (eIF5A-2) in Arabidopsis thaliana: Effects of Wounding and Pathogen Attack
(University of Waterloo, 2004) Gatsukovich, Yulia
Plants respond to wounding and pathogen ingress by synthesizing defence proteins that facilitate wound healing, cell death or provide protection against further pathogen attack. The accumulation of these proteins is mainly due to rapid transcriptional activation of corresponding genes, though preliminary evidence implies that translational control may also have a role in the stress response. This study focuses on the function of Arabidopsis thaliana eukaryotic translation initiation factor 5A (eIF5A) in mechanical wounding and during infection with the phytopathogen Pseudomonas syringae. Recent evidence suggests that eIF5A acts as a nucleo-cytoplasmic shuttling protein that facilitates mRNA translation through selective transport from the nucleus. Three isoforms of eIF5A have been identified and isolated in Arabidopsis thaliana, suggesting that each distinct isoform may be involved in transport of different subsets of mRNA required for a specific physiological event. The analysis in this study revealed that the eIF5A-2 isoform appears to be involved in the signal transduction pathways that result in cell death following virulent infection, systemic acquired resistance and wounding in Arabidopsis thaliana. Transgenic plants with reduced expression of eIF5A-2 were developed using antisense T-DNA insertion. These lines have shown marked resistance to Pseudomonas syringae pv tomato DC3000, exhibiting up to 99% decrease of bacteria in planta and cell death associated with disease. The capacity to effectively limit growth of various pathogens is important for the design of strategies to improve disease resistance in crops. Development of disease-resistant lines allows efficient crop production with reduced reliance on environmentally undesirable toxic agrichemicals.
Phytoplankton Production In Lake Victoria, East Africa
(University of Waterloo, 2004) Silsbe, Gregory
This thesis develops, validates and applies an empirical model that provides the first spatially explicit estimates of gross and net phytoplankton production in Lake Victoria. Gross and net phytoplankton production are in turn used to estimate the maximum sustainable yield (MSY) of Lake Victoria's fishery following an empirical formula and the carbon efficiency transfer method. Chapter 2 presents results from three inshore areas where diurnal and sub-seasonal gross and net phytoplankton production was derived using an adapted version of the phytoplankton production model developed by Fee (1990). Spatial and temporal trends of chlorophyll (chl), PI parameters, the vertical attenuation of PAR (kPAR), Secchi depths (SD) and respiration rates are identified. kPAR and SD are highly correlated to chl within the euphotic zone, as well as to each other. Furthermore, the two PI parameters, PBM and aB, exhibit a strong linear relationship and both decline along an increasing chl gradient, presumably due to increased light-limitation, a taxonomic shift from diatoms to cyanobacteria with increasing chl as well as an increased need for biologically fixed nitrogen. These hypotheses are supported by observed synchronous changes in the PSII:PSI ratio of phytoplankton and changes in the chl-specific attenuation of PAR (kchl). Relationships are also derived between biomass-specific respiration rates (RB) with chl and PBM; similar to PI parameters RB decreases with increasing chl. Owing to these correlative trends, only one parameter is required to estimate gross phytoplankton production through the empirical model developed in this thesis. The empirical model predicts that gross phytoplankton production increases in a near linear fashion between chl of 0 to 10 mg. m-3, begins to flatten out as chl approaches 20 mg. m-3 and then slightly decreases when chl exceeds 40 mg. m-3 where the maximum PPG of 13. 1 g O₂. m-2. day-1 is reached and is in close agreement with a theoretical argument proposed by Talling (1965). Areal respiration and consequently net phytoplankton production are sensitive to chl within the mixed layer as well as mixed layer depths. Overall, the lakewide averages of gross and net phytoplankton production are 9. 68 and 2. 2 g O₂. m-2. day-1 respectively. Significant temporal variability was observed on sub-seasonal scales within the inshore of Lake Victoria, and changes in limnological parameters coincided with changes in water column temperatures in each of the three bays. In Fielding Bay, the availability of meteorological data revealed that strong nocturnal wind events decreased both the water column temperature and chl, while both parameters generally increased in the absence of any such wind event. Lateral exchange of water with deeper areas through strong wind events essentially flushes Fielding Bay causing the observed decreases in both the water column temperature and chl; this hydrodynamic event also influences other limnological parameters according to their respective correlative regression equations with chl. Spatial trends were also observed between inshore areas. The deepest area, Napoleon Gulf, has the lowest values of chl while the shallowest area, Inner Murchison Bay, has the highest chl as the mean depth of a bay sets an approximate upper limit on chl. With respect to diurnal variability, PI parameters decline through the day, kPAR increases over the day and no statistically valid trends were ascertained for chl and RB. Chapter three examined spatial and seasonal patterns of chlorophyll fluorescence, temperature, dissolved oxygen and water transparency from four lakewide cruises. Significant spatial variability of each parameter confirmed that lakewide data is required to generate spatially explicit estimates of phytoplankton production. Complex patterns in the thermal structure during each cruise illustrated that physical processes in Lake Victoria are at times more complex that a previously stated unidirectional hypothesis of warm water in the north and cool water in the south (Spigel and Coulter 1996), and these patterns influence spatial patterns in dissolved oxygen and Secchi depths. Similar to Chapter 2, estimates of chl within the mixed layer were highly correlated to mixed depths, while lakewide averages of chl are lower than previously reported offshore values (Mugidde 1993, 2001).
The production and fate of picoplankton and protozoa in the pelagic food web of Napoleon Gulf, Lake Victoria, East Africa
(University of Waterloo, 2004) Jackson, Victoria S.
The importance of the microbial food web and how it interplays with the classical food chain has gained considerable attention in temperate lakes. However its role in carbon transfer from pico- and nanoplankton to zooplankton and planktivores is relatively unknown in tropical lakes. Sampling of the microbial food web and experiments to estimate the growth rate and fate of its components were performed in Lake Victoria, East Africa, during the mixing season (May to August) 2002. Bacterioplankton and ciliate densities in Napoleon Gulf ranged from 6. 2 to 14. 9 cells x 10⁶•mL^-1 and 51. 9 to 75. 2 cells•mL^-1, respectively. Flagellate abundance was high, ranging from 70. 4 to 127. 9 cells x 10³•mL^-1. Small flagellates, tentatively called Choanoflagellida, dominated the flagellate community by abundance and biomass. Bacterial growth rates were low, yet high abundance and cell size resulted in high bacterial production representing 24 to 38% of phytoplankton production. Protozoan growth rates and production are similar to values reported for other African lakes and the Laurentian Great Lakes. Protozoa were the dominant grazers of bacteria with grazing pressure switching from protozoa > 5 µm in June to protozoa < 5 µm (presumably flagellates) in July. In July, grazing on flagellates was from predators < 40 µm, probably ciliates, while the ciliate community was grazed by > 40-µm plankton. Given that plankton of Lake Victoria is dominated by colonial cyanobacteria and raptorial zooplankton, protozoa could be an important pathway in the pelagic food web of Lake Victoria, East Africa.
Environmental Photoinduced Toxicity of Polycyclic Aromatic Hydrocarbons: Occurrence and Toxicity of Photomodified PAHs and Predictive Modeling of Photoinduced Toxicity
(University of Waterloo, 2005) Lampi, Mark
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants known for their photoinduced toxicity. There are two mechanisms through which this may occur: photosensitization and photomodification. Photosensitization generally leads to the production of singlet oxygen, a reactive oxygen species (ROS), which is highly damaging to biological molecules. Photomodification of PAHs, usually via oxygenation, results in the formation of new compounds (oxyPAHs), and can occur under environmentally relevant levels of actinic radiation.

PAHs and oxyPAHs readily adsorb to the organic phase of particulate matter in the environment such as sediments. It is logical to conclude that sediment transport will also facilitate the transport of these contaminants, and it has been shown that in the course of transport, degradative processes evoke a change in the profile of the PAHs present. Sediment samples taken along a transect from Hamilton Harbour were fractionated, and analyzed using a 2D HPLC method. All sediments contained intact and modified PAHs, although a marked change was noted in the profile of compounds present in the samples, which differ in distance from shore. Fractions of sediment extract were tested for toxicity using a bacterial respiration assay. Toxicity was observed in fractions containing modified PAHs, and was similar to that of intact PAH-containing fractions.

Subsequently, the toxicities of 16 intact PAHs were assessed to Daphnia magna under two ultraviolet radiation (UV) conditions. The toxicity of intact PAHs generally increased in the presence of full spectrum simulated solar radiation (SSR), relative to visible light plus UVA only. To expand the existing data on the effects of PAH photoproducts to animals, fourteen oxyPAHs were also assayed with D. magna, most of which were highly toxic without further photomodification. The data presented highlight the effects of UV radiation on mediating PAH toxicity. The importance of the role of photomodification is also stressed, as several oxyPAHs were highly toxic to D. magna, a key bioindicator species in aquatic ecosystems.

A QSAR model previously developed for Lemna gibba showed that a photosensitization factor (PSF) and a photomodification factor (PMF) could be combined to describe toxicity. To determine whether it was predictive for D. magna, toxicity was assessed as both EC50 and ET50. As with L. gibba and Vibrio fischeri, neither the PSF nor the PMF alone correlated to D. magna toxicity. However, a PSF modified for D. magna did in fact exhibit correlation with toxicity, which was further improved when summed with a modified PMF. The greatest correlation was observed with EC50 toxicity data. This research provides further evidence that models that include factors for photosensitization and photomodification will likely be applicable across a broad range of species. To gain further knowledge of the roles that the variables contributing to the photosensitization and photomodification, a structural equation model was constructed based on the D. magna QSAR. This model accounted for a high amount of variance in six sets of toxicity data, as well as insight into the mechanisms of phototoxicity affecting different aquatic organisms.
Modeling the growth dynamics of Cladophora in eastern Lake Erie
(University of Waterloo, 2005) Higgins, Scott
Cladophora glomerata is a filamentous green alga that currently forms extensive blooms in nearshore areas of Lake Ontario, eastern Lake Erie, Lake Michigan, and isolated locations in Lake Huron. The biomass, areal coverage, algal bed characteristics, and tissue phosphorus concentrations of Cladophora glomerata were measured at 24 nearshore rocky sites along the northern shoreline of Lake Erie?s eastern basin between 1995-2002. Midsummer areal coverage at shallow depths (≤5m) ranged from 4-100 %, with a median value of 96%. Peak seasonal biomass ranged from <1 to 940 g m^-2 dry mass (DM), with a median value of 171 g m^-2 DM. Tissue phosphorus varied seasonally, with initial high values in early May (0. 15 to 0. 27 % DM; median 0. 23 % DM) to midsummer seasonal low values during peak biomass (0. 03 to 0. 23 % DM; median 0. 06 % DM). A numerical Cladophora growth model (CGM) was revised and field-tested at 5 sites in eastern Lake Erie during 2002. The CGM is useful for: 1) Predicting Cladophora growth, biomass, and tissue phosphorus concentrations under non-point source P loading with no depth restrictions; 2) providing estimates of the timing and magnitude of the midsummer sloughing phenomenon; 3) determining the contribution of Dreissena invasion to the resurgence of Cladophora in eastern Lake Erie; and 4) developing management strategies for Cladophora abatement. The CGM was applied to investigate how the spatial and temporal patterns of Cladophora growth were influenced by the natural variability in environmental parameters in eastern Lake Erie. Seasonal patterns in Cladophora growth were strongly influenced by temperature, and peak depth-integrated biomass was strongly influenced by both available light and phosphorus. The photosynthetic capacity of field collected Cladophora was a poor predictor of the mid-summer sloughing phenomenon. The CGM, however, predicted that self-shading within the dense Cladophora mats would have caused negative growth rates at the base of the dense mats for 14 days prior to the sloughing event. The metabolic imbalances at the base of the Cladophora mats were driven primarily by the availability of light and were exacerbated by intermediate water temperatures (~23°C). The excellent agreement between model simulations and field data illustrates the ability of the CGM to predict tissue P and growth over a range of sites and depths in eastern Lake Erie and suggests potential for the model to be successfully applied in other systems.
The carbon and nitrogen composition of suspended particulate matter in Lake Erie, selected tributaries, and its outflow
(University of Waterloo, 2005) Upsdell, Brynn
Since their introduction to Lake Erie, dreissenid mussels may have reengineered the cycling of nutrients in the lake so that the nearshore benthic community intercepts, retains, and recycles greater quantities of nutrients. This study traces particulate matter on a basin scale by characterizing the chemical composition (POC and PN concentrations, POC/PN mass ratios, δ¹³C and δ¹⁵N) of suspended particulate matter in Lake Erie, three tributary inflows, and the lake outflow between May and October, 2002. The data are used to 1) determine the relative contributions of allochthonous and autochthonous sources to suspended particulate matter, 2) identify possible sources of suspended particulate matter, and 3) compare suspended particulate matter in the eastern basin of Lake Erie with that in the central and western basins. Mean POC concentrations range from 175 to 4494 µg/L and mean PN concentrations range from 33 to 812 µg/L in this system. Mean POC/PN mass ratios are similar across all sampling locations, ranging between 4. 5 and 6. 9, and indicate that suspended particulate matter at these sites is mainly derived from autochthonous sources, particularly plankton. The ranges of δ¹³C (-34 to -22 ?) and δ¹⁵N (1 to 12 ?) identify terrestrial plants and soil matter, aquatic macrophytes, phytoplankton, and sewage as possible sources of suspended particulate matter at all sites. Plankton is probably the dominant source of suspended particulate matter at each site, with smaller contributions from allochthonous and other autochthonous sources. Significant differences in the concentration and isotope data between inflow and lake or outflow sites indicate that tributary inflows may receive greater contributions from terrestrial plants and soils and aquatic macrophytes than the lake and outflow. δ¹⁵N signatures also identify animal manure as a possible source of suspended particulate matter at the inflows. PN concentrations and δ¹⁵N signatures suggest that the shallowest nearshore sites close to Peacock Point in the eastern basin receive PN from a source that is not present at the other eastern basin sites or at the sites in the central and western basins. This source may be related to dreissenid mussels at these nearshore sites recycling nitrogen back into the water column.
cDNA?GFP Fusion Libraries for Analyses of Protein Localization in Mouse Stem Cells
(University of Waterloo, 2005) Murray, Heather
Stem cells have great potential value for treating a number of diseases and conditions, including diabetes, Parkinson's, and spinal cord injuries. Applying stem cells for therapeutic purposes will require an in-depth understanding of their biology, not only of the genes they express, but also the functions of the proteins encoded by the genes. The goal of the project presented in this thesis was to develop a method for high-throughput analyses of protein localization in mouse stem cells. Localization information can provide insight into the functions and biological roles of proteins.

One means of studying protein localization involves creating proteins with a green fluorescent protein (GFP) reporter gene and analyzing their localization using fluorescence microscopy. The research outlined in this thesis focused on developing a system to create a large number of GFP-tagged proteins by constructing a cDNA?GFP fusion library. This involved exploring methods for optimizing cDNA synthesis, designing a retroviral vector (pBES23) for the expression of cDNA?GFP fusions in mouse stem cells, and constructing a cDNA?GFP fusion library in this vector using R1 mouse embryonic stem cell mRNA. The library constructed was not successfully delivered to target cells for GFP-tagged protein expression; it was therefore not possible to characterize protein localization in mouse stem cells. Suggestions are given as to how the methods used in this thesis might be optimized further.
Characterization of HSP47 Expression in Xenopus Laevis Cell Culture and Embryos
(University of Waterloo, 2005) Hamilton, Amanda
The heat shock or stress response is a transient response to stressful stimuli that protects vital cellular proteins from damage and irreversible aggregation. Heat shock proteins (Hsps) are molecular chaperones that bind to unfolded protein and inhibit their aggregation, thereby maintaining their solubility until they can be refolded to their native conformation. Hsp47 is an endoplasmic reticulum (ER)-resident protein that serves as a molecular chaperone during collagen production. Collagen is the major class of insoluble fibrous protein found in the extracellular matrix and in connective tissues. It is the single most abundant protein of the animal kingdom; at least 14 different forms exist, each with distinct structures and binding properties. The various types of collagen all possess protein regions with the distinct triple helical conformation. This complex physical structure requires very organized assembly and HSP47 has been established as an integral component of this process for collagen types I-V. Most of the previous studies examining the expression and function of hsp47 have been conducted with mammalian cultured cells. The present study represented the first investigation of the expression of hsp47 in the poikilothermic vertebrate, Xenopus laevis. Full-length Xenopus hsp47 nucleotide and amino acid sequences were obtained from Genbank and compared with hsp47 from chicken, mouse, rat, human and zebrafish. Xenopus HSP47 protein had an identity of approximately 77% with chicken, 73% with mouse, 72% with rat and human, and 70% with zebrafish. Most of the sequence identity between HSP47 from all investigated organisms occurred centrally in the amino acid sequence and in several carboxyl terminal regions. Three key features were conserved between HSP47 proteins from most species investigated: a hydrophobic leader sequence, two potential glycosylation sites and the ER-retention signal, RDEL. A partial cDNA clone encoding Xenopus hsp47 was obtained from the American Type Culture Collection (ATCC) and used to generate hsp47 antisense riboprobe for the purpose of investigating hsp47 mRNA accumulation in Xenopus A6 kidney epithelial cells and embryos. Northern blot analysis detected hsp47 mRNA constitutively in A6 cells. The expression pattern for hsp47 mRNA was compared with two other Xenopus heat shock proteins that have been previously characterized in our laboratory: hsp70, a cystolic/nuclear hsp and BiP, an ER-resident hsp. The results of hsp47 mRNA accumulation in A6 cells suggested that the expression pattern for Xenopus hsp47 was unique but, with respect to some stressors, resembled that of a cytosolic hsp rather than an ER-resident hsp. HSP47 protein levels were also examined in A6 cells. Heat shock, sodium arsenite and b-aminopropionitrile fumerate treatments enhanced hsp47 accumulation. In some experiments, western blot analysis revealed the presence of two closely sized protein bands. It is possible that minor differences in HSP47 protein size may be due to post-translational modification, namely phosphorylation or glycosylation. The present study also examined the accumulation and spatial pattern of hsp47 mRNA accumulation during Xenopus laevis early development. Hsp47 was constitutively expressed throughout Xenopus early development. Constitutive levels of hsp47 mRNA in unfertilized eggs, fertilized eggs and cleavage stage embryos indicated that these transcripts were maternally inherited. Constitutive hsp47 mRNA accumulation was enhanced in neurula and tailbud embryos compared to earlier stages. This finding may be explained by the shift towards organogenesis during these stages. Whole mount in situ hybridization revealed hsp47 message along the dorsal region of the embryo, in the notochord and somites, as well as in the head region including the eye vesicle. Hsp47 mRNA induction in Xenopus embryos was also examined in response to heat shock. Hsp47 mRNA accumulated in response to heat shock immediately following the midblastula transition (MBT). In tailbud stages, hsp47 mRNA accumulated in the notochord, somites and head region. Northern blot analysis and whole mount in situ hybridization results revealed an expression pattern that coincided well with the development of collagen-rich tissues thereby substantiating the proposed role of HSP47 as a procollagen molecular chaperone.
Paleolimnological evidence of the effects of recent cultural eutrophication and climatic variability during the last 300 years in Lake Malawi, East Africa
(University of Waterloo, 2005) Puchniak, Megan
Lake Malawi is the second largest lake in Africa, supporting diverse populations of endemic cichlids and supplying essential water resources to Malawi, Mozambique and Tanzania. However, population growth, rapid deforestation and intensive agriculture, especially in the southern catchments, have accelerated soil erosion during the last half century. These anthropogenically-disturbed catchments have caused rivers to transport greater sediment loads into Lake Malawi than rivers within forested catchments. Lake Malawi?s immense size and oligotrophic nature may retard detection of inputs of external contaminants. Reversing the effects of increased nutrient loading to Lake Malawi once observed would likely take generations, as the residence time of water is over 140 years. Therefore, sensitive metrics are required to assess the effects of land use change and climate variability in Lake Malawi in advance of deleterious effects. In this study, paleolimnological analyses of four sediment cores collected in 1997 and 1998 along a longitudinal transect of Lake Malawi, dated with ²¹⁰Pb analyses and analyzed for biogenic silica and sedimentary diatom assemblages, were used to create a long-term water quality dataset. These four sites span gradients of land use and latitude in order to reconstruct limnological conditions over the whole lake during the last 300 years. Paleoecological results indicate that patterns of diatom assemblage change are not uniform lake wide. Southern cores contain evidence of nutrient enrichment starting as early as ca. 1940, indicated by increased silica, carbon and nitrogen burial. By ca. 1970, increased rates of sedimentation, diatom influx and changes in diatom community composition, characterized by increased percent abundance of eutrophic diatom taxa, are attributable to accelerated enrichment by terrestrial soil erosion. The succession of diatoms in southern Lake Malawi begins with high percent abundance of Aulacoseira nyassensis and Fragilaria africana, which thrive in nutrient-rich waters, followed by a shift towards diatom taxa with reduced silica requirements by ca. 1980 (e. g. Stephanodiscus nyassae, S. minutulus, S. muelleri, Cyclostephanos and small Nitzschia species. ), a pattern comparable to the eutrophication-induced decline in silica to phosphorus ratios in Lake Victoria. In Lake Malawi, evidence of eutrophication extends to the mid lake as indicated by similar diatom assemblage changes in the sediment core from the central region. Diatom stratigraphies from the north end of the lake indicate no observable impacts of land use change on the northern basin of Lake Malawi during the past 350 years. However, a nine-meter rise in water level ca. 1860 AD appears to have resulted in elevated diatom influxes at that time comparable to the recent eutrophication-induced diatom influxes of the southern cores. The effects of this rise in water level was recorded in all three measured sites, southern, central and northern Lake Malawi, indicating lake-wide increased productivity, yet changes to the diatom community composition were imperceptible. This study shows evidence of recent cultural eutrophication altering limnological conditions with impacts to the biogeochemical cycling of silica, the available silica to phosphorus ratios and the biotic communities of a large portion of Lake Malawi. Thus, providing an early warning that proper stewardship of Lake Malawi requires effective management of land-use practices within the catchment to reduce soil erosion and avoid widespread water quality deterioration of this great lake.
An Assessment of Hydro-ecological Changes at Two Closed-drainage Basins in the Peace-Athabasca Delta, Alberta, Canada
(University of Waterloo, 2006) Sinnatamby, Ramila Niloshini
Diatom analyses were carried out on sediment cores collected from two low-lying, closed-drainage basins (PAD 9 - 58º46. 46?N, 111º19. 48?W; PAD 12 - 58º57. 29?, 111º19. 74?) in the Peace sector of the Peace-Athabasca Delta (PAD), Alberta, Canada, to provide >1000 year long records of hydro-ecological change. Results from diatom analyses were compared with macrofossil and stable isotope records from the same cores and assessed within the framework of an Athabasca River headwater climate record inferred from isotope dendroclimate data. Results from PAD 9 and PAD 12 sediment cores indicated closed-drainage conditions during the Medieval Warm Period (MWP) and the post-Little Ice Age and high water conditions during the Little Ice Age (LIA). High water levels at PAD 9 and PAD 12 reflected high water conditions on Lake Athabasca and the Rivière des Rochers or possibly the Peace River during the LIA (~AD 1600-1900). High water conditions were also observed at low-lying sites in the central and southern regions of the PAD (PAD 31 and PAD 37), and corresponded with evidence of high streamflows on the North Saskatchewan River. In contrast, desiccation evident at PAD 5, a site largely isolated from river influence, reflected atmospherically dry conditions during the LIA. Consistent with changes observed at PAD 5, sediment records at PAD 15, an oxbow lake off the Revillion Coupé, demonstrated low flood frequency during the early to mid-1700s. Increased water levels evident at low-lying sites located in proximity to the central open-drainage network of lakes and rivers were likely due to higher flows on the Athabasca River and potentially on the Peace River. High flows on rivers of the PAD may be attributed to snowmelt-dominated runoff during the LIA relative to the rainfall-dominated runoff during MWP (prior to ~AD 1600) and the post-LIA period (~AD 1900 to present).
Obesity associated colon tumorigenesis: An assessment of tumor phenotype
(University of Waterloo, 2006) Saxena, Swati
Colon cancer and obesity are two significant and related pathological states with multiple etiological factors. In this dissertation, it was hypothesized that tumor growth is accelerated in the altered state of obesity due to their resistance towards tumor necrosis factor-alpha (TNF-alpha) mediated cytotoxicity. Physiologically elevated TNF-alpha in an obese state induces increased nuclear transcription factor-kB (NF-kB) activity, known to transcribe genes crucial to cell survival. Insulin resistance, oxidative stress, and a pro-inflammatory environment are few of the biological consequences of TNF-alpha and NF-kB pathway activation, and further contribute to disease progression.

Three major studies were conducted to investigate phenotypical changes in obesity associated tumors. Firstly, characteristics of the TNF-alpha resistant phenotype were preliminarily assessed by evaluating the effects of exogenous TNF-alpha treatment to HT-29 cells. Elevated levels of NF-kB in response to exogenous TNF-alpha gave an indication that this pathway is critical for cell survival. Furthermore, upregulation of TNF-alpha receptor 2 (TNFR2) suggested another strategy by which the cells were utilizing exogenous TNF-alpha for a survival advantage. Inhibition of NF-kB via St. John?s Wort treatment demonstrated that HT-29 cells may be sensitized towards TNF-alpha mediated cytotoxicity.

Zucker obese (Zk-Ob), Zucker lean (Zk-Ln), and Sprague Dawley (SD) animal models were used to assess tumor phenotype in vivo. Remarkable physiological differences between genotypes were observed. Zk-Ob rats had significantly higher body and organ weights as well as plasma TNF- alpha, insulin, leptin, and oxidative markers than Zk-Ln and SD animals. Tumor incidence and multiplicity were also notably higher in Zk-Ob rats. Protein analyses demonstrated increased levels of TNF-alpha, TNFR2, NF-kB, IkB kinase beta (IKKbeta), insulin receptor (IR), insulin like growth factor-I-receptor (IGF-IR), and mitogen activated protein kinase (MAPK) in Zk-Ob tumors than Zk-Ln counterparts. In all groups, tumors generally had higher protein expression than surrounding, normal appearing colonic mucosa. It is well known that these molecules are involved in signaling pathways that influence and co-operate with each other in rendering growth autonomy to tumor tissue.

A higher number of lesions in the distal than proximal colon in Zk-Ob rats was observed, supporting the emerging concept that genotype/physiological state of the host affects development and distribution of tumors. Thus, a third study was conducted to explore differences between distal and proximal tumor phenotype. Results demonstrated that expression of TNFR2, NF-kB, IR, IGF-IR, and MAPK p44 were significantly higher in distal than proximal tumors. This observation suggested that development of tumors in different regions of the colon varied under the same physiological conditions. Moreover, phenotype of distal tumors appeared to be upregulating survival pathways in comparison to proximal lesions, possibly explaining the higher tumor incidence in the distal colon.

Research documented in this thesis supported the hypothesis that the physiological status of the host intricately affects tumor phenotype. In particular, the TNF-alpha resistant phenotype was most prominent in Zk-Ob tumors, and appeared to be associated with upregulation of multiple signaling pathways cooperating towards tumorigenesis.
Chemical Composition of Soybean Root Epidermal Cell Walls
(University of Waterloo, 2006) Fang, Xingxiao
The root epidermis, being the outermost cell layer of the organ, is in contact with the soil environment. The position of the epidermis determines its important roles, such as taking up water and ions from the surrounding soil, and defending against harmful microorganisms. What is the chemical composition of the walls in this layer? The chemical nature of the soybean epidermal wall modifying substance was investigated in this study with the use of histochemical tests coupled with electron microscopy, and chemical depolymerizations in combination with chromatography. Soybean (Glycine max) was used as a test species in the present studay. Results of histochemical and electron microscopical studies indicated that the epidermal walls are modified with suberin. The suberized epidermal walls were permeable to apoplastic tracers, differing from those of cells with suberized Casparian bands, possibly due to the spatial distribution or chemical components of the suberin. Suberin may occur in a diffuse form linked with other wall components in the epidermis. What is the chemical nature of this modification, and does it play a role in pathogen resistance? The root epidermal wall compositions of two soybean cultivars were compared; one (cv. Conrad) is resistant to Phytophthora sojae and the other (cv. OX 760-6) is susceptible to this root-rot oomycete. Their epidermal walls were isolated enzymatically and subjected to two different degradation methods, i. e. BF₃-MeOH transesterification and nitrobenzene oxidation. The compositions of depolymerisates of the cell walls determined by GC-MS indicated four dominant suberin monomers varying in chain length from C16 to C24. In all epidermal cell walls, ω-hydroxycarboxylic acids were more abundant than diacids, carboxylic acids and alcohols. Two of the monomers detected (hydroxycarboxylic acid and a,ω-dicarboxylic acid) are known to be characteristic suberin markers. The quantitative chemical compositions significantly differed in the epidermal cell walls of the two soybean varieties. Walls of the resistant cultivar (Conrad) had a greater quantity of both the aliphatic and aromatic components of the polymer than the susceptible cultivar (OX760-6), providing evidence to support the hypothesis that preformed suberin plays a role in plant defense.
Characterization of Genes involved In Development and Senescence
(University of Waterloo, 2006) Hopkins (nee Kaup), Marianne
Plant development is complex and highly regulated. Tens of thousands of genes have been sequenced for the model plant Arabidopsis thaliana, yet few have been functionally annotated and characterized. This thesis describes the expression analysis and characterization of four genes in Arabidopsis. Three of these belong to the eukaryotic translation initiation factor 5A (eIF5A) gene family, and the fourth encodes diacylglycerol acyltransferase 1 (DGAT1). Putative roles for these genes in the development of Arabidopsis thaliana are described.

eIF5A is the only known protein to contain the amino acid hypusine. It has been demonstrated previously that eIF5A acts as a shuttle protein, moving specific mRNAs from the nucleus to the cytoplasm for translation. In Arabidopsis thaliana (At), there are three isoforms of eIF5A, and it is clear from the present study that they each have a unique temporal and spatial expression pattern. AteIF5A-1 and -2 are up-regulated during natural senescence and wounding/pathogenesis, respectively, and it is proposed that they regulate the onset of programmed cell death during these events. AteIF5A-3 is up-regulated in elongating meristem of the root, and it is proposed that this isoform is involved in cell growth.

Over-expression of the individual AteIF5A isoforms in planta resulted in pleiotropic phenotypes. When AteIF5A-1 or AteIF5A-2 was over-expressed, the phenotypes observed were indicative of their putative roles in the translation of proteins required for programmed cell death. When AteIF5A-3 was over-expressed, the phenotypes were indicative of a role for this protein in the regulation of cell and tissue elongation.

Lipid analysis of rosette leaves from Arabidopsis thaliana revealed an accumulation of triacylglycerol with advancing leaf senescence coincident with an increase in the abundance and size of plastoglobuli. The terminal step in the biosynthesis of triacylglycerol in Arabidopsis is catalyzed by DGAT1. When gel blots of RNA isolated from rosette leaves at various stages of development were probed with the Arabidopsis EST clone, E6B2T7, which has been annotated as DGAT1, a steep increase in DGAT1 transcript levels was evident in the senescing leaves coincident with the accumulation of triacylglycerol. The increase in DGAT1 transcript correlated temporally with enhanced levels of DGAT1 protein detected immunologically. Two lines of evidence indicated that the triacylglycerol of senescing leaves is synthesized in chloroplasts and sequesters fatty acids released from the catabolism of thylakoid galactolipids. First, triacylglycerol isolated from senescing leaves proved to be enriched in hexadecatrienoic acid (16:3) and linolenic acid (18:3), which are normally present in thylakoid galactolipids. Second, DGAT1 protein in senescing leaves was found to be associated with chloroplast membranes. These findings collectively indicate that DGAT1 plays a role in senescence by sequestering fatty acids de-esterified from galactolipids into triacylglycerol.
Molecular characterization of potential geosmin-producing cyanobacteria from Lake Ontario
(University of Waterloo, 2006) Gill, Andrea
Geosmin is an odorous secondary metabolite produced by some cyanobacteria during growth and released from the cells. Little is known about the biosynthesis of geosmin and the gene(s) required for its production have not been characterized. During late August and early September geosmin episodes due to planktonic cyanobacteria frequently occur in the northwest basin of Lake Ontario waters resulting in taste and odour episodes in drinking water that serves more than 5 million people. At high concentrations geosmin evades traditional drinking water treatment and reaches the tap. These episodes often elicit consumer concern and are wrongly construed to reflect impaired drinking water safety. Water quality managers in the region have generally been unable to prevent or control taste and odour episodes via a proactive approach due to the lack of knowledge of cyanobacterial communities in offshore waters as well as the inability to predict when geosmin will reach intake pipes due to downwelling, the process by which the surface waters mix with the hypolimnion. This study evaluated denaturing gradient gel electrophoresis (DGGE) as a molecular tool for proactive monitoring of potential taste and odour-causing cyanobacteria in environmental samples. The 16S rRNA gene was assessed for its ability to distinguish among geosmin-producing and non-producing strains. This study also examined the evolutionary relationships among geosmin-producing cyanobacteria using the full-length 16S rRNA gene and compared phylogenies with current taxonomy.

A DGGE standard using the V3 hypervariable region of the 16S rRNA gene was developed using geosmin-producing and non-producing isolates of cyanobacteria. Included in the standard was the suspected primary contributor to Lake Ontario taste and odour, Anabaena lemmermannii Richter. This standard was then applied to various environmental collections from Lake Ontario (August 2005) to examine the cyanobacterial community composition. DGGE profiles were consistent with the presence of An. lemmermannii at locations with increased geosmin concentrations (determined using gas chromatography-mass spectrometry), supporting hypothesis that An. lemmermannii is the primary contributor to northwestern Lake Ontario taste and odour. In addition, the application of DGGE in the identification of potential geosmin-producing species of cyanobacteria was deemed to be a potentially useful approach to monitoring cyanobacterial communities in source waters. The 16S rRNA-V3 region alone did not distinguish among geosmin-producing and non-producing strains, however with additional data (actual geosmin concentration) it was showed relationships.

In the phylogenetic analyses, geosmin-producing cyanobacteria did not group monophyletically and it was not possible to state that a single evolutionary event has led to the acquisition of the geosmin-producing trait. Phylogenies also showed that the taxonomy of the Cyanobacteria is largely unresolved. All five Sections (bacteriological classification)/four orders (Komárek & Anagnostidis classification) were paraphyletic, however the heterocystous cyanobacteria (Sections IV and V/Nostocales and Stigonematales) grouped separately from the non-heterocystous cyanobacteria (Sections I, III/Chroococcales and Oscillatoriales). Although both systems of classification compared in this study were similar, nomenclature and groupings were occasionally different among the groups. This demonstrates the incongruity between bacteriologists and phycologists and emphasizes the need for a consensus system of classification for the Cyanobacteria.
Molecular Phylogeography and Species Discrimination of Freshwater Cladophora (Cladophorales, Chlorophyta) in North America
(University of Waterloo, 2006) Ross, Sara J.
Cladophora is a widespread freshwater filamentous cholorophyte genus and is frequently observed in eutrophic waters where it can produce large nuisance blooms. These blooms can have direct impacts on water intake for power generation, irrigation canals and can be aesthetically unpleasant. Much of the ecological and physiological studies on Cladophora have assumed that the populations of this genus in North America belong to the species Cladophora glomerata. However, this has never been tested despite that it is well documented that identifying freshwater Cladophora to the species level is difficult due morphological variability under different ecological conditions. In addition, the species epithets for freshwater Cladophora are based on European collections and it is not clear if these should be applied to North America. This study examines approximately 40 collections of Cladophora from the Laurentian Great Lakes and 43 from various locations in North America ranging from the Northwest Territories to Puerto Rico. Initially we determined the nucleotide sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal cistron and observed sequence divergence to be low (0-3%), demonstrating an inability for this marker to resolve species delineation as divergence of this region was low. Amplification of the inter-simple sequence repeat (ISSR) regions were used to analyze microsatellite motif frequency throughout the genome to evaluate the biogeography relationships, including diversity, of freshwater Cladophora sp. five different primers were used on 70 individuals. UPGMA analyses of the presence/absence of bands demonstrate that each of the Great Lake populations separate into groups according to the Lake they were initially sampled from. However, collections from North America are highly variable and do not form well supported biogeographic clades. In addition, these collections appear to be distinct from type cultures of freshwater Cladophora from Europe. Supplementary morphological analysis using suggested taxonomically valid criterion (length and diameter of main axis, ultimate branch, and apical cell) none were able to differentiate Great Lake populations.
Determining food web impacts on experimental aquatic systems from the disposal of oil sands process-affected waste materials.
(University of Waterloo, 2006) Elshayeb, Monalisa
Current mining operators in the Athabasca oil sands deposit of Alberta, Canada have made commitments to zero discharge of oil sands process-affected waste materials (OSPM) from the mine site and rehabilitation of mined lands to a pre-mining state. As part of aquatic reclamation efforts, experimental test sites that contain a range of OSPM (solid and liquid components) were constructed to monitor the evolution and viability of aquatic habitats used as disposal sinks for OSPM produced by mining activities. In the present study, stable isotopes of carbon, nitrogen and sulphur were used to gauge some of the potential effects of OSPM site construction methods on aquatic food webs. Carbon and nitrogen isotopic signatures of sediment, dissolved inorganic carbon, dissolved organic carbon, particulate organic matter, periphytic material, plants, plankton, aquatic invertebrates and fish were used to assess differences related to the naphthenic acid (NA) concentration in OSPM and reference sites. For statistical analyses, sites were grouped into low (0 to 4 mg/L), medium (4 to 15 mg/L) and high (> 15 mg/L) NA concentrations. There were no significant differences in food web area or food web length among the low, medium and high NA concentration sites. In most cases, sample carbon isotope analyses of low, medium and high NA concentration sites were not significantly different, suggesting food web carbon sources did not include significant contributions from OSPM materials at OSPM sites. Significant differences, however, were found in the sample nitrogen isotope signatures between low, medium and high NA concentration sites. Ammonia from OSPM is suggested to be the main contributor to δ¹⁵N enrichment.

To determine the potential effects of site construction and OSPM within experimental test sites, carbon and sulphur stable isotopes of water, plankton, aquatic invertebrates and fish were analyzed. With the exception of Chaoborus and Haliplus, all carbon isotope signatures were not significantly different in constructed and reference sites. Also with the exception of Haliplus, sulphur isotope values for aquatic organisms from constructed and reference sites were significantly different. Aquatic organisms and water samples from constructed sites built in, or close, to the boundary of Kcw clays typically had δ³⁴S < 0 ?. Coinciding with depleted δ³⁴S signatures found in these aquatic systems were elevated sulphate concentrations. The waters at experimental test sites are in direct contact with the soil materials that facilitate the accumulation of sulphates as a result of the oxidation of substrate sulphide minerals. In general the results of the study suggest that aquatic food web structure and function do not change with the introduction of OSPM. Shifts in isotopic signatures suggestive of changes in food web structure, however, do occur when site construction exposes Kcw clays in the substrate.
A study of innate antiviral mechanisms using fish cell lines
(University of Waterloo, 2006) DeWitte-Orr, Stephanie
Understanding basic antiviral mechanisms in vertebrates is essential for developing methods to enhance antiviral responses and promote human and animal health. In fish these antiviral mechanisms are poorly understood, but are important to understand because of the devastating impact of viral diseases on aquaculture. Therefore, the antiviral responses of a rainbow trout macrophage-like cell line, RTS11, and two non-immune cell lines, the rainbow trout fibroblast RTG-2 and Chinook salmon embryo CHSE-214 were studied. Three antiviral responses were first characterized using the viral mimic, synthetic double-stranded RNA (poly IC), and then their induction was investigated using Chum salmon reovirus (CSV). The responses were: 1) apoptosis, which is programmed cell death and a primitive antiviral defense; 2) homotypic aggregation (HA), which is clustering of like immune cells; and 3) expression of Mxs, which are antiviral proteins belonging to GTPase super-family. Some of these antiviral mechanisms were investigated using a novel continuous cell line, PBLE, developed from a peripheral blood leukocyte preparation of the American eel, Anguilla rostrata.

RTS11 was exceptionally susceptible to apoptosis. The cells died at lower concentrations of poly IC and other agents, including the translation inhibitor, cycloheximide (CHX), and fungal metabolite, gliotoxin. Death was predominantly by apoptosis, as judged by DNA ladders, nuclear fragmentation, and protection by caspase inhibitors. By contrast, the other two cell lines died most commonly by necrosis, when death did occur. Co-treating RTS11 with CHX greatly sensitized the cells to poly IC. Based on the protection afforded by inhibitors of dsRNA-dependent protein kinase (PKR), RTS11 apoptosis induced by poly IC with CHX co-treatment but not gliotoxin was mediated by PKR. As macrophages are likely among the first cells to contact viruses during an infection in vivo and are mobile, the sensitivity of RTS11 to dsRNA killing could reflect a protective mechanism by which virus spread is limited by the early death of these first responders.

HA of RTS11 was induced by poly IC. HA required divalent cations and was blocked by CHX and by PKR inhibitors. This suggested that HA induction was PKR-mediated and involved the synthesis of new cell surface molecule(s), possibly galectins. As an antiviral mechanism, HA induction by dsRNA could be interpreted as an initial protective response, allowing cell localization at the site of infection, but once translation becomes inhibited, apoptosis ensues.

Mx was induced by poly IC in RTS11 and RTG-2 as judged by RT-PCR. Western blotting revealed constitutive Mx expression more consistantly in RTS11, but induction by poly IC in both cell lines. Medium conditioned by cells previously exposed to poly IC and assumed to contain interferon also induced Mx transcripts in RTS11 but not RTG-2. In RTS11, poly IC activated PKR activity, and PKR inhibitors blocked Mx induction, which is the first demonstration of PKR mediating Mx expression.

The dsRNA virus, CSV, also induced apoptosis, HA, and Mx expression, but in some cases contrasting with poly IC experiments. CSV induced apoptosis in RTG-2 and CHSE-214 but not in RTS11, and HA induction by CSV in RTS11 was not dependent on PKR. Mx induction was sustained in RTG-2 and transitory in RTS11; however, both cell lines supported CSV replication.

The novel cell line, PBLE, was also characterized in this study. PBLE was derived from an adherent culture of peripheral blood leukocytes from the American eel, Anguilla rostrata. PBLE were found to grow over a wide range of temperatures and fetal bovine serum (FBS) concentrations. This cell line was able to undergo apoptosis in response to gliotoxin. PBLE was also susceptible to a number of viruses, including CSV; however, CSV infection did not lead to apoptosis.

This study suggests that antiviral responses are likely numerous and overlapping and depend on cell type and virus. Understanding them should lead to novel methods for protecting fish from viral diseases. More specifically, using cell lines such as PBLE may aid in the understanding of species specific and perhaps even cell type specific antiviral mechanisms.

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