Kapron, Carolyn

Impacts of embryo incubation temperature on ontogenic thermal tolerance of brook trout (Salvelinus fontinalis)

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Creator (cre): Lechner, Emily Rebekka, Thesis advisor (ths): Raby, Graham D, Thesis advisor (ths): Wilson, Chris C, Degree committee member (dgc): Kapron, Carolyn, Degree granting institution (dgg): Trent University
Abstract:

I investigated the climate vulnerability of a cold-water salmonid by observing the upper thermal tolerance of brook trout (Salvelinus fontinalis) across multiple life stages. Using hatchery- and wild-ancestry fish, I assessed variation in thermal tolerances and carryover effects within and among brook trout life stages and populations that were reared under varying winter thermal regimes as embryos. Embryonic, fry, and gravid adult upper thermal tolerance were tested via CTmax. Warm acclimation was associated with an increase in embryonic upper thermal tolerance. CTmax variability was markedly higher in embryos than juvenile or adult salmonids. Effects of thermal incubation treatments varied by life stage, with incubation temperature and life stage both influencing body size and thermal tolerance. Collectively, these results suggest that brook trout only exhibit short-term carryover effects from thermal stressors during embryo incubation, with no lasting effects on phenotype beyond the first few months (10 weeks) after hatch.

Author Keywords: acclimation response, climate change, ontogeny, salmonid, stress, thermal plasticity

2024

Concentration-Dependent Effects of Cadmium on Mouse Angiogenesis In Vitro

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Creator (cre): Knight, Caitlyn, Thesis advisor (ths): Kapron, Carolyn, Degree committee member (dgc): Tobin, Stephanie, Degree committee member (dgc): Huber, Robert, Degree committee member (dgc): West, Sarah, Degree granting institution (dgg): Trent University
Abstract:

Cadmium is a toxic metal that has detrimental effects on blood vessel development and function. To examine the effect of varying concentrations of cadmium on angiogenesis, two in vitro assays were used. First, developing yolk sac blood vessels were studied in gestation day 8 mouse embryos exposed to medium alone, 1.25, or 1.75 μM cadmium chloride (CdCl2). Embryos exposed to 1.25 μM cadmium experienced a significant increase in the number of vessels formed; however, they were smaller in size. Vessel morphology and signalling pathways were also investigated using the mouse aortic ring assay, with exposures of 0.0, 0.5, 1.0, 5.0, or 10.0 μM CdCl2. Samples exposed to 10 μM experienced a significant increase in vessel length. However, no significant differences in phosphorylated PTEN and AKT were observed. The results of this study suggest that low levels of cadmium may disrupt angiogenesis, particularly the development of the embryonic vasculature in the yolk sac.

Author Keywords: Angiogenesis, Cadmium, Embryonic Development, Teratogenicity, Vascular Development, Vasculogenesis

2024

Mfsd8 regulates growth and multicellular development in Dictyostelium discoideum

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Creator (cre): Yap, Shyong Quan, Thesis advisor (ths): Huber, Robert J, Degree committee member (dgc): Kapron, Carolyn, Degree committee member (dgc): Morrison, Erin N, Degree granting institution (dgg): Trent University
Abstract:

The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of inherited neurodegenerative lysosomal storage disorders. CLN7 disease is a subtype of NCL that is caused by mutations in the MFSD8 gene. MFSD8 encodes a lysosomal transmembrane protein that is predicted to play a role in transporting small substrates across membranes. However, little is known about its role and substrate specificity. Previous work identified an ortholog of human MFSD8 in the social amoeba Dictyostelium discoideum and reported its localization to endocytic compartments. In this study, the effects of mfsd8 loss during Dictyostelium growth and multicellular development were further characterized. Dictyostelium mfsd8- cells displayed increased rates of proliferation and pinocytosis in liquid media. During growth, loss of mfsd8 altered lysosomal enzymatic activities and reduced the intracellular and extracellular levels of autocrine proliferation repressor A. mfsd8- cells grown on a lawn of bacteria formed plaques in a shorter period of time compared to WT cells, providing additional support for the enhanced growth of mfsd8- cells. Upon starvation, the aggregation of mfsd8- cells was delayed, and mfsd8- cells formed more mounds that were smaller in size, which may be attributed to the reduced cell-substrate adhesion and altered lysosomal enzymatic activities observed for mfsd8- cells. Following aggregation, tipped mound formation was delayed, however, loss of mfsd8 did not affect the timing of slug/finger and fruiting body formation. Additionally, slug migration was reduced in mfsd8- cells. These aberrant phenotypes, excluding fruiting body formation, were effectively or partially rescued when Mfsd8-GFP was introduced into mfsd8- cells. Overall, these results show that Mfsd8 plays a role in regulating growth and developmental processes in Dictyostelium via lysosomal-associated functions.

Author Keywords: CLN7, Dictyostelium discoideum, Lysosomes, MFSD8, Neuronal Ceroid Lipofuscinoses

2022

The Effect of SP600125 JNK Inhibitor on Cadmium-Treated Mouse Embryo Forelimb Bud Cells In Vitro

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Creator (cre): Alhoesh, Afnan M., Thesis advisor (ths): Kapron, Carolyn, Degree committee member (dgc): Brenner, Ingrid, Degree committee member (dgc): Brunetti, Craig, Degree granting institution (dgg): Trent University
Abstract:

This study investigated the role of the JNK signaling pathway in cadmium-treated mouse embryo forelimb bud cells in vitro. Primary cultures of forelimb bud cells harvested at day 11 of gestation were pre-treated with JNK inhibitor SP600125, and incubated with or without CdCl2 for 15, 30, 60, 120 minutes and 24, 48 hours or 5 days. Endpoints of toxicity were measured through cell differentiation by Alcian Blue Assay and phosphorylation of JNK proteins by Western blot. The results demonstrated that, in the cell differentiation assay, inhibiting JNK activation by 20 μM SP600125 causes an enhanced toxic effect in limb cells and inhibits cell differentiation, whereas 2 μM decreases differentiated nodule numbers under both cadmium stress and normal conditions. In conclusion, the JNK pathway has an essential role in the differentiation processes of limb bud cells in normal growth conditions.

Author Keywords: Cadmium, Cell Signaling, JNK, Limbs, Mouse Embryo, Teratology

2015

The role of Cln5 in autophagy, using a Dictyostelium discoideum model of Batten disease

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Creator (cre): McLaren, Meagan, Thesis advisor (ths): Huber, Robert J., Degree committee member (dgc): Kapron, Carolyn, Degree committee member (dgc): Brunetti, Craig, Degree granting institution (dgg): Trent University
Abstract:

This thesis investigated the role of the neuronal ceroid lipofuscinosis protein, Cln5, during autophagy. This was accomplished by performing well-established assays in a Dictyostelium cln5 knockout model (cln5-). In this study, cln5- cells displayed a reduced maximum cell density during growth and impaired cell proliferation in autophagy-stimulating media. cln5- cells had an increased number of autophagic puncta (autophagosomes and lysosomes), suggesting that autophagy is induced when cln5 is absent. cln5- cells displayed increased amounts of ubiquitin-positive proteins but had no change in proteasome protein abundance. During the development of cln5- cells, fruiting bodies developed precociously and cln5- slug size was reduced. Lastly, when cln5- cells were developed on water agar containing ammonium chloride (NH4Cl), a lysosomotropic agent, the formation of multicellular structures was impaired, and the small slug phenotype was exaggerated. In summary, these results indicate that Cln5 plays a role in autophagy in Dictyostelium. The cellular processes that regulate autophagy in Dictyostelium are similar to those that regulate the process in mammalian cells. Thus, this research provides insight into the undefined pathological mechanism of CLN5 disease and could identify cellular pathways for targeted therapeutics.

Author Keywords: Autophagy, Batten disease, Cln5, Dictyostelium discoideum, NCL

2020

Characterization of frog virus 3 and its binding partner LITAF

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Creator (cre): Lacerda, Andressa, Thesis advisor (ths): Brunetti, Craig R, Degree committee member (dgc): Saville, Barry, Degree committee member (dgc): Kapron, Carolyn, Degree granting institution (dgg): Trent University
Abstract:

Iridoviruses are large (120-200nm) double stranded DNA viruses that contain an icosahedral capsid. The iridoviridae family is composed of five genera that infect a wide range of poikilothermic vertebrates (Lymphocystivirus, Ranavirus and Megalocyivirus) and invertebrate hosts (Iridovirus, Chloriridovirus). Frog virus 3 (FV3) is a member of the Ranavirus genus, and is commonly used as a model system to study iridoviruses. I was interested in understanding virus-host interaction in FV3. I studied two viral genes, FV3 97R and FV3 75L. Here I demonstrate that 97R localizes to the endoplasmic reticulum (ER) at 24 hours post-transfection. However, at 35 hours post-transfection 97R localizes to the ER but also begins to form concentrated pockets, continuous with the nuclear membrane This study found that 97R possess a unique phenotype and that its localization to the ER is mediated through its C-terminus transmembrane domain. FV3 75L encodes an 84 amino acids protein. I showed that FV3 75L localizes to the early endosomes, while its cellular binding partner, LITAF, localizes to late endosome/lysosome. Interestingly, when FV3 75L and LITAF are co-transfected into cells, LITAF can alter the subcellular localization of FV3 75L to late endosome/lysosomes. A physical interaction between LITAF and FV3 75L was demonstrated through a pull-down assay and that a highly conserved domain found in both proteins may mediate the interaction. LITAF has been proposed to function in protein degradation, but there is still uncertainty on LITAF's specific role. I was interested in further characterizing LITAF and its implications in protein degradation and a neurodegenerative disorder. At least 9 mutations of LITAF are associated with Charcot-Marie-Tooth disease type 1C (CMT1C), which belongs to the group of most common heritable neuromuscular disorders, affecting approximately one in 2500 people. We show that LITAF mutants G112S and W116G mislocalize from the late endosome/lysosome to the mitochondria while the T49M and P135T mutants show partial mislocalization with a portion of the protein present in the late endosome/lysosome and a portion of the protein localized to the mitochondria. Since LITAF is believed to play a role in protein degradation, it is possible that the specific characteristics of CMT1C may occur though impaired degradation of Schwann cell membrane proteins, such as PMP22. I was able to show that when WT LITAF is present, there is a decrease in the PMP22 intracellular levels, which suggest that LITAF plays an important role in protein degradation, and also in other types of CMT. Insight into how mutations in LITAF cause CMT1C may not only help better understand cellular pathways, but also further elucidate the role LITAF's viral homolog FV3 75L during viral infection.

Author Keywords: 75L, Charcot-Marie-Tooth, CMTC1, ER, FV3, LITAF

2015

Nutrient Metabolism of an Aquatic Invertebrate and its Importance to Ecology

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Creator (cre): Wagner, Nicole D., Thesis advisor (ths): Frost, Paul C, Degree committee member (dgc): Kapron, Carolyn, Degree committee member (dgc): Brunetti, Craig, Degree granting institution (dgg): Trent University
Abstract:

Aquatic consumers frequently face nutritional limitation, caused in part, by imbalances between the nutrients supplied by primary producers and the metabolic demands of the consumers. These nutritional imbalances alter many ecological processes including consumer life-history traits, population dynamics, and food web properties. Given the important ecological role of organismal nutrition, there is a need to have precise and specific indicators of nutritional stress in animals. Despite this need, current methods used to study nutrition are unable to distinguish between different types of nutritional limitation. Here I studied nutritional metabolism in the freshwater zooplankter, Daphnia. A greater understanding of nutritional metabolism would allow for the development of dietary bio-indicators that could improve the study of the nutritional ecology of animal consumers. Specifically, I addressed the question: What affects the biochemical composition of a generalist aquatic consumer? My overall hypothesis was that the quantity and quality of the diet affects the biochemical composition in a nutrient specific manner. To test this hypothesis, I examined various response variables involved in nutrient metabolism such as alkaline phosphatase activity, whole metabolome, and free amino acid composition. For each response variable, I grew Daphnia under various nutritional stressors and determined if responses are nutrient specific or are a general stress response. I found the current method of measuring alkaline phosphatase was not a phosphorus specific indicator, as activity increased in all nutrient stressed treatments. Analyzing the whole metabolome resulted in nutritional stressors being separated in multivariate space, with many identified metabolites being significantly different from nutrient rich Daphnia. Upon further examination the daphnids free amino acids profiles are caused by differences between the supply of amino acids from the algae and the demand within the Daphnia. These differences in supply and demand resulted in the ability to classify the nutritional status of Daphnia with the use of discriminant analysis, a classification multivariate model. In addition to a deeper understanding and advanced knowledge of the physiological changes caused by nutrient limitation, this research has provided strong evidence for the application of nutritional biomarkers/profiles to identified the nutritional status of Daphnia.

Author Keywords: Bio-indictor, Ecological stoichiometry, Metabolism, Nutritional limitation, Nutritional status

2015