Undergraduate Honours Theses (Biology)
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Item Chronic states of iron deficiency and excess in combination with erastin: An in-vitro study of acute monocytic leukemia (THP-1 cells)(2025) Fenniri, Zachariah A. H.Iron is essential for numerous biological processes, due to its inherent capacity to transfer electrons. This chemical feature is a double-edged sword, as excess iron catalyzes the formation of reactive oxygen species (ROS) that have potential to cause cellular damage and induce ferroptosis – an iron-dependent form of cell death. Dysregulated iron homeostasis, in favour of accumulation, is a central feature of highly proliferative leukemias, as it enables the required energy metabolism and biosynthesis for unrestrained proliferation. Iron deprivation through the use of iron chelators and the induction of ferroptosis are two potential avenues of clinical antileukemic treatment. The present study aimed to examine the effects of chronic states of iron deficiency and excess in THP-1 cells, an in-vitro model of acute monocytic leukemia (AMoL). Moreover, this study sought to examine the effects of these chronic iron states on THP-1 cell resistance to erastin, a compound that induces ferroptosis in many cells, but is largely ineffective in leukemic cell lines. THP-1 cells were cultured in various concentrations of ferric citrate or deferoxamine (DFO), a clinical iron chelator, for 96 hours to simulate chronic states of iron excess and deficiency, respectively. Following 72 hours of treatment, cells were administered erastin for the final 24 hours of treatment. Cell death, metabolic activity, and intracellular glutathione (GSH), a pivotal antioxidant, were quantified. Moderate to high doses of DFO induced marked cell death, as well as a notable reduction in metabolic activity and intracellular GSH. THP-1 cell resistance to erastin was partially attenuated by ferric citrate treatment, evidenced by a small but consistent iron-dose dependent increase in cell death. Similarly, intracellular GSH showed a subtle, insignificant iron-dose dependent reduction in erastin-treated cells. Metabolic activity in the surviving cells was unaffected. DFO is shown to act synergistically with erastin in THP-1 cells, inducing a significant increase in cell death, and in the surviving cells, a reduction in metabolic activity. Likewise, intracellular GSH increases in the surviving cells of this cotreatment. Taken together, the present study demonstrates the robustness of THP-1 cells in states of iron excess and their sensitivity to iron deficiency. It also provides novel evidence of a synergy between DFO and erastin, and of ferric citrate attenuating THP-1 cell resistance to erastin. Supervisors: Patrick B. Walter and Jürgen EhltingItem Establishing a new protocol to investigate sensory adaptation in a mouse model of Rett Syndrome(2022) Kaban, TaylorThis project is a continuation of Farhoomand (2021). Dr. Farhoomand studied sensory adaptation in the hindlimb to primary somatosensory cortical pathway in a mouse model of Rett syndrome to better understand the loss of MeCP2 function in neuronal circuit and sensory processing. Cortical evoked responses (CERs) to vibratory tactile stimulation of the hind limb were assessed via intrinsic optical imaging (IOS) and intracortical local field potential recordings (LFPs) before, during, and after 1 hour of repetitive vibratory stimulation at 100hz. After 1 hour, the CER was reduced by approximately 40% in both Rett mice (RTT) and wild-type mice (WT). Reduced responses persisted for at least 60 minutes in wild-type mice but recovered to 90-100% of baseline within 15 – 30 minutes in Rett mice. Analysis of this phenomenon via LFP within the test train indicated that the reduced CER was due to an increase in short term adaptation during the 7-stimulus train that was retained in the WT mice but reversed rapidly in the RTT mice. We therefore propose that the persistent sensory adaptation differences between WT and RTT that are mediated by increased short-term adaptation may reflect enhanced feedback by inhibitory elements of circuits within the sensory pathway. The lack of the adaptation to persist after continuous stimulation in the RTT mice may therefore reflect a deficit in the capacity for activity dependent plasticity to consolidate. To determine if this is the case, this project sought to establish a new protocol to further probe this phenomenon of sensory adaptation with a larger cranial window that allows for the simultaneous recording of the CER from both hemispheres. We also seek to replace the piezo device with a new vibratory device that does not leave an artifact in the LFP data. To establish this new protocol this project sought to answer three main questions: (1) Can we reliably replace the piezo vibratory device with a motor device that is cheaper and doesn’t leave an artifact in the data. We found that while there was a significant difference in the timing of the CER after stimulus offset with the motor device, there was no significant difference in the variability of that timing, suggesting consistency with the motor device. We also found no significant difference in the sum of the peaks (mV) between the piezo device and one of the motor settings tested, and finally, we found no significant difference in the paired pulse ratio. In summary, the motor device can reliably replace the piezo device. (2) Do we see evidence for adaptation with the motor device? We found that there was evidence for adaptation, though there may be differences in the persistence of that adaptation. (3) Is there evidence for cross adaptation? We found that while there was a reduction in the CER after the adaptation phase, it wasn’t statistically significant (likely due to the small sample size of N=2). In summary, this project has established many successful aspects of a new protocol to further probe sensory adaptation. It has also paved a clear direction for the next steps in further progress towards our goal of providing a platform to understand some of the circuit level substrates of the sensory, learning, and cognitive deficits in RTT patients.