College of Science, Engineering and Food Science - Masters by Research Theses
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Item A model of SPRY3 - VGCC interactions relating to autism(University College Cork, 2023) Bharatham Vijayaraghavan, Sashank; Moore, Thomas F.; Burk, KatjaAutism Spectrum Disorder (ASD) is a neurobehavioral condition characterized by impaired social interaction and communication resulting from irregular brain development during infancy and adolescence. Globally, over 168 million cases of ASD have been diagnosed, with Ireland ranking sixth in prevalence (583.69 cases per 100,000 people). Heritability, estimated between 40% and 80% in twin and family studies, underscores the importance of identifying susceptibility genes in ASD research. The ASD susceptibility candidate gene SPRY3 encodes a receptor tyrosine kinase inhibitor and plays a regulatory role in branching morphogenesis. SPRY3 is highly expressed in cerebellar Purkinje cells. Spry3 and p75NTR have opposite expression patterns in the cerebellar vermis of the mouse and it has been hypothesized that reactivation of the epigenetically silenced Y-linked SPRY3 copy in the human might interact with the TrkB and p75NTR signaling pathways to cause Purkinje cell pathology. The current work evaluates the potential that SPRY3-GFP colocalizes with neurotropic receptors in HEK293 cell line model and found that SPRY3-GFP colocalizes with TrkB-RFP and p75NTR-RFP. Significant colocalization of SPRY3 with EGFR-GFP and TrkA-RFP was also observed. This investigation detected no colocalization between SPRY3-RFP and CasR-GFP, which is a G-protein-coupled extracellular calcium-sensing receptor. The involvement of voltage-gated calcium channels (Cav) have been implicated in ASD and their role in the regulation of branching morphogenesis in the brain and lung, and the fact that calcium acts as a secondary messenger to modulate various signaling pathways involved in branching morphogenesis, we hypothesized a functional relationship between SPRY3 and calcium signaling mediated through Cav receptors. SPRY3 plays an important role in regulating axon branching of motor neurons. Similarly, growth cones rely on calcium signaling to respond to guidance cues and adjust their behavior accordingly and it is critical for proper axon extension and guidance. Using calcium imaging in SH-SY5Y cells, we found that adding KCl to SH-SY5Y cells expressing SPRY3-GFP significantly altered the function of Cav 1.2, Cav 1.3, and Cav 2.2. SPRY3-GFP did not alter the levels of calcium when the L-type inhibitor nifedipine was added to SH-SY5Y cells, shows that SPRY3 interacts with Cav 1.2 and Cav 1.3. Cav2.2 is a prominent VGCC in IMR-32 cells, where calcium levels are not altered. Also, we found that adding BDNF to SH-SY5Y cells transfected with SPRY3-GFP shown no changes in intracellular calcium levels. Overall, this study suggests an interaction between SPRY3 and L-type VGCCs, which may be relevant to ASD pathogenesis. In a pilot study, there was no significant difference in the mRNA expression of FMR1-, a known ASD gene in SPRY3-GFP transfected SH-SY5Y cells compared to GFP transfected SH-SY5Y cells.Item Dynamics of adaptive recurrent neural networks(University College Cork, 2023) Fox, David; Amann, Andreas; Keane, Andrew; University College CorkIn this thesis a simple, phenomenological model of a neural network with plasticity is presented in the form of a slow-fast adaptive dynamical recurrent neural network. The plasticity rule is chosen from the class of Hebbian learning rules, in which the synaptic connection between two neurons evolves continuously as a function of their correlation in the recent past. Initially an analysis of networks of two neurons is presented, which exhibit relaxation oscillations in which one neuron switches between an ’off’ state, where it takes a negative value, and an ’on’ state, where it takes a positive value, while the other neuron stays in one on/off state. Then, by means of an example with a nine neuron network, the system is shown to exhibit both stable frequency cluster synchronization and transient frequency cluster synchronization.Item Neurobiological effects of food fermentation-derived metabolites for metabolic and mental health(University College Cork, 2023) Carey, Nathan; Schellekens, Harriet; O'Mahony, Siobhain M.Nutrition and diet are becoming increasingly popular therapeutic interventions as we discover more about the complex roles the foods we consume play in maintaining our health status. It is now clear that foods we ingest daily and their metabolites interact with systems both within and outside the gastrointestinal tract including the gut microbiome, the nervous system, immune system and hormonal system. Each of these play essential roles in the bi-directional communication pathway of the microbiota-gut-brain axis. Interactions between our food and this axis can potentially influence centrally mediated processes such as cognition, mood and even appetite. While several foods have been identified as being beneficial to our health, there is one food group that remains under investigated and holds promise as a reservoir of both beneficial bacteria and bioactive compounds – fermented foods. Fermented foods are created through the controlled enzymatic conversion of foods to simpler organics substances by microorganisms. Common examples include foods like kimchi (a fermented cabbage product), kefir (a fermented milk product) and kombucha (a fermented sweet tea beverage). While human studies on fermented foods remain sparce, one recent finding recorded lower perceived stress in human adults who underwent a dietary intervention that included fermented food intake. Recent findings in rodents suggest that fermented foods can alter social behaviour, reduce body weight and lead to reduced anxiety in animals. The mechanism by which fermented foods act is still unknown but it likely due to a number of factors such as their probiotic bacteria content, their metabolite content including short chain fatty acids (SCFA), and the ability to breakdown their starter compounds into simpler molecules and increase their bioavailability such as phenolic compounds in fruits and vegetables. The research conducted in this thesis aims to investigate the ability of food-fermentation derived metabolites, with a specific focus on SCFAs and polyphenols, to alter the neurobiological functions associated with central appetite regulation (hypothalamus) and cognition (hippocampus). Using in vitro assays, we tested the selected panel of metabolites shown to be found in fermented foods, and capable of crossing the blood brain barrier, on both immortal cell lines (hypothalamic and hippocampal) and on primary neurosphere cultures (hippocampal). A panel of SCFA were administered to hippocampal and hypothalamic cell lines and were capable of altering brain-derived neurotrophic factor (BDNF) gene expression. These metabolites were also tested on primary hippocampal cells using a neurosphere assay of proliferation. Positive trends were observed across many of the treatments, however these trends were not significant. Perhaps most interesting were our findings when submitting the same hippocampal neurosphere assay to a panel of phenolic compounds. Apigenin and kaempferol (both flavonoids) significantly increased hippocampal cell proliferation. Moreover, to enhance the efficiency of data analysis, a semi-automatic quantification pipeline was developed for high-throughput screening of primary neurosphere cultures. This pipeline offers a systematic and reliable method for evaluating neurosphere proliferation, providing a valuable tool for future studies in the field. Our results highlight the modulatory effects of SCFA and phenolic compounds on hypothalamic and hippocampal cells in vitro, emphasising the potential role of dietary metabolites and fermented foods as a whole on brain function related to metabolic and mental health. These findings also highlight the need for more in depth analysis of fermented foods and their neuromodulator effects both in vivo.Item Differential regulation of the phosphorylated and dephosphorylated forms of the ubiquitin-conjugating enzyme Ubc6e(University College Cork, 2024) Burns, Stephanie; Fleming, John V (Eoin)The ubiquitin proteasome system (UPS) is a key stress response employed by cells to remove damaged and misfolded proteins that accumulate in the endoplasmic reticulum (ER) during energy deprivation, hypoxia, or viral infection. Ubc6e is an ER-localised ubiquitin-conjugating enzyme that plays an important role in the proteasomal degradation of misfolded proteins and can be phosphorylated at serine residue 184. Recent studies have demonstrated that Ubc6e is co-localised with p62/sequestrosome, which is a cargo receptor that delivers ubiquitinated cargo to autophagosomes for degradation. To further investigate the possible role that Ubc6e may play in autophagy, we aimed to investigate any protein-protein interactions between p62 and Ubc6e and we also aimed to investigate the effect of autophagic-related proteins, p62 and ULK1 on Ubc6e. From our research, we saw that Ubc6e levels are reduced when co-expressed with proteins that promote autophagy. Specifically for the autophagy inducer ULK1, we saw differential regulation between the phosphorylated and dephosphorylated forms of Ubc6e, suggesting that the S184 phosphorylation may protect the protein from autophagic degradation. Degradation of the dephosphorylated form, on the other hand, did not depend on S184 phosphorylation, or the catalytic activity of Ubc6e. Neither was it dependent absolutely on ER localisation. Finally, although ULK1 co-expression did not appear to specifically alter the ER/ perinuclear localisation of Ubc6e, it was noted that amino-terminal tagging with GFP led to a ULK1-related localisation of Ubc6e to intracellular vesicles. Together our results suggest that Ubc6e does play a role in autophagy however it is not mediated by the direct interaction with p62.Item Dissolvable microneedle manufacturing methods and the application of three-dimensional printing to pharmaceuticals(University College Cork, 2024) Wilkstein, Katerina; Moore, Anne; Vucen, Sonja; HEA AIVRTDissolvable microneedles are an intradermal drug delivery technology with exciting potential for widespread distribution of minimally invasive, self-administered medicines and vaccines. The most popular manufacturing method for medical dissolvable microneedles is micromoulding, a process which can have drawbacks of low material efficiency and high regulatory requirements. The UCC-patented “ImmuPatch” process is a micromoulding method which significantly improves on the material efficiency of traditional micromoulding. In this study, a specific method based upon the ImmuPatch process was developed for aseptic manual fabrication of two-layered dissolvable microneedles capable of incorporating at 2 μg protein dose. The developed method was not ideal, with high variability in the patches produced and significant challenges in verifying the protein dose. Fabrication of dissolvable microneedles is well positioned for the application of automation methods. One proposed option for the automation of dissolvable microneedle manufacture is additive manufacturing, which in addition to automation has the advantage of eliminating the regulatory challenges associated with micromoulding. A systematic literature review of methods applying additive manufacturing to the fabrication of solid dosage formats was conducted and identified processing parameters and format properties associated with different technologies. Additive manufacturing has been applied to micromould-free dissolvable microneedle fabrication in limited examples and should be explored further.