Ph.D. Biology
http://hdl.handle.net/10464/2238
2024-03-18T19:12:07ZInvestigating the Effect of Cell Culture Compositions on Mitochondrial Metabolism, Dynamics, and Transcriptome and Proteome of cells
http://hdl.handle.net/10464/15595
Investigating the Effect of Cell Culture Compositions on Mitochondrial Metabolism, Dynamics, and Transcriptome and Proteome of cells
Moradi, Fereshteh
The phytoestrogen Resveratrol (RES) is a natural polyphenol that has been detected in more than 70 plant species. RES has structural similarity to mammalian estrogens and can bind to estrogen receptors, eliciting genomic and non-genomic effects. Both RES and physiological estrogens like 17-β-estradiol (E2) have wide-ranging effects on mitochondria. In this thesis, I began by investigating RES’s effects on mitochondrial network dynamics (Chapter 2) and discovered a pro-fusion activity apparently mediated by the mitofusin enzyme Mfn2. RES stimulated mitochondrial network hyper-fusion morphology in all three cell lines investigated (C2C12 (mouse myoblast), PC3 (prostate cancer), and MEFs (mouse embryonic fibroblast)), but the effect was absent in Mfn2-null MEFs. As this work was being completed; several research groups introduced ‘physiologic cell culture media’ that are modeled on the human plasma metabolome. I co-authored a study (not in this thesis) demonstrating that RES’s effects on mitochondrial dynamics are dependent on cell culture conditions. To follow up on this, I investigated whether E2’s mitochondrial effects might also be dependent on the cell culture environment, and showed conclusively that this is indeed the case, using C2C12 cells as a model system (Chapter 3). These results and those published by others in 2017-2019 suggested that medium composition can profoundly affect cellular functions. In Chapter 4, I followed this up by studying how culture conditions affect mitochondrial bioenergetics and network morphology using four cancer cell lines and showed that this is a significant issue. Finally, to gain a more complete understanding of this phenomenon, I completed a full transcriptomic and proteomic analysis of media effects using MCF7 breast cancer cells as a model (Chapter 5). I showed that hundreds of transcripts and proteins are affected according to culture conditions. Taken together, the results presented in this thesis emphasize the significant extent to which the cell culture environment affects experimental outcomes, particularly with respect to mitochondrial bioenergetics and dynamics. This information contributes to the development of cell culture experiments providing results that can be translated in vivo.
Thermal tasting: methodological considerations and implications for alcohol behaviour.
http://hdl.handle.net/10464/15156
Thermal tasting: methodological considerations and implications for alcohol behaviour.
Thibodeau, Margaret
Thermal tasting is a phenomenon whereby some individuals perceive thermally-induced taste sensations when their tongue is warmed or cooled. These individuals, known as thermal tasters (TT), report a variety of thermally-induced tastes and the tastes reported can vary with temperature regime used and location on the tongue tested. TT are typically compared to thermal non-tasters (TnT), individuals who do not experience thermally-induced sensations. The literature suggests that TT give higher intensity ratings to orosensory stimuli than TnT; however, small sample sizes and differences in classification schemes between studies confound our understanding of TTS (thermal taste status). It is unknown whether the increased orosensory responsiveness of TT is universal or whether it is driven by a subgroup of TT. Furthermore, up to 50% of individuals are non-classifiable (NC). The largest database of individuals who have undergone TTS screening was compiled to address the literature gaps. Findings indicate that TT are more responsive than TnT to orosensory stimuli, regardless of the classification scheme used. The orosensory responsiveness of NC is not homogeneous, suggesting that NC are not a separate group but rather misclassified TT and TnT. Sweet TT are more likely than non-sweet TT to experience thermally-induced sensations during lingual warming. Similarly, sour TT are more likely than non-sour TT to report thermally-induced tastes during cooling. However, no differences in orosensory responsiveness based on these or other subgroups are identified, suggesting that the heightened orosensory responsiveness of TT is universal across this phenotype. The final study sought to characterize the binary interactions between ethanol and four orosensory stimuli (fructose, quinine, tartaric acid and alum sulphate) both overall and by comparing TT and TnT. In general, TT are more responsive than TnT to all stimuli in the study. Few interactions between TTS and stimulus intensity exist suggesting that TT and TnT perceive the sensations elicited by alcoholic beverages similarly, albeit at different intensities. Together, the thesis helps inform best practices for TTS screening and classification, provides insights into TTS mechanisms and furthers our understanding of alcoholic beverage perception.
Metarhizium robertsii interactions with Phaseolus vulgaris (Haricot Bean)
http://hdl.handle.net/10464/15131
Metarhizium robertsii interactions with Phaseolus vulgaris (Haricot Bean)
Hu, Shasha
Metarhizium is an insect pathogenic fungus, as well as a plant root symbiont. During symbiotic interactions, it can benefit the plant by improving plant growth, antagonizing plant pathogens and herbivores, and enhancing plant tolerance to abiotic stresses. In this thesis, the interactions between Metarhizium robertsii and Phaseolus vulgaris (haricot bean) were studied from two aspects. First, a phenotypically degenerated (low conidia production) strain of Metarhizium was serially passaged through bean plant. Second, the immune responses of haricot bean during endophytic colonization were assessed.
Commercial application of Metarhizium for insect biocontrol requires optimal production of conidia as infective propagules. It was demonstrated that conidial production and virulence of phenotypically degenerated Metarhizium were restored by serial passages through bean roots, as well as switchgrass roots, and wax moth larvae. A decrease in the expression of fungal DNA methyltransferase was observed in the phenotypically degenerated Metarhizium strain through bean passages. Whole genome bisulfite sequencing analysis showed differences in the distribution of differentially methylated regions in the degenerated and subsequently recovered strains.
Metarhizium can antagonize the plant pathogen, Fusarium solani f. sp. phaseoli during bean root colonization. Using comprehensive plant hormone analysis, transcriptional expression, and stomatal size analysis, bean immune responses to colonization by Metarhizium and/or Fusarium were assessed. In comparison to un-inoculated bean, root colonization by Metarhizium resulted in reduction of abscisic acid (ABA), increased stomatal size, and decreased expression of plant immunity genes in bean leaves, which is different from those in bean colonized by Fusarium. Furthermore, exogenous application of ABA resulted in reduction of bean root colonization by Metarhizium but increased colonization by Fusarium, compared to corresponding plants without ABA application. Therefore, ABA was implicated in differential responses of bean plants to root colonization by Metarhizium and Fusarium.
In conclusion, this thesis provided new insights into the study of the interactions between Metarhizium and haricot bean. Some novel findings were that fungal DNA methyltransferase was implicated in the recovery of phenotypically degenerated Metarhizium and a plant hormone, abscisic acid was implicated in differential interactions of endophytic colonization by Metarhizium when compared to a pathogenic interaction by Fusarium.
Investigating the Role of MicroRNAs in Regeneration and Axonal Pathfinding
http://hdl.handle.net/10464/14880
Investigating the Role of MicroRNAs in Regeneration and Axonal Pathfinding
Walker, Sarah
During both development and regeneration, axons must navigate through a complex and changing environment to reach their proper synaptic target. To do so, axons utilize a specialized structure, the growth cone, which senses and interprets guidance cues in its surrounding environment to change the direction of axonal outgrowth. MicroRNAs, which regulate mRNA translation, have recently been shown to regulate both neurite outgrowth and growth cone guidance in response to classical guidance cues during vertebrate development. However, little is known of their regulation of neuronal regeneration in an invertebrate. Thus, the main aim of this thesis was to study the role of microRNAs during CNS regeneration of the pond snail, Lymnaea stagnalis. Specifically, I determined the expression patterns and relative abundance of microRNAs in the regenerating CNS in response to retinoic acid (RA). Using miRNA-Sequencing, I identified one neuronally enriched microRNA, miR-124, that was up-regulated in RA-induced regenerating CNS. Using PCR and in situ hybridization, I characterized its distribution in the snail CNS, and discovered it shared similar expression patterns to that of vertebrates. In cell culture, I found miR-124 was abundant within regenerating motorneurons and was localized to their growth cones. I next determined that miR-124 contributed to RA-induced growth cone turning behaviour. During attractive growth cone turning to RA, the abundance and distribution of miR-124 was altered, in both a cue and context-dependent manner. Finally, I demonstrated that miR-124 targeted the Rho kinase, ROCK, during turning responses to RA, likely to promote the formation of a neurite shaft, or to maintain growth cone polarity. Together, these findings provide the first evidence for a role of microRNAs in mediating growth cone behaviours to RA in regenerating motorneurons.