Biology

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The Role of p107 in Myogenic Stem Cell DNA Methylation
(2024-07-18) Fine, Kyra Rachel; Scimè, Anthony
Myogenic stem cells have the potential to either self-renew or differentiate into muscle fibers. These stem cell fate choices are influenced by metabolic changes which affect the epigenetic landscape, including DNA methylation which alters gene expression patterns. Because we have found that a mitochondrial function of p107, a transcriptional co-repressor, is involved in regulating SC metabolism and fate decisions, we investigated whether p107 influences the mitochondrial DNA methylation signature. For this we used a portable long read sequencer MinION (Nanopore), to sequence and compare the mitochondria DNA methylation pattern of wildtype and p107 genetically deleted (p107KO) C2C12 myogenic cell line. We found that p107KO mitochondrial DNA had decreased methylation levels for OXPHOS genes compared to wild type. Thus, suggesting that p107 might suppress ND4 gene expression. We also compared a novel method of mtDNA isolation to three existing methods in order to determine the most efficient approach for sequencing.
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Role of MEKK2 Phosphorylation at Threonine 263 on SMYD3-Mediated Methylation of Lysine 260
(2024-07-18) Corridore, Stephanie Amelia; Scheid, Michael
MEKK2 is a protein serine/threonine-kinase involved in the activation of many MAP-kinase signalling pathways. In a previous study, SMYD3-mediated methylation at K260 of MEKK2 was demonstrated to promote aberrant input downstream of oncogenic Ras-signaling, promoting Ras-driven PDAC and LAC progression. Our lab has previously characterized the role of MEKK2 phosphorylation at T283 and has recently discovered a novel second phosphorylation site at T263. We show that together, these sites form the bipartite binding group for 14-3-3 adapter proteins. This study focuses on characterizing T263 and T283 as regulatory phosphosites in K260 methylation and implicates 14-3-3 as a promoting factor in facilitating SMYD3-mediated methylation at K260. Our findings provide a potential mechanism of MEKK2 oncogenic function, whereby 14-3-3 preserves phosphorylation at T263 and T283, together promoting K260 methylation and MEKK2 activation. Our study characterizes T263 and T283 of MEKK2 and 14-3-3 phosphoadapter proteins as potential therapeutic targets in PDAC and LAC.
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The Metabolic Regulation of the TOR Pathway and the Circadian Rhythmicity of Neurospora crassa
(2024-07-18) Fayyazi, Maryam; Lakin-Thomas, Patricia
Eukaryotic circadian rhythms are often attributed to transcription-translation feedback loops (TTFLs). However, rhythms can still persist in the absence of functioning TTFLs. Our laboratory has discovered that mutations in the components of the TOR pathway compromise the non- TTFL rhythms, implying the potential involvement of this pathway in regulating the rhythmicity of Neurospora crassa. The TOR pathway is a nutrient-sensing pathway that its modulation in Neurospora remains enigmatic. Here, I investigated the metabolic regulation of the TOR pathway to delineate its relationship with circadian rhythmicity. Using the phosphorylation state of S6 ribosomal protein, I found that different metabolites, including amino acids and lipids affect the TOR pathway only after a prolonged glucose-starvation period, suggesting that glucose alone is sufficient to activate the TOR pathway. Interestingly, my findings indicate that factors perturbing the TOR activity also influence the period of conidiation, thereby establishing a connection between the TOR pathway and circadian rhythmicity.
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Multiple Interactions and Plant Reproduction: The Intersection of Herbivore, Pollinator, and Plant-Plant Interactions
(2024-07-18) Haas-Desmarais, Stephanie Margaret; Paluzzi, Jean-Paul
Plants interact with a variety of organisms. Both positive and negative interactions exist between plants and other plant species as well as animals. Because these plants and animals exist in communities simultaneously, these interactions will in turn interact. In this dissertation, I examine both the costs and benefits of interactions between plants, herbivores, pollinators, and other plants and how these interactions impact plant reproduction. To start I synthesize the literature on herbivore-pollinator interactions, followed by updating a meta-analysis on the same topic. I then examine both direct and indirect interactions between shrubs and the surrounding desert annual plant community before testing how the same shrub benefactor can change the impact of pollination and herbivory on the reproductive output of one of its proteges. I found that plant reproductive output was dependent on all three types of interactions (pollination, herbivory, and plant-plant interactions) to varying degrees. I compared damage to different types of plant tissues in the literature as well as in the field using artificial damage. I found that damage to flowers and leaves generally decreased plant reproductive output in the literature, but that the relationship was more complex in the field, depending on the presence of other factors. While the interaction between both types of damage were not commonly studied in the literature, I found that applying both types of damage was required to consistently observe decreases in reproduction in response to damage. When testing the effects of shrubs on annuals, shrubs had a mixed effect on plant species density and floral density that depended on annual species identity. Indirect effects of shrubs through the plant community were more consistent between protege species. However, when examining individual plants (as opposed to populations or communities) of one species that was associated with the shrub species in an experimental setting, facilitation was only observed in the presence of damage. I show that herbivory is integral in driving the coevolution between plants and pollinators as well as facilitators and their proteges.
Open Access
The seasonal nutritional physiology of polar bears (Ursus maritimus) under human care
(2024-07-18) Ouellette, Veronica Tate; Thiemann, Gregory
Despite the seasonal ecology of polar bears, zoos feed their captive bears a high-protein, high-calorie diet year-round. This approach to polar bear husbandry has been associated with poor welfare and death by liver/kidney disease. I assessed the effects of diet – calories and macronutrients – on the physiology and behaviour of 5 bears at The Toronto Zoo. Diet varied seasonally in caloric intake, and bears were fed a high-protein diet from January 2018 to February 2019, and a high-fat diet from January 2022 to June 2023. During both periods, the body mass and fat, blood chemistry, and body temperature of bears were recorded. Behavior was observed under the high-fat diet. Diet had significant effects on body and blood variables. Calories had significant effects on activity and body temperature, while macronutrients had no effect on these variables, suggesting thermal stress and laziness are not relevant welfare concerns for captive bears on this diet.
Open Access
Identifying Novel Substrates of Ubiquitin-Specific Protease 7 (USP7) And Speckle-Type POZ (Pox Virus and Zinc Finger) Protein (SPOP)
(2024-07-18) Singh, Sukhdeep Kaur; Saridakis, Vivian
Speckle-type POZ protein (SPOP), an E3 ubiquitin ligase adaptor protein, and ubiquitin-specific protease 7 (USP7), a deubiquitinating enzyme, have previously been implicated in prostate cancer pathogenesis. The objective of this project was to identify novel substrates of SPOP and USP7 to provide insight on the molecular mechanisms contributing to prostate cancer. Employing biochemical and cellular biology techniques, we identified, murine double minute 2 (MDM2), an E3 ubiquitin ligase, as a potential substrate of SPOP. We demonstrated that SPOP interacts with MDM2 through its MATH domain in vitro and negatively regulates its stability in vivo. Moreover, we identified sirtuin 2 (SIRT2), an NAD+-dependent deacetylase, as a novel substrate of USP7. We demonstrated that USP7 interacts and regulates the stability of SIRT2 in vivo. Collectively, our results suggest that SPOP and USP7 through the regulation of MDM2 and SIRT2, may contribute to prostate cancer, however further studies are required to verify this.
Open Access
A Role for p107 in Muscle Satellite Cell Self-Renewal
(2024-03-16) Shah, Vicky; Scime, Anthony
It is well established that skeletal muscle homeostasis is dependent on the activity of the muscle resident stem cells termed satellite cells (SCs). Many neuromuscular dystrophies and complications brought on from ageing are associated with decline to the SC population. Thus, there is a critical need to investigate the control mechanisms that dictate SC fate decisions for self-renewal that are crucial to maintain the SC population. A fundamental regulator of SC fate decisions is mitochondrial metabolism and thereby mitochondrial ATP generation via oxidative phosphorylation (OXPHOS). In our study we uncover a potential novel role for the transcriptional co-repressor retinoblastoma susceptibility protein like 1(Rbl1 or p107) in manipulating the self-renewal capacity of SCs through its mitochondrial localization under the control of the NAD+/NADH ratio. An investigation of this role for p107 function establishes a new mechanism to target SC decline and improve muscle regeneration that is required in muscular dystrophies and ageing.
Open Access
The B-Catenin Interactome in Myogenic Cells
(2024-03-16) Farhat, Fatima; McDermott, John Charles
Beta-catenin is a versatile protein implicated in a wide range of cellular processes including cell fate determination, proliferation, differentiation, adhesion, and cell survival. Beta-catenin performs its cellular functions through protein-protein interactions since it lacks the ability to interact directly with DNA. Our group has characterized the beta-catenin protein interactome in myogenic cells using a GFP-Nanotrap based affinity purification approach followed LC-MS/MS analysis which produced a comprehensive list of established and potential beta-catenin interactors. The objective of this study was to investigate the beta-catenin interactome dataset to better understand the molecular mechanisms underlying the function of beta-catenin in myogenic cells. To address this objective, we have used bioinformatic and biochemical approaches directed at identifying one potential beta-catenin binding partner and investigating its effect on beta-catenin’s function.
Open Access
Pre-migration behaviour and survival of juvenile Wood Thrushes (Hylocichla mustelina) in a fragmented forest landscape
(2024-03-16) Hayes, Susan Mary; Stutchbury, Bridget J.
Due to the difficulty in tracking juvenile songbirds once independent and dispersed from their natal areas, little is known about survival during the first year of life, despite being important for understanding population dynamics in migratory songbirds. Using technology advancements with the Motus Wildlife Tracking System and the miniaturization of radio-tags (~1-year battery), I tracked 189 Wood Thrush nestlings from the nest to provide survival estimates at 3 key life stages: fledgling survival, pre-migration survival, and migration/wintering survival. As small forest fragments have been identified as lower quality habitat, I tested if natal fragment size was a strong predictor of either survival or the extent of pre-migratory movement, and whether juveniles from small fragments suffered from carry-over effects that delayed fall migration departures or lowered annual survival. Lastly, I was able to test hypotheses that could explain the function of pre-migratory movements: habitat optimization, prospecting for future breeding territories, homing target; and for the first time, a flight performance hypothesis that proposes that the extent of pre-migratory movement improves first migration flight performance. Natal forest fragment size was not a strong predictor of fledgling, pre-migration, or apparent annual survival and onset of fall migration was best predicted by fledge date. Weekly survival was lowest for fledglings on their natal territory, high for juveniles as they explored the landscape prior to fall migration, and lower during their first migration/wintering season. Most juveniles that were known to survive wintering/migration, returned to the study area (81%, 25/31) within ~12km of their natal site. Long-distance exploration of the landscape occurred for a high proportion (78%) of juveniles prior to onset of fall migration and these pre-migratory movements occurred at night, mainly 2 hours before sunrise. My results best supported the homing target hypotheses as pre-migratory movements were common in juveniles but not adults, were random in orientation, and a relatively large proportion of juveniles returned the next spring. Overall, my results suggest that even small forest fragments on the breeding grounds are important as they can support high juvenile survival for Wood Thrushes and that survival is driven primarily by factors outside of the breeding grounds.
Open Access
Influence of a visual landmark shift on memory-guided reaching in the monkey
(2024-03-16) Lin, Jennifer Yi Xuan; Crawford, John Douglas
Reach and gaze data were collected from one female Macaca mulatta monkey (ML) trained to perform a memory-guided reaching task to determine the influence of allocentric cue shifts on reaching responses in the non-human primate. A landmark (4 ‘dots’ spaced 10° apart forming the corners of a virtual square) was presented at 1 of 15 locations on a touch screen. The landmark either reappeared at the same location (stable landmark condition) or shifted by 8° in one of 8 directions (landmark shift condition). ‘No-landmark’ controls were the same, but without the landmark. The presence of a stable landmark increased the accuracy of both gaze and touch responses and the precision of gaze. In the landmark shift condition, reaches shifted partially (mean = 29 %) with the landmark. Overall, these data suggest that the monkey is influenced by visual landmarks when reaching to remembered targets in a similar way as humans.
Open Access
Exploring Gene Expression Strategies in Potato Leafroll Virus
(2024-03-16) Arrigo, Julia; White, K. Andrew
Potato leafroll virus (PLRV) is a positive-sense single-stranded RNA plant virus in the genus Polerovirus (family Solemoviridae). This virus is known to use many gene expression strategies during viral infection, including subgenomic messenger RNA (sg mRNA) transcription and non-AUG initiation. In addition, it is known to produce a small viral RNA (svRNA) degradation product by cellular exoribonuclease digestion. The goal of this thesis was to investigate PLRV gene expression strategies and corresponding translational products. Two conserved structural elements, a sg mRNA promoter element, and a downstream stem loop (dSL), were identified. A previously defined RNA element involved in generating svRNA was also investigated. The results uncovered a sg mRNA transcriptional promoter element, a potential coding function for svRNA and no role for the dSL in modulating viral protein translation. These findings contribute to understanding how PLRV expresses its viral proteins during infections.
Open Access
Male mating effort and female preference in relation to male dominance rank, tenure length, and age in wild vervet monkeys.
(2024-03-16) Fane, Taylor Courtney; Schoof, Valerie
In this study, we aimed to evaluate the effect of male dominance rank, tenure, and age on male mating effort and female choice while quantifying four sociometric variables of male dominance hierarchy (stability, linearity, directional consistency, steepness) in two groups of wild vervet monkeys (Chlorocebus pygerythrus) at Lewa-Borana Conservancy, Kenya. We collected a total of 305 hours of behavioural data across a 4-month field season in 2022 on 13 adult/subadult males. Overall, adult, high-ranking males who had been in the group for a shorter amount of time, generally put forth more mating effort, copulated more frequently, and were preferred by females. The male dominance hierarchies for both groups were stable (mean S = 0.9844 ± 0.001), quasi-linear (mean h’ = 0.7554 ± 0.018), unidirectional (mean DCI = 0.7017 ± 0.121), and moderately steep (mean Dij = 0.6437 ± 0.044).
Open Access
Evaluating the extent and drivers of dietary specialization in polar bears (Ursus maritimus) across Western Hudson Bay
(2024-03-16) King, Kiersten Christina Kelva; Thiemann, Gregory
Climate warming has caused significant reductions in the extent of annual sea ice. As an ice-dependent species, the polar bear (Ursus maritimus) relies on the ice to hunt, making it susceptible to large-scale changes in sea ice and prey availability. My thesis sought to investigate individual-level foraging habits of polar bears in Western Hudson Bay from 2004-2021. I used fatty acid analyses and the proportional similarity index to investigate the extent and drivers of individual dietary specialization and interindividual differences in foraging habits. My results indicated a significant proportion of specialization, with the degree of specialization differing across age and sex classes. Annual snow depth and the North Atlantic Oscillation (NAO) were strong predictors of specialization, resulting in increased specialization with increasing snow depth and NAO. Continued monitoring efforts are necessary to ensure interindividual variation in foraging is considered when modelling the responses of polar bears to future climatic warming.
Open Access
Intestinal Immunolocalization and Insight on the Role of Tachykinin-Related Peptides in the Yellow Fever Mosquito, Aedes aegypti
(2024-03-16) Kohandel, Zeynab; Paluzzi, Jean-Paul
This study investigates a family of neuropeptides known as tackykinin-related peptides (TKRPs) in the mosquito Ae. aegypti, which is an insect of medical concern owing to its transmission of several arboviruses. As knowledge on TKRPs is limited in this mosquito, this study aimed to create a tk knockout line using CRISPR/Cas9 and characterize the distribution of TKRP immunoreactivity in the midgut over different physiological conditions including starved, sucrose-fed and blood-fed, and across developmental stages. The results demonstrate that TKRP immunoreactivity in the Ae. aegypti midgut is greatest in adult stage mosquitoes. Further, starvation significantly reduced TKRP immunoreactivity in the midgut compared to sugar fed adult mosquitoes, but no change was observed in relation to blood-feeding by females. Overall, this study established the intestinal distribution of TKRPs in Ae. aegypti and identified functional sgRNAs to disrupt the tk gene so that the physiological role of TKRPs can soon be characterized.
Open Access
Elucidating the Role of Syntaxin-1A in Cardiac Excitation-Contraction Coupling and Hypertrophic Remodeling in the Adult Mouse Heart
(2024-03-16) Anser, Fahad; Tsushima, Robert G.
Syntaxin-1A (STX1A) is a member of the SNARE family which is recognized for its role in membrane exocytosis and ion-channel regulation. Although the physiological properties of STX1A has been assessed in many tissues, its role within the heart remains unelucidated. To address this, a cardiomyocyte specific STX1A knockout (KO) model was generated in adult C57BL/6J mice. Through utilization of both in vivo and in vitro approaches, STX1A haploinsufficiency was demonstrated to induce transient contractile dysfunction, a global electromechanical delay, and reduction in Ca2+ handling gene expression. The depressed cardiac function was supported by volumetric overload of the left-ventricle and cardiomyocyte/sarcomeric restructuring. Furthermore, pathological hypertrophy was observed as evident by the re-expression of fetal genes and left-ventricular fibrosis. Collectively, the results suggest an important role of STX1A in cardiac E-C coupling, whereby its KO impairs the heart’s electromechanical function and induces ventricular remodelling to compensate for the elevated hemodynamic volume overload.
Open Access
Elastic tethers remain functional during anaphase arrest in partially-lysed crane-fly spermatocytes, and in non-lysed spermatocytes treated with microtubule inhibitors
(2024-03-16) Adil, Aisha; Forer, Arthur H.
Elastic tethers connect corresponding arms (telomeres) of partner chromosomes during anaphase segregation; they exert anti-poleward (backward) forces on the poleward-moving chromosomes. This thesis examined how arresting anaphase chromosomal segregation affected elastic tether function. Various dilutions of a standard immunofluorescent lysis buffer were used to partially lyse anaphase-I spermatocytes of crane flies. Partial cell lysis deactivated the anaphase spindle apparatus and arrested anaphase chromosomal segregation. Elastic tethers remained functional, and backward tether forces acting on chromosomes were able to cause backward chromosomal movements. Backward-moving chromosomes were attached to their kinetochore microtubules (kMTs) which might have been slowing them down. To test whether detaching chromosomes from their kMTs could enable their faster backward movements, anaphase spermatocytes were treated with various microtubule inhibitors to disassemble kMTs. The inhibitors led to anaphase segregation arrest, and backward chromosomal movements. However, the drugs were unable to disassemble the stable, acetylated kMTs, and allow faster backward movements.
Open Access
A non-canonical role for p107 in muscle stem cell fate decisions
(2024-03-16) Hsiung, Justin Wei-Liang; Scime, Anthony
The regenerative potential of skeletal muscle is attributed to the presence of resident muscle stem cells known as satellite cells (SCs). After activation SCs have two fates, either to differentiate into new muscle or self-renew to replenish their population. Dysregulation in favor of one outcome over the other has been implicated in loss of muscular regenerative capacity. Mitochondrial metabolism has recently emerged as a regulator of SC fate decisions. We uncovered a non-canonical mitochondrial role for retinoblastoma-like protein 1 (Rbl1, p107) in manipulating fate decisions through its effect on mitochondrial dynamics. We find that in the absence of mitochondrially localized p107, SCs display a higher rate of self-renewal, while also exhibiting higher mitochondrial connectivity. This was associated with increases in the mitochondrial fusion protein OPA1, as well as a loss in cellular acetylation. Taken together, these findings suggest that non-canonical p107 function in SCs controls their cell fate decisions.
Open Access
Electrophoresis- Driven Lateral Flow Immunoassay and Aptamer Selection
(2024-03-16) Teclemichael, Eden Tesfagiorgis; Krylov, Sergey N.
Electrophoresis has become an indispensable tool in Biochemistry and Molecular Biology, essential for analyzing proteins and nucleic acids. My work focuses on new bioanalytical applications of electrophoresis: lateral flow immunoassay (LFIA) and the selection of oligonucleotide aptamers. Electrophoretically-driven LFIA (eLFIA) is a new technique aiming to enhance diagnostic sensitivity of LFIA in both antigen and serological tests. While previously applied to Hepatitis B and C, I aimed to extend eLFIA's scope to analyze the SARS-CoV-2 spike protein, demonstrating a 77% reduction in the limit of detection compared to conventional LFIA. Shifting focus to aptamers, I utilized capillary electrophoresis (CE), with the highest partitioning efficiency, to address challenges in aptamer selection. I determined the optimum target concentration and developed bulk affinity assays workflow that quantitatively assesses the progress of selection. Understanding these parameters can significantly influence aptamer selection efficiency and can guide researchers in designing assays and developing novel diagnostic tools.
Open Access
The Diversity and Distribution of Avian Communities in Relation to the Shrubs Ephedra Californica and Larrea Tridentata in the Central California Desert
(2023-12-08) Hillier-Weltman, Zoe Emma; Lortie, C. J.
Interactions between key landscape features in desert ecosystems can influence avian community assembly. Bird species may use resources provided by shrubs, including as thermal refuges and as a food source. Citizen science data, such as eBird, is broadly accessible and has been underutilized in the study of fine-scale avian populations and distributions. eBird data offers opportunities for examining avian diversity and abundance across ecological gradients. Using citizen science data, I tested the hypothesis that shrub density and aridity predict the abundance and diversity of bird communities throughout the Central California desert. Shrub density and aridity were important predictors of avian diversity and abundance, but this effect was not constant across species. eBird data offers promise for testing predictions at fine spatial scales, but limitations in the quality and availability of data across locations must be taken into consideration.
Open Access
The stress response of pokeweed and phylogeny of a defense gene family in plants
(2023-12-08) Dougherty, Kyra; Hudak, Katalin A.
Pokeweed (Phytolacca americana L.) is a non-model plant known for its resistance to a variety of biotic and abiotic stressors. Part of the reason for this is the presence of ribosome inactivating proteins (RIPs), which can hydrolyze adenine bases from nucleic acids. These proteins are upregulated in pokeweed by jasmonic acid, a plant hormone involved in stress response. The goal of this research was to gain a better understanding of how plants respond to stress and so two different, but complementary, approaches were taken. Firstly, an in-depth look at the diversity and evolution of RIPs in plants was undertaken by curating a dataset of RIPs from publicly available data and using computational approaches to characterize their domain architecture, identify conserved amino acids, and construct a gene tree. This research revealed that despite the damage that RIPs can potentially cause to the plant’s own nucleic acids, RIPs are common among plants and their diversity indicates the potential for a multi-faceted impact on plant defense. Looking more closely at how pokeweed responds to stress, we applied jasmonic acid to leaves and analyzed changes in gene expression, through RNA sequencing (RNA-Seq). Identification of gene clusters involved in defense was aided by the generation of a pokeweed genome assembly. This research revealed that there is a variety of strategies that plants can implement to respond to stress, and that these strategies are applied differently by different species. Overall, this research contributes to a better understanding of the diversity and nuance present in the ways plants defend themselves.