Theses (Biology)

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Theses from the Dept. of Biology.

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Impacts of heatwaves and hypoxia on gene expression in the pacific oyster and the development of monitoring and mitigation tools for summer mortality
(2025) Bickell, Andrew; Pearce, Christopher Michael; Bates, Amanda
Marine heatwaves and coastal hypoxic events are increasing in frequency and intensity under anthropogenic climate change, resulting in widespread mass mortalities of the Pacific oyster (Crassostrea gigas). Those mortality events threaten the economic stability of global aquaculture, yet strategies to monitor oyster health and mitigate losses during periods of environmental stress are largely limited. Changes in gene expression of C. gigas in response to laboratory-simulated heatwaves and hypoxic events were assessed to identify candidate monitoring genes and explore artificial aeration as a potential mortality mitigation strategy. Two laboratory experiments were performed, exposing farmed C. gigas to simulated 10-day heatwave and hypoxic conditions similar to a 2021 marine heatwave that triggered farmed oyster mortality in Baynes Sound, British Columbia, Canada. Gill tissues were periodically sampled during the experiments and total RNA was extracted to explore patterns of gene expression via RNAseq and qPCR. Five candidate genes were consistently differentially expressed in both experiments— death-associated inhibitor of apoptosis 2 (A2I), high mobility group protein DSP1 (DSP1), high mobility group box 1 (HMGB1), heat shock protein 90 (HSP90), and peptidyl-prolyl cis-trans isomerase (PPCTI)—demonstrating potential for monitoring summer mortality. No significant differences in expression of the general stress marker genes heat shock protein 70 (HSP70) and heat shock protein 20 (HSP20) were detected, suggesting that genes related to immune function and regulation of transcription may be more appropriate for monitoring summer mortality. In addition, the presence of artificial aeration resulted in significantly lower HSP90 relative expression, suggesting some potential utility in stress mitigation during heatwaves. The present work provides insights into the role of heatwaves and hypoxia in Pacific oyster summer mortality and will inform effective monitoring and mitigation practices to support the adaptation of shellfish aquaculture to the growing impacts of climate change.
Monitoring deep-sea MPAs: Functional and trait-based approaches for adaptive management in changing oceans
(2025) Davies, Megan A.; Bates, Amanda; Du Preez, Cherisse
Changing ocean conditions are disrupting marine ecosystems, posing significant challenges for monitoring and managing biodiversity in remote, offshore marine protected areas (MPAs). Effective conservation depends on biological assessment tools that can overcome the logistical constraints of deep-sea monitoring while detecting long-term ecological changes. This thesis evaluates two possible approaches for assessing and monitoring species in offshore MPAs. First, I assess the feasibility of a functional-group monitoring approach, using cold-water corals and sponges (CWCS) in the Northeast Pacific as a case study. I analyze inter- and intra-seamount variability in depth-occupancy patterns using data extracted from remotely operated vehicle (ROV) transects performed by Fisheries and Oceans Canada (Chapter 2). Additionally, I identify potential indicator species that could be used to streamline long-term monitoring efforts. Second, I develop a species-level vulnerability framework using functional traits, with molluscs in the Azores Marine Park as a case study. I conduct a comprehensive literature review to determine which traits are most relevant for assessing vulnerability to climate change. Using species trait data from the FUN Azores Trait Database, I integrate oceanographic models to quantify species-specific exposure, sensitivity, and adaptive capacity to ocean acidification and warming (Chapter 3). My findings show that while functional groups capture broad CWCS distribution patterns, species-level assessments remain necessary for detecting ecological changes and refining monitoring strategies in the NE Pacific. In the Azores, I find that bivalves in northern MPAs are particularly vulnerable due to their high sensitivity and low adaptive capacity, while cephalopods exhibit greater resilience to climate change. Together, these studies highlight the strengths and limitations of biological assessment tools for long-term deep-sea MPA monitoring, offering insights into their role in adaptive conservation strategies. By integrating functional-group and trait-based approaches, this thesis contributes to a more adaptive and effective conservation framework for managing marine species in a changing ocean.
Quantification of melanopsin gene expression during sablefish (Anoplopoma fimbria) development and spatial mapping of Opn4m proteins on a newly established brain atlas
(2025) Mokariasl, Niloufar; Taylor, John Stewart
In this research, I investigated opsins in sablefish (Anoplopoma fimbria). Sablefish is a fast-growing teleost species that experiences a unique developmental environment, traveling to the surface from a kilometer deep in the ocean across embryonic and larval stages. Teleost fish, in particular, have a high number of opsins due to the third genome duplication event and the retention of opsins after duplication. I uncovered 38 opsins in sablefish despite the fact that they spend most of their life in the ocean’s aphotic zone. I focused on melanopsin/Opn4, an evolutionarily conserved subfamily of non-visual opsins, with a well-established role in the maintenance of circadian rhythm in mammals. Despite this well-researched role, melanopsin shows expression in a diversity of cell and tissue types, suggesting the possibility of other functional roles. I examined these genes across four distinct life stages using qPCR and identified gene expression in whole larva at 30 days post-fertilization (dpf), in the larval brain at 82 dpf, and in the brain, eye, and heart of juveniles, as well as in the brain and heart of adults. Using fluorescent immunolabeling on sablefish optic tectum sections, I localized Opn4m proteins for the first time in the endfeet of radial glial cells and likely in neuroepithelial cells within the juvenile optic tectum. The immunolabeling data on juvenile sablefish eye also revealed the expression of these proteins in all layers of the retina, including outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), and ganglion cell layer (GCL). Using qPCR, I have also quantified the expression of these genes throughout sablefish development from unfertilized eggs through all embryo stages and to the oldest larval stage. Furthermore, I examined the localization of Opn4m protein(s) in brain and eye of the four larval stages (18, 29, 36, and 47 dpf) to identify the cells that express these proteins. In this study, we report changes in expression of five melanopsin genes over embryonic and larval development, from unfertilized egg to 47 dpf. Using fluorescent immunolabeling and immunogold labeling, we also report for the first time, the expression of Opn4m proteins in proliferative neural cells, radial glial cells, and potentially astroglia in the brain over larval development. Unsurprisingly, we also found these proteins expressed in amacrine, bipolar, horizontal, and photoreceptor cells in the retina of 47 dpf sablefish, a similar pattern of expression to that observed in zebrafish larvae, sablefish juveniles and adult zebrafish. While melanopsin is best known for expression in intrinsically photosensitive retinal ganglion cells (ipRGCs) and for its role in circadian rhythm entrainment, an often-neglected observation is that when first described, melanopsin was detected in X. laevis retinal photoreceptors. It has since been found in photoreceptors of reptiles and ray-finned fish. In zebrafish (Danio rerio) an immunoprobe that binds to three of the five Opn4 paralogs showed a ring-like pattern of expression in cone cell inner-segments (Davies et al., 2011). No explanation for this was offered. I revisited this unusual pattern of expression using transmission electron microscopy and we looked for evidence of melanopsin expression in photoreceptors of a distant relative, the sablefish (Anoplopoma fimbria). Immunogold labeling was observed in outer segments and in ellipsoid megamitochondria of cone cells in both species Additionally I created the brain atlas for early sablefish brain development to guide my immunolabeling findings in larval stages. Using hemotaxylin and eosin staining (H&E) and ultramicrotomy, I have provided a developmental series of sablefish eye and brain sections, and created the partial brain atlas. The eye and brain atlas reported here includes three- and four-micron sections (942 in total) from 18, 29, 36, and 47 days dpf sablefish. Sections start at the olfactory bulb and run to the posterior medulla. Brain atlases with similar resolution are available for only a few other fishes, including zebrafish (Danio rerio) and medaka (Oryzias latipes). This resource is developed to provide a histological perspective on opsin gene expression data, but also makes an important contribution to the understanding of neurodevelopment in ray-finned fish. Sablefish take 47 days to reach the same stage of eye and brain development as 5 dpf zebrafish. Apart from this difference in rate, development is similar to both zebrafish and medaka, despite large differences in life history, morphology and behavior.
Using iron deficiency stress in plants as a tool for discovery of novel iron chelators for human use
(2024) Lane, Sarah; Ehlting, Jürgen; Walter, Patrick B
Iron is an essential micronutrient for life, but has harmful effects when dysregulated, especially when this leads to its accumulation in the body. Iron overload in humans contributes to the pathology of diseases like thalassemia, cancer and Parkinson’s Disease (PD). Treatments for iron overload can include chelation therapy to bind iron and remove it from tissues, but options authorized for clinical use are limited and can cause severe side effects. Plants are a good source of specialized, bioactive metabolites, and may be an untapped resource for novel iron chelators, as they produce these in roots to acquire environmental iron. This research uses a directed screening approach to find new chelators, taking advantage of plant biology by reducing iron in the plant growth environment to stimulate the production of iron-related metabolites, then testing them in a cell culture model of systemic iron overload. Of three species explored in this research, Populus trichocarpa x P. deltoides (poplar) was shown to be the most promising candidate for iron chelator production. It had a widespread response to iron reduction, including increased production of sideretin, a known iron chelator, and trichocarpin, a salicinoid not previously associated with iron deficiencies. In phytochemical screening, it was mainly non-toxic and had dose-dependent antioxidant activity in unstressed THP-1 monocytic cells. When used in a model of systemic iron overload, it did not significantly decrease intracellular iron in these cells, but biological variation in source plants had a considerable effect on bioactivity. When a component of poplar root extract, chlorogenic acid, was used instead, it exerted a significant dose-dependent reduction of intracellular iron content. Modeling brain iron overload and chelation therapy in vitro is more challenging, and the final component of this research was to explore induced pluripotent stem cells as dopaminergic neurons in monolayer and 3D cell constructs for future modelling of chelation therapy. Patient-derived iPSC were successfully established in 3D culture as a stepping stone to investigating the use of iron chelators in 3D printed tissues. This work contributes to our understanding of iron acquisition strategies in plants, and presents novel ways to discover new therapeutic agents through plant-biology directed screening and innovative technology.
Nutrient physiology of siliceous phytoplankton under warming and acidification in Arctic and subtropical oceans
(2024) Wyatt, Shea; Varela, Diana Esther
Steadily rising atmospheric CO2 concentrations have the potential to impact marine ecosystems by increasing the temperature and acidity of the world’s oceans. In the sunlit upper ocean, phytoplankton affect elemental cycling and contribute to nutrient export to deeper waters by incorporating nutrients into biomass and supporting higher tropic levels. One unique group of phytoplankton, diatoms, are characterized by their typically larger size and heavy silica frustules, and they make considerable contributions to global primary productivity. Diatoms are expected to be impacted by oceanic change in various ways, but the degree of this effect is still uncertain. The overall objective of this thesis is to improve our understanding of how marine diatom physiology, specifically the utilization of silicon (Si), and the contribution by diatoms to the cycles of carbon (C) and nitrogen (N), are affected by climate-induced increases in temperature and acidification. I investigated the impact of mesoscale physical processes on diatom contributions to utilization rates of C (ρC) and nitrate (ρNO3) in the Sargasso Sea in the North Atlantic subtropical gyre, an ecosystem impacted by increased stratification due to ocean warming. Diatoms played a minor role in nutrient utilization and biomass during the lowest-productivity time of year, but they dominated nutrient utilization rates in the deeper euphotic zone of the Sargasso Sea when nutrient concentrations were enhanced by eddy-driven upwelling. In the contrasting environment of the Bering and Chukchi Seas, I investigated the effects of a warming ocean on diatom physiology and elemental composition as part of an on-going oceanographic time-series in the Pacific Arctic Region (PAR). I found significant trends in ocean temperature and sea ice breakup dates for different regions of the PAR, and evidence for declining diatom biomass in one area of the northern Bering Sea. Anomalously low particulate C:N values were observed across the PAR during the 2019 MHW, but otherwise the response of diatom assemblages in the PAR to a sustained warming period and marine heatwave (MHW) in 2019 varied substantially. Estimates of diatom contributions to ρC and ρNO3 in the PAR were improved compared to previous studies, demonstrating that diatoms were responsible for most of the nutrient utilization in all regions. Ocean acidification experiments were conducted with a model diatom species and two natural phytoplankton assemblages to assess the effects of decreased pH on nutrient physiology. Overall, diatom Si utilization and silicification in laboratory and field culture experiments were unaffected by pH. I found that the cell size of a model species of diatom, Thalassiosira rotula, decreased under OA, while in subtropical and Arctic phytoplankton assemblages, OA had no conclusive meaningful impacts on other measures of physiology, or assemblage composition. This dissertation provides valuable insights into how siliceous phytoplankton, particularly diatoms, interact with marine cycles of Si, C, and N across cold and warm marine ecosystems. It also deepens our understanding of how these dynamic systems may respond to oceanic change, and sets the stage for future research on the evolving impacts of climate-driven physical processes.
AMPA and NMDA receptor localization and recruitment in a central circuit of the mammalian retina
(2024) Chundekkad, Pavitra; Awatramani, Gautam
Retinal direction-selective ganglion cells (DSGC) have the ability to encode direction of moving objects over a wide range of contrasts. In part, the DSGC response is shaped by graded excitatory information mediated by glutamatergic bipolar cells, via specialized ribbon synapses. Glutamate signals to DSGCs are known to be mediated via two types of receptors: AMPA and NMDA receptors (AMPARs & NMDARs, respectively). The main aim of my thesis was to investigate how AMPARs and NMDARs are distributed at the sub-cellular and synaptic levels, in an effort to understand how DSGCs encode responses over a range of conditions. My research employed patch-clamp electrophysiology and pharmacological techniques to examine the voltage-dependence and contrast-dependence of glutamate input to DSGCs arising from presynaptic bipolar cells. First, I observed that the proportion of AMPARs and NMDARs across the DSGC dendritic arbor appeared constant when probed with small light spots. Interestingly, however, NMDARs and AMPARs were recruited over distinct contrast ranges. NMDARs primarily mediated responses to low contrast stimuli, while AMPARs were recruited at higher contrast levels. This indicated that AMPARs and NMDARs do not necessarily reside within the same bipolar cell synapses. I also observed that NMDARs measured in pharmacological isolation could encode a wide range of stimulus contrast. Thus, the graded glutamate signals from bipolar cells appear to be effectively detected by NMDARs present on DSGCs. This was somewhat surprising because NMDARs are generally prone to saturation owing to their high affinity for glutamate. Furthermore, pharmacological investigation revealed that GluN2A and GluN2B subtypes of NMDARs contribute equally to the light-evoked responses, over the entire range of contrasts. Interestingly, currents triggered by spontaneous vesicle release from bipolar cells, which indicate the involvement of receptors directly opposite the release site, were almost entirely inhibited by the GluN2A antagonist. These findings point to a distinct localization of GluN2A and GluN2B in synaptic and extrasynaptic regions, respectively. My results suggest that NMDARs encode a large range of stimulus contrasts and are able to evade saturation by employing distinct bipolar cell types as well as incorporating a combination of GluN2A and GluN2B receptors positioned at specific distances from the vesicle release site. Together, my work provides novel insights into how AMPARs and NMDARs contribute to the ability of DSGCs to respond to diverse stimuli.
Introgression and subtle population structure in Copper and Quillback rockfishes
(2024) Sykes, Nathan; Owens, Gregory Lawrence
Genomic methods are increasingly being applied in fisheries to promote effective management and sustainability. Pacific rockfishes, genus Sebastes, inhabit inshore, shelf, and slope habitats along the North American west coast. Among these, Copper and Quillback rockfishes (abbreviated to Copper and Quillback) are closely related species known to hybridize, particularly within the Salish Sea in North America’s Pacific Northwest. Here, I investigate genetic population structure and introgression patterns in Copper and Quillback rockfishes from Alaska to California. Using low-coverage whole-genome resequencing (lcWGS) across a broad geographic range, I seek to (1) describe genetic differentiation between the rockfish species, (2) assess population structure within each species, (3) identify regions of the genome with unique patterns of differentiation between species, and (4) look for signatures of introgression in the genomes of both species. My analyses reveal that Copper exhibit higher levels of population differentiation compared to Quillback, especially between coastal and Salish Sea populations. In contrast, Quillback populations appear to be more panmictic, with lower overall differentiation. Several large haploblocks are found to be segregating between the species, with introgression patterns varying across genomic regions. These findings provide novel insights into the range-wide genetic structure of these species and highlight the role of genomic architecture in local introgression.
Improving our understanding of vertebrates in nutrient recycling and ecological stoichiometry
(2024) May, Emily M.; El-Sabaawi, Rana
Nutrient-rich, large, long-lived, and mobile, vertebrates may uniquely impact nutrient recycling and thus ecosystem function by storing copious nutrients within their bodies, by producing large quantities of waste, and by transporting nutrients long distances within and across ecosystems. Currently, vertebrates face myriad threats, leaving many ecosystems to face a future with reduced vertebrate biomass and diversity; understanding how vertebrates affect nutrient cycles thus provides valuable insight into how vertebrate extinctions will impact ecosystems. Broadly, I characterize variation in how vertebrates store phosphorus (P) within their bodies and recycle P from their diets in their excreted and egested wastes. To accomplish this, I link bone investment, bodily elemental content, and waste production. Bone, a P-rich tissue (mean of ~12%) unique to vertebrates, can alter vertebrate-driven nutrient recycling both by increasing P storage within vertebrate bodies and by changing P demand and thus P release in waste. Given this, my dissertation has three goals: (1) quantify how vertebrates vary in bone content and whole-body %P, (2) determine how this variation affects dietary intake and waste production, and (3) evaluate how bone may affect P demand differently from other P-rich bodily components. To fully characterize this, I used ecological stoichiometry (ES), a framework that uses mass-balance to conceptualize organismal and ecological processes, including nutrient uptake and processing by animals. I therefore begin my dissertation by developing methods to measure all factors effectively and simultaneously, which is rarely achieved in current literature. Using these methods, I then performed a field study to examine how intraspecific bone variation in the threespine stickleback (an evolutionary model fish species with well-characterized bone variation) affected whole-body %P, dietary %P, and P release in excreta and egesta. ES predicts that bone increases whole-body %P and should therefore increase P demand and decrease P release. Counter to these predictions, I found that although stickleback with more bone indeed had higher whole-body %P, bone did not clearly impact P demand. Stickleback with more bone showed similar diets, nutrient absorption, and egestion to those with less bone. Additionally, stickleback with more bone excreted more P, perhaps implying that bone mitigates P demand rather than causing it. More broadly, I found considerable variation in bone, whole-body %P, and waste production within and across sites, most of which was unexplained by size, sex, or environmental characteristics. Bone also causes interspecific and ontogenetic differences. In a systematic review of vertebrate elemental content, I found whole-body %P ranging from 0.39 – 6.91% P, suggesting strong bone variation across taxa (interspecific) and life stages (intraspecific). Given my previous findings, this variation may impact P demand and release in unexpected ways. Thus, I finish by integrating osteological and stoichiometric literature to examine how bone’s unique physiology affects its relationship with P demand and release. While ES implicitly assumes all tissues impact demand equally, bone’s unique characteristics change how it affects elemental demand. Primarily, bone’s ability to self-destroy and provide mineral nutrients to the rest of the body allows bone to directly mitigate a vertebrate’s increased P or calcium demands. This may make vertebrates with more bone more resilient to dietary P limitation, rather than the opposite. Ideally, future research will build on these findings to improve our overall understanding of vertebrate-driven nutrient recycling.
Molecular characterization of the Neodiprion abietis nucleopolyhedrovirus
(2002) Young, Aaron Michael
Baculoviruses are a large, diverse family of viruses that are pathogenic for Arthropods, particularly members or the Order Insecta. These viruses are comprised of large enveloped rod-shaped capsids that contain supercoiled, double-stranded DNA genomes. Baculoviruses are of particular interest because of their use as gene expression vectors and insect biological control agents. Most of the information currently available on baculoviruses comes from studies of the Autographa californica nucleopolyhedrovirus and closely related species. Very little information is available on baculoviruses from other insect Orders, including Hymenoptera. The balsam fir sawfly (Neodiprion abietis, Order:Hymenoptera) is a native insect species that is currently causing severe, annual damage to economically valuable balsam fir forests in eastern Canada. Due to environmental regulations, standard chemical pesticides cannot be used to control this pest and as a result, biological control agents have been proposed as a potential alternative. A nucleopolyhedrovirus (NPV) has recently been isolated from N abietis and has been shown to be able to control balsam fir sawfly population outbreaks in controlled field experiments, however, before the N abietis nucleopolyhedrovirus (NeabNPV) can be registered for use as a biological control agent, research must be conducted to characterize the virus. I have partially characterized NeabNPV at the nucleotide and protein sequence levels and have concluded that NeabNPV is highly diverged from other members of the Family Baculoviridae. DNA libraries were constructed for NeabNPV using sheared viral genome fragments and HindIII restriction fragments. An EcoRI restriction library was obtained from the Canadian Forestry Centre, Atlantic Division, and was also included in this study. Analysis of the restriction endonuclease profiles obtained for NeabNPV indicate that NeabNPV has a genome approximately 94.7 kilobase pairs in size. Nucleotide sequence data obtained from these DNA libraries was used to perform comparative sequence analyses and demonstrated that NeabNPV contains many putative baculovirus gene homologues. From the sequence data, four putative complete open reading frames (ORF) were predicted: odv-e56, vlf-1, p74, and polh. The putative polyhedrin gene homologue was amplified from the NeabNPV genome and sequenced. Analysis of the polyhedrin gene sequence showed that it was 738 nucleotides long and encoded a protein of a predicted molecular weight of 29 .5 kDa. The promoter region of the gene was determined to contain two copies of the baculovirus specific late transcription motif, TAAG. The NeabNPV polyhedrin gene is highly diverged from Lepidopteran NPVs and GVs (Granulovirus) and shares its highest sequence identity with the polyhedrin gene of the Hymenopteran baculovirus Neodiprion sertifer nucleopolyhedrovirus. Phylogenetic analyses of several putative NeabNPV gene homologues suggested that NeabNPV is highly diverged from the previously described Group I and Group II NPVs and appears in a unique evolutionary clade separate from both the Lepidopteran NPVs and GVs. Phylogenies which included the mosquito (Order: Diptera) baculovirus Culex nigripalpus nucleopolyhedrovirus (CuniNPV) and that used the GV s as a sister outgroup to the NPV s placed N eabNPV and CuniNPV into a unique evolutionary clade within the NPV group, separate from the Group I and Group II NPV s. I have also investigated NeabNPV at the protein level and have putatively identified the major nucleocapsid protein, VP39. Structural proteins from budded (BV) and occlusion-derived virions (ODY) were separated on polyacrylamide gels using SDS PAGE and several well-defined proteins were isolated and partially sequenced. The amino acid sequence data was used to perform comparative sequence analyses against our NeabNPV DNA libraries and determined that the genomic DNA library contained several putative overlapping partial vp39 sequences. These sequences were assembled to create an 864 nucleotide long, partial NeabNPV vp39 sequence. Analysis of the inferred amino acid sequence indicated that the NeabNPV VP39 protein shares the most identity to the GVs, but is most similar to the NPVs. The partial VP39 protein demonstrates a predicted secondary structure and hydrophilic profile that is conserved among the Baculoviridae.
Infectivity of Agrobacterium tumefaciens and proteins of crown gall tumors in Helianthus annuus
(1985) Wu, Hsiao-Chiang
A qualitative and quantitative study of the infectivity of Agrobacterium tumefaciens on sunflowers (Helianthus annuus) and other species (peas, beans and corn) was made by measuring the frequency of tumor induction in the greenhouse. According to the results, sunflowers and peas were sensitive to tumor induction . The effects of concentration of agrobacteria, and flowering stage or age of sunflower on tumor induction frequency were significant. After flowering, sunflower was resistant to tumor induction. This could be an aging effect. The effects of bacteria culture time after 2 days, and plant age before anthesis on tumor induction were not significant. But plant age may have had some effect on tumor development; the younger the plant, the more rapid the tumor development. The effect of inoculation position on the sunflower stem on tumor induction was not significant, but no tumors could be induced on leaves of any plants studied. The light and temperature differences of plant growth conditions between the greenhouse and growth chambers were significant on tumor development . In greenhouses, tumors developed more slowly than in growth chambers. No tumor was induced on maize or on two cultivars of beans (Phaseolus vulgaris cv. Improved Tendergreen and cv. Kentucky Wonder Green). Soluble proteins of crown gall tumors in Helianthus annuus were analyzed by methods of SDS-Polyacrylamide gel electrophoresis and spectrodensitometry. The chlorophyll-containing complexes were the same in tumor and control tissues. It was found that the migration distance of these complexes was negatively affected by the sample volume loaded. Sometimes a good separation of these complexes was not obtained at the same time as good separation of soluble proteins. A sample buffer with 5% SDS and 8 M urea was good for preventing aggregation of proteins from sunflowers which probably contained a high amount of interfering compounds , and increased the resolving power of SDS-PAGE.
Characterization of the multigene family encoding endopolygalacturonase in the basidiomycete Chondrostereum purpureum
(2001) Williams, Holly Louise
Chondrostereum purpureum is a white-rot basidiomycete fungus which is being developed as a biocontrol agent of hardwoods for use in reforestation sites and hydroelectric rights-of-ways. It produces several plant cell wall-degrading enzymes that are suspected virulence factors, including endopolygalacturonase (endoPG). To search for new endoPG genes in C. purpureum, degenerate oligonucleotide primers were designed based on conserved regions of seventeen ascomycete endoPG genes and two published endoPG sequences from C. purpureum. These primers were used to amplify endoPG gene fragments from C. purpureum 2128u genomic DNA. Fifty-six of the amplification products were analyzed by sequence determination or restriction digestion to reveal segments of three previously uncharacterized endoPG genes, as well as epgA, an endoPG gene sequenced from C. purpureum 2128u. The three new genes were named epgB, epgC, and epgD, and the full length sequences of two copies of epgB (epgB1 and epgB2) were retrieved from a genomic library of C. purpureum 2128u. The identities between the deduced polypeptide sequences for epgA , epgB1, epgC and epgD ranged from 61.8% to 80.0%. The two epgB genes shared 97.6% nucleotide identity and 97.1 % amino acid identity, with the majority of differences between them near the 3' ends of the genes. Phylogenetic analysis of these five basidiomycete endoPGs, seventeen ascomycete endoPGs, four ascomycete exoPGs, two bacterial endoPGs, and two bacterial exoPGs separated the C. purpureum endoPGs into their own monophyletic clade; whereas the endoPGs from ascomycete species having multiple endoPGs were divided between different monophyletic groups within the ascomycete endoPG clade. This split between the ascomycete and basidiomycete endoPGs suggests the two fungal phyla diverged before the duplication of existing endoPG genes. Mechanisms regulating the expression of the endoPG genes in C. purpureum were examined by northern analyses using mycelia grown in media with different carbohydrate sources. The epgA gene was expressed at high levels when glucose, sucrose or pectin was used as the carbon source; whereas both epgB genes, epgC, and epgD were expressed at low or non-detectable levels on all media types. The expression of epgA appeared to be constitutive and not repressed by simple sugars, suggesting EPGA may be the most important of the five endoPGs in the plant cell wall degradation caused by C. purpureum.
Some aspects of the reproductive biology of Fusitriton oregonensis (Redfield) (Gastropoda, Prosobranchia)
(1976) Williams, Daphne Eiri
Little is known of t he reproductive biology of the prosobranch Fusitriton oregonensis (Redfield). Therefore this thesis describes the morphology and histology of the male and female reproductive tracts, the spermatogenesis of all types of sperm and their structure. Both the male and female reproductive systems are in an advanced mesogastropod state, approaching the neogastropod condition. Primitive features of the male system are an open prostate gland and an open sperm groove extending along the phallus. The least advanced feature of the female system is the seminal receptacle which is merely a dilation of the distal portion of the oviduct. Three types of sperm develop in the testes of F. oregonensis continually throughout the year. Maximum production however, occurs during February, March and April. Two sperm lines develop, (1) the eupyrene or fertilizing sperm and (2) the apyrene anucleate sperm, of which there are two types, the carrier and the lancet. Development of eupyrene sperm is typical but complex; one spermatogonium produces four haploid sperm after two meiotic divisions. In contrast, development of apyrene is atypical and direct; one spermatogonium produces one sperm. The nucleus of the spermatogonium fragments and degenerates within the cell. Cellular differentiation is not complex. The most numerous and active of all three types are the eupyrene sperm. They are long and filamentous, similar to other gastropod sperm. The acrosome is conical and hollow. A rod extends through the subacrosomal space which also contains a subacrosomal granule. Posterior to the acrosome is a modified centriole from which extends an axoneme of the standard 9+2 pattern. The nucleus is cylindrical, encircling the axoneme distal to the acrosome. Distal to the nucleus is a mitochondrial spiral also encircling the axoneme and further distal is the glycogen region. Grouped glycogen rosettes having a characteristic pattern extend to the end of the tail piece where the axoneme emerges free for a short distance. The slow-moving carrier apyrene sperm is short and fusiform. Through its centre runs a core of axonemes of standard configuration, each of which extends from an anterior modified centriole. Encircling the core are tightly packed large droplets containing acid mucopolysaccharide complexed with protein. Dispersed amongst these are glycogen rosettes and a few mitochondria. Fifty or more eupyrene sperm attach to one carrier sperm forming a spermatozeugma. Carrier sperm are probably a nutrient source for eupyrene sperm. There may be other less obvious functions for the spermatozeugrnata. Lancet apyrene sperm are long and so named because of their shape. They are less active than the carrier sperm and eupyrene sperm do not attach to them. Several standard axonemes arise from apically placed modified centrioles and extend beneath the plasmalemma distally to the end of the sperm; single microtubules lie in a similar position between the axonemes. Disposed anteriorly in the cytoplasm are mitochondria and numerous small droplets containing acid mucopolysaccharides complexed with protein. Posterior are several large mucus - containing droplets. Scattered throughout the cytoplasm are glycogen rosettes. The roles of the lancet sperm are less obvious than those of the carrier. After copulation, the three types of sperm are stored for a short time in the female copulatory bursa. Most eupyrene sperm move to the seminal receptacle for some time prior to fertilization. Apyrene sperm remain in the viscous fluid in the copulatory bursa. Lancet sperm appear to begin degeneration and entrap any other sperm remaining in the bursa, producing plasmodial-like formations. The significance of this is uncertain.
Life histories and post-glacial origins of tundra caddisflies (Trichoptera) from the Tuktoyaktuk Peninsula, Northwest Territories
(1984) Winchester, Neville Norman
Life histories, species composition and post-glacial origins of Trichoptera from the Tuktoyaktuk Peninsula, Northwest Territories were examined. Nineteen species were identified while eight others could not be identified to species. Eighty-two percent of this fauna represented new Canadian arctic reports and all species represented Nearctic range extensions. Sphagnophylax meiops, gen. et sp. nov. was discovered. Time available for insect growth in arctic regions is short and in this study was confined to early June - late September. Life cycles were completed in temperatures that consistently remained above 0c. For the remainder of the year complete freezing of the stream and shallow areas of the surrounding drainage basin occurred. Trichoptera overwintered in several larval instars and survived ice encasement. These larvae were probably freezing tolerant, although more than one mechanism may exist to allow survival at sub-zero temperatures. Thawing, which began in mid-May, was completed by mid-June and created a mosaic of flooded lowland marsh. This continuous littoral zone dominated by rooted macrophytes provided large areas for caddisfly aggregations. A small portion of the total fauna resided in the stream channel. This limited stream habitat contributed to the exclusion of strict lotic Trichoptera. Life history information for 10 of 19 species suggested that all species have a one year life cycle. Adult emergence coincided with the onset of drought. Developmental rates and number of instars were constant among all species. Rates of increase varied between 1.13 and 1.44. All rates agreed with Dyar's Rule except that of Sphagnophylax meiops. Three patterns of flight period were recognized, defined on the basis of flight duration. Flight periods did not show a progression towards short and highly synchronized emergence with increased latitude. The systematic and biogeographic relationships within the Trichoptera of northwestern North America were affected by past glacial events. The post-glacial origin of this fauna was composed of three groups that included Trichoptera that spent the glaciation: (1) in Beringia, (2) south of the ice sheets, or (3) in both areas. The Beringian element was subdivided into 3 groups based on species distributional patterns. Group A was found only in Beringia (in North America). Group B was Beringian and widespread in North America but absent in the Palearctic. Group C included Amphi-Beringian species with Nearctic and Palearctic distributions. Populations may have been split and were present in both areas simultaneously. The probable post-glacial sources of this fauna were: Beringia, 47%; south of the ice sheets, 23.5%; and both of these areas, 29.5%. The composition of the Beringian elements including those species thought to have re-colonized from both areas was: Group 1, 31%; Group 2, 31%; and Group 3, 38%. This study notes that a diverse caddisfly assemblage exists in the low arctic. Species survive extended cold periods, periods of drought, and are able to complete their life cycles in one year. Beringia is considered an important source area contributing to the observed species diversity.
Biochemical responses based on cytochrome P450 induction in chinook salmon (Oncorhynchus tshawytscha) exposed to bleached kraft mill effluent on the Fraser River
(1996) Wilson, Joanna Yvonne
Juvenile chinook salmon were collected from sites on the upper Fraser River downstream of bleached kraft pulp mills and municipal outfalls. Ethoxyresorufin-O-deethylase activity (EROD), CYP I Al density, and DN A adduct concentrations were measured in liver tissue. Liver histopathology was performed. Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) were measured in carcasses. Polyaromatic hydrocarbon (PAH) metabolites were measured in bile. Biochemical, but not histopathological, responses were significantly different from those in controls at nearly all sites. Biochemical responses were not correlated with any of the contaminants analyzed. Fish sampled closest to effluent discharge showed the weakest responses. In fish experimentally exposed to effluent, significant increases in biological effects were seen at 2% (and higher) effluent concentrations. These results indicate that fish at this site may not be exposed to effluent concentrations previously thought, perhaps suggesting that chinook are mobile during winter months. Biological responses are not caused by the organic contaminants measured.
Age determination of individual garter snakes (Thamnophis spp.) using skeletochronology
(1995) Waye, Heather Louise
Individual and population-specific patterns of growth, and variations in these patterns, can be determined if age structure is known; this allows the dynamics of populations to be more accurately modelled and projected into the future. Besides contributing to our understanding of the fundamental ecological issue of limitation of distribution and abundance, such knowledge is critical to management or conservation plans for many species. Skeletochronology, the reading of growth rings in bony structures, has been used extensively to determine the ages of individual fish and, to a lesser extent, reptiles and amphibians. This study evaluates the use of skeletochronology to determine the ages of garter snakes (Thamnophis spp.) with emphasis on the development of techniques that allow the sampling of bone structures from live animals. Rings were observed in the caudal vertebrae of three species of garter snakes and were consistent in number within individual snakes. The validity of growth rings as indicators of age was established using snakes raised in the laboratory under differing hibernation regimes, and through recapture and resampling of snakes caught the previous year. The age structure of this population of garter snakes is discussed, and a preliminary life table based on age was constructed. Skeletochronology has the potential to be an important and useful technique for the study of age in snakes, but is very labour-intensive and best used as part of a large, long-term sampling project.
Gaucher disease : expression of human Glucocerebrosidase in Pichia pastoris and evolution of Glucocerbrosidase gene and pseudogene in primates
(2000) Wafaei, Julie Rachel
Gaucher disease, the most common lysosomal storage disorder, is caused by insufficient levels of the enzyme glucocerebrosidase (GBA). This thesis examines two aspects of glucocerebrosidase: (1) the heterologous expression of glucocerebrosidase in Pichia pastoris, and (2) the molecular evolution of glucocerebrosidase functional gene and pseudogene in primate species. The development of recombinant GBA by economical methods is of importance for treating Gaucher patients by enzyme replacement therapy. Here I explore two variables that may inhibit GBA expression in Pichia pastoris, proteolytic instability and gene dosage. These variables were found to have minimal impact on the consistently low levels of enzymatically and immunologically active recombinant GBA expressed in P.pastoris. The GBA genes duplicated approximately 40 million years ago and subsequently diverged. Here I investigate the molecular evolution of GBA and its pseudogene by sequencing approximately 1.1 kb of the C-terminal region of nine GBA genes in chimpanzee, gorilla, orangutan, baboon, and squirrel monkey. These data indicate that gene conversion has affected the evolution of GBA and its pseudogene, as well as provide information on GBA gene copy number and the functionality of the GBA genes.
Characterization of gonadotropin-releasing hormone (GnRH) from lake whitefish (Coregonus clupeaformis) : structure, function and location
(2002) Vickers, Elaine Denise
Gonadotropin-releasing hormone (GnRH) is a small peptide hormone that is essential for reproduction in vertebrates and some invertebrates. GnRH is synthesized in the brain and acts on the pituitary gland stimulating the synthesis and release of gonadotropins into the blood. These gonadotropins circulate to the gonads where they stimulate gametogenesis and the synthesis and release of reproductive steroid hormones such as estrogen and testosterone. Seventeen GnRH forms have been isolated and named after the species in which they were first identified. The structure of this ten-amino-acid hormone is highly conserved among vertebrates, except for the four amino acids located in positions five to eight. To date, species contain either two or three GnRH forms. Lake whitefish was chosen as the study animal because it is a representative of the earliest subfamily of salmonids ( Coregoninae). All salmonids studied so far express only two GnRH forms: salmon (s)GnRH and chicken (c)GnRH-II. However, our previous protein work done on lake whitefish identified three GnRH forms : sGnRH, cGn.RH-II, and the third form called whitefish (wf)Gn.RH. There are three objectives in this study: ( 1) to obtain the cDNA sequences of each form of GnRH in lake whitefish, (2) to determine the releasing potential of wfGnRH on pituitary cells, and (3) to detem1ine the location of each GnRH form in the whitefish brain using in situ hybridization. An examination of the structure (both protein and DNA), specific brain function, and location of each GnRH form within a species provides clues to the evolution of the hormone. The cDNA sequence of each form of GnRH in lake whitefish was isolated using 5' and 3' rapid amplification of cDNA ends (RACE) and reverse transcription polymerase chain reaction (RT- PCR). Each sequence consists of a 5' untranslated region (UTR), signal peptide, GnRH-encoding region, cut site, GnRH-associated peptide (GAP), and 3 'UTR. The lake whitefish cGnRH-II precursor has 88% nucleotide and 79% amino acid identity with rainbow trout cGnRH-II. The lake whitefish sGnRH precursor shows 93% nucleotide and 79% amino acid identity when compared to Atlantic salmon sGnRH. As seen in other vertebrate GnRH sequences, the regions that are most conserved encode the GnRH peptide and the cut site. The novel wfGnRH cDNA reveals little nucleotide sequence identity to any known GnRH sequences but shows 60% amino acid identity with Haplochromis burtoni seabream (sb)GnRH. This suggests the ancestral forms ofwfGnRH and sbGnRH were closely related. Whitefish GnRH is biologically active, as it stimulated cultured pituitary cells to increase the expression of the a-subunit of gonadotropin mRNA. This suggests that wfGnRH has a role in gonadotropin-release from the pituitary in lake whitefish. Also, cGnRH-II had some biological activity, but is unlikely to cause gonadotropin-release in vivo due to its location in the midbrain. Localization of brain GnRH by in situ hybridization revealed an overlap of regions expressing sGnRH or wfGnRH in the anterior brain from olfactory bulb to preoptic area (POA). The only form located in the hypothalamus was wfGnRH and in the midbrain was cGnRH-II. It is concluded that wfGnRH plays the main role in gonadotropin release in lake whitefish and that sGnRH may have a minor role. Also, both sGnRH and cGnRH-II probably have neuromodulatory roles as well.
Production of antiserum to a goldfish visual pigment opsin
(1996) Veldhoen, Kathleen Maria
Characterization of visual opsins can be achieved with the use of specific polyclonal or monoclonal antibodies. In this study, antiserum has been raised against a conjugate protein including an N-terminal sequence of the goldfish short wavelength sensitive visual pigment opsin. Results of immunocytochemical assays reveal that the antiserum contains antibodies specific for an opsin which resides in single cone photoreceptors of the goldfish, rainbow trout and bluehead wrasse. The results confirm earlier studies of goldfish and rainbow trout retinae, which localized short wavelength sensitivity to single cones. Cross reactivity of an anti-bovine rhodopsin antibody with rainbow trout rods and cones was observed. The antiserum specificity to teleost SWS opsins demonstrates its usefulness as a tool to reliably identify these opsins in teleost retinae.
Detection and distribution of estrogen receptor in human breast cancer by a combined biochemical/immunohistochemical multiple microsample assay
(1987) Thornton, Ian George
The relationship between high estrogen receptor (ER) levels and the regression of tumor growth following estrogen ablative therapy is well-established in human breast cancer. Generally however, even with treatment, tumors may become ER-negative and unresponsive to further hormonal therapy. This has traditionally been explained as a clonal expansion of ER-negative cells that have a selective growth advantage over ER-positive ones in the absence of estrogen. According to this hypothesis, once such a clone is established the tumor's growth can proceed unimpeded, after which reversal to a hormone-controlled state is improbable. Commonly, the measurement of ER for clinical screening of patients for hormone therapy is done by steroid-binding assay (SBA) which measures the uptake of radiolabelled estradiol by a single sample of tumor. However, this method measures only previously-unoccupied receptor sites. Furthermore, if the sample does not consist entirely of tumor cells and has a high proportion of connective tissue, fluid, or serum contaminants, the quantitation of ER may be invalidated and tumors having a small proportion of highly ER-positive cells susceptible to endocrine response could remain undetected. In order to further understanding of ER distribution in breast cancer a comparative multiple microsample assay was performed on 21 tumors . Four 13 X 1.5 X 1.5 mm (approx. 40 mg) microsamples from each tumor were divided equally and longitudinally, one half used for SBA by cold-agar gel electrophoresis (CAGE), and the other half for an estrogen receptor immunochemical assay (ER-ICA) employing monoclonal antibodies against ER. For SBA , cytosols of microsamples were prepared by sequential homogenization and centrifugation , then incubated with tritiated 17B- estradiol. CAGE was used to separate receptor-bound hormone from unbound hormone . Uptake of estradiol was quantitated by scintillometry . Albumin , which binds estradiol nonspecifically, and in high concentrations interferes with ER estimation , was measured by radial immunodiffusion assay . For ER- ICA 6 ~m frozen sections were incubated with monoclonal anti -human ER antibody. Following this, a peroxidase: anti-peroxidase (PAP) immunohistochemical procedure was applied to all sections in order to localize ER- bound antibody . Each stained section was examined by four independent observers to determine per cent carcinoma (PCC) and per cent specific ER staining (PSS) . SBA-derived ER concentrations were then corrected for PCC. The proportion of tumor cells stained specifically for ER in each microsample was estimated and adjustment made for differences in staining intensity (SI). The resulting histoscore is an arbitrary value that allows these data to be ranked and compared with SBA-derived data. Qualitatively, 68 % of all microsamples were ER-positive , and 28 . 5 % were negative by both methods, with an overall correlation of 0 . 97 . Quantitatively, there was a correlation of 0.75 between SBA-derived corrected ER and ER-ICA derived histoscores for corresponding microsamples. The high correlation between SBA and ER- ICA data demonstrates the potential value of combined biochemical and immunohistochemical methods for screening breast cancer patients most likely to benefit from endocrine therapy, and thus avoiding the complications associated with other forms of therapy, such as radiation and chemotherapy. The distribution of ER within the 21 tumors studied revealed a variety of staining patterns, both homogeneous and heterogeneous, the latter demonstrating various degrees of intermixing of ER- positive and ER-negative tumor cells . Within individual tumors heterogeneous, as opposed to homogeneous , ER distribution patterns were seen in 12 of the 15 (80 %) ER-positive tumors . The checkerboard staining pattern observed in 9 of these might represent a variant type of tumor in which ER-negative status is attained randomly throughout the tumor, rather than by means of a focal, clonal expansion of ER-negative cells. If so , it may be possible that appropriate manipulation of the cell environment could reverse the tendency for breast tumor cells to become hormone-independent.
Habitat selection by marten (Martes americana) in managed mixedwood forests of the boreal white and black spruce biogeoclimatic zone of northeastern British Columbia
(2002) Therrien, Steve
I studied macro and micro-habitat selection by marten (Martes americana) in a mixedwood forest of the Boreal White and Black Spruce Biogeoclimatic zone. I also examined the short-term effects of partial-cut systems on marten in the study area. The mean home range area (kernel 80 %) for male (n=7) and female (n=4) marten was 5.4 and 2.4 km2, respectively. At the macro-habitat scale of selection, eight of the radio collared marten avoided the inclusion of habitat-types characterised by either sparse or shrubby vegetation, or pole trees within their core use areas (kernel 50 %). Five marten preferably incorporated late- successional mixedwood of trembling aspen (Populus tremuloides) and white spruce (Picea glauca) habitat-types within their core areas. Core areas of male and female marten contained an average proportion of 47 and 70% of mature forests, respectively, while the availability was 37 % in the study area. The study martens used the partial-cuts to their availability at the stand scale within their home ranges. At the micro-habitat scale of selection, structural habitat characteristics at marten locations within core areas were compared to random plots within the study area. Compared to random sites, marten locations had greater stockings of large diameter trees, greater stockings of large diameter snags, and greater abundance of large coarse woody debris. Marten activity in the partial-cut treatments was compared to that in control stands, during early and late winter by snow-tracking sampling. Marten hunting activity was greater in the partial-cuts during early winter, but decreased to a level similar to the controls in late winter. In controls, marten foraging activity was not affected by winter progression. During snow-tracking, resting sites were found at comparable occurrences in treatments and controls. All resting sites were associated with large coarse woody debris covered with snow. The partial-cut systems examined represent a valuable alternative to clearcutting, to preserve marten habitat in mixedwood forests. In my study area, the physical structures intrinsic to late successional forests appeared to be determinant in the ecology of marten. If forest management strategies aim at maintaining minimal levels of marten habitat within harvest units, the retention or the re-construction of physical structure must represent an integrated silviculture practice.

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