Biochemistry and Cell Biology - 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 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.Item Impact of endosomal recycling inhibitors on drug resistant and immune tolerant breast cancers(University College Cork, 2024) Chembukavu, Suraj Narayanan; Lindsay, AndrewBreast cancer (BC) is the most common and frequent of all the cancers, accounting for about 25% of all the cases of cancer detected in women annually. There are a number of approved drugs on the market that have been developed or repurposed for the treatment of BC. However, a recurrent problem faced in the clinic is the emergence of drug resistance, which can be intrinsic or acquired. Prolonged periods of chemotherapeutics and targeted therapy have resulted in the development of acquired resistance through a number of different adaptations. This project focuses on resistance developed by various sub-types of breast cancer cells through drug-therapy-induced senescence. Previous studies have reported the cytoprotective nature of this cell cycle arrest phenomenon upon prolonged exposure to targeted- and chemotherapeutics. This study investigates specific molecular mechanisms governing therapy-induced senescence, the impact it has on the membrane trafficking of iron transporters, and the potential repurposing of endosomal recycling inhibitors (ERIs) to target these drug-tolerant cells. Additionally, we examined the effects of ERIs on the total protein and surface levels of immune checkpoint proteins. This was done by targeting the recycling of the glycoprotein PDL1, which is expressed on the surface of some tumour cells and binds to its cognate receptors on the surface of T cells. PDL1-PD1 binding leads to T cell inactivation. A number of ERIs were used to determine their impact on the levels of PDL1.Item Novel RNA-based biomarkers for ovarian cancer – uncovering how LINC01132 is regulated by the tumour suppressor protein, p53(University College Cork, 2023) Hartigan, Shaun; Dean, Kellie; McKenna, Sharon L.; Irish Research CouncilOvarian cancer is one of the deadliest female cancers worldwide. Most cases are detected in advanced stages, as it is difficult to diagnose ovarian cancer early due to nonspecific symptoms. Presently, there are no sensitive biomarkers to identify early-stage disease. Over 60% of ovarian cancer cases have a mutation in p53, a tumour suppressor transcription factor which counteracts cell stress and oncogenic signals. Most mutations occur within p53’s DNA-binding domain, a vital region which facilitates its anchorage to target gene promoters. Previous data found numerous long-non-coding RNAs (lncRNAs) are differentially expressed in ovarian cancer cells with mutant TP53, compared to those with the wildtype gene. Here, we show that three p53 mutants (R175H, I195T and R248Q) commonly found in ovarian cancer patients, were unable to activate firefly luciferase expression from a synthetic p53-responsive promoter, reflecting the impact of p53 core domain mutations on its ability to bind target promoters and transactivate gene expression. In silico genome-wide ChIP-seq analysis of differentially expressed lncRNAs identified three (MEG3, LINC01132, and LINC2960) containing regions within -1 kb of their transcription start sites, showing interaction with p53. Another four (EMX20S, PRICKLE2-DT, LINC00887 and LINC02610 contained p53-interacting regions within -5 kb. Division of the region up to -6,995 bp of the LINC01132 transcription start site into a distal, middle, and proximal segment, and subsequent cloning upstream of firefly luciferase, allowed us to assess p53 activity at the LINC01132 promoter. Western blot analysis could not detect luciferase expression from either segment under wildtype p53 overexpression, despite the proximal segment containing six p53-interacting sites. To determine the true response of wildtype and mutant p53 binding to the LINC01132 promoter, future quantitative reverse-transcriptase PCR and luciferase assays should be conducted, given their higher sensitivity compared to Western blot analysis. To improve patient prognosis in ovarian cancer, there is vital necessity to discover specific biomarkers, which can diagnose and monitor disease progression. LncRNAs can be detected in blood, so linking expression of differentially expressed lncRNAs to the mutational status of p53 in ovarian cancer, and studying how these change with the therapies, is novel, previously unexplored, and may aid in biomarker discovery.Item Interrogating regulated intramembrane proteolysis of IGF-1R, and its role in cell migration(University College Cork, 2023) O'Donoghue, Jordan Christopher; McCarthy, Justin V.; O'Connor, Rosemary; University College CorkThe IGF-1R has long been implicated in numerous malignant characteristics of cancers including enhanced proliferation, insensitivity to apoptosis, chemoresistance, increased migratory and invasive capacity, epithelial-to-mesenchymal transition, and metabolic augmentation. IGF-1R, much like other receptor tyrosine kinases, has been characterised as a substrate for gamma-secretase-mediated regulated intramembrane proteolysis (RIP). This proteolytic pathway begins with ectodomain shedding, mediated by sheddases, which produces a soluble fragment in the extracellular space. Following ectodomain shedding, a membrane anchored C-terminal fragment (CTF) is produced which is then cleaved by gamma-secretase at the internal juxtamembrane region to produce a soluble intracellular domain (ICD). Classically, RIP was thought to be a means by which cells would terminate receptor signalling, however contemporary research indicates a more nuanced role. It is now understood that the fragments generated through RIP can retain signalling of the full-form receptors and, in some instances, may acquire novel functionality. Although the RIP of IGF-1R has been outlined, the underlying dynamics of this pathway and the consequences of such remain to be elucidated. With this in mind, we endeavoured to identify the regulatory mechanisms underlying IGF-1R RIP and subsequently aimed to identify potential functions of the fragments produced. Firstly, we generated a GFP-tagged K1003 IGF-1R point mutant, termed a kinase dead mutant, which lacks the ability to catalyse trans-autophosphorylation of the IGF-1R kinase domains thus preventing downstream signal transduction. Trans-autophosphorylation is a critical step in the activation of receptor tyrosine kinases during which the kinase domain of one intracellular domain catalyses the phosphorylation of tyrosine residues within the other intracellular domain. With this tool, we validated the cleavage of IGF-1R and the production of its CTF via transient transfection of HEK293T cells with plasmids expressing either wild-type or K1003R kinase dead mutant forms of IGF-1R C-terminally tagged with GFP. We subsequently identified receptor kinase activation as a potential driver of IGF-1R RIP and determined that ligand stimulation significantly catalyses the accumulation of IGF-1R CTF. This was accomplished via transient transfection of HEK293T cells with plasmids expressing the aforementioned IGF-1R forms. Following this, we elucidated clathrin-mediated endocytosis as a probable regulatory pre-requisite step for IGF-1R RIP by gamma-secretase. Furthermore, our data indicates that ectodomain shedding of IGF-1R is likely catalysed in a metalloprotease dependent fashion, consistent with other receptor tyrosine kinases. We also confirmed that the CTF can undergo nuclear translocation in a clathrin-mediated endocytosis dependent manner, consistent with the translocation of the holoreceptor. Utilising wound healing assays, conducted in Hela cells, our data indicates that gamma-secretase inhibition by DAPT (a small molecule gamma-secretase inhibitor) alone does not antagonise IGF-1-induced cell migration. Similarly, the administration of Pitstop alone does not impinge upon IGF-1 induced cell migration. Interestingly, the administration of batimistat (a broad spectrum metalloprotease inhibitor) significantly suppresses cell migration in the presence of IGF-1. Collectively, our data provides a framework for the construction of the IGF-1R RIP cascade. Following ligand-binding it appears that IGF-1R undergoes metalloprotease-dependent ectodomain shedding leading to loss of the extracellular alpha-chains. The CTF, which remains in the plasma membrane, then undergoes clathrin-mediated endocytosis and packaging into early endosomes. Following internalisation, the CTF is cleaved by gamma-secretase to generate a soluble ICD. Our data also demonstrates that IGF-1R CTF undergoes nuclear translocation in a clathrin-mediated endocytosis dependent fashion. Lastly, data gathered from our wound healing assays indicates that IGF-1R ectodomain shedding is likely a required event for the efficient IGF-1-induced migration of cells, possibly through the proteolytic augmentation of focal adhesion complexes containing IGF-1R.