Medicine - Masters by Research Theses

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    An investigation on proteolysis of the ACE2 receptor and its involvement in the cellular uptake and transmission of SARS-CoV-2
    (University College Cork, 2021-12-21) Wakerlin, Samantha Leigh; McCarthy, Justin V.; Lindsay, Andrew; Coleman-Vaughan, Caroline
    The renin-angiotensin system (RAS) is a key physiologic signalling network in blood and tissue homeostasis in humans. Angiotensin-converting enzyme 2 (ACE2) is a key regulator of the protective axis in RAS signalling as it antagonises the mechanisms of Angiotensin II (Ang II), the major vasoactive peptide of the RAS that can be dysregulated and overactive in disease states. The type I transmembrane protein can also function as a viral receptor, as ACE2 has recently been identified as the host receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 undergoes ectodomain shedding by ADAM17 and other host cell proteases, resulting in the release of its catalytic ectodomain into the extracellular space and the C-terminal fragment (CTF) secured in the membrane. Ectodomain shedding is a prerequisite for further cleavage by gamma-secretase in a two-step mechanism known as regulated intramembrane proteolysis, a common fate of many known receptors that function as viral targets. Given the structural similarity of ACE2 to other known substrates of gamma-secretase, the hypothesised role of ACE2 as a substrate for the protease was explored. Here, we show that the ACE2 CTF product of ADAM17/TMPRSS2 shedding is subsequently cleaved by the gamma-secretase protease to produce an intracellular domain (ICD) that is released within the cell. Pharmacological inhibition of gamma-secretase prevents generation of the ACE2 ICD and leads to the accumulation of membrane-anchored ACE2 CTF lacking the catalytic ectodomain. These observations demonstrate that ACE2 is a substrate for gamma-secretase proteolysis, providing a novel pathway for cellular trafficking of ACE2 that may have therapeutic potential in protective RAS signalling or antiviral immunity.
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    Single dose challenges in the diagnosis and management of cow's milk allergy in infants
    (University College Cork, 2021-06-28) d'Art, Yvonne; Hourihane, Jonathan O'B.; Gibson, Louise; Byrne, Aideen; National Children's Research Centre
    Background: Cows milk protein allergy (CMPA) is one of the most common food allergies in infancy. While it usually resolves slowly over time in most cases, it significantly disrupts family life and compromises affected childrens’ nutrition and growth. Parents often display significant anxiety about this condition and we speculated if this anxiety predates or develops in response to the onset of CMPA in their child. Single dose challenges are a new method of assessing dose reactivity in food allergic children. We recruited children referred for evaluation of CMPA to a randomised, controlled trial of single dose exposure to cows milk, using the validated dose of milk that would elicit reactions in 5% of CMPA subjects - the ED05, before implementation of graded exposure to CM (using the 12 step IMAP Milk Tolerance Induction Ladder) at home. Methods: 60 infants were recruited from referrals to 2 tertiary allergy centres and 1 secondary level allergy clinic. Inclusion criteria were age <12 months, a convincing CM allergic reaction <2 months before assessment and positive skin prick test (SPT) to milk +/- raised SpIgE to milk. Children were randomised 2:1 to a single dose of the ED05 for CM - (0.5mg milk protein) given as liquid CM (0.015mls) and observed for 2 hours post ingestion - or to no dose. Results: 60 patients were recruited, 57 (95%) were followed to 6 months, 3 intervention subjects were lost to follow up. By 6 months 27/37 (73%) intervention subjects had reached step 6 or above on the milk ladder compared to 10/20 (50%) control subjects (p=0.048). By 6 months 11/37 (30%) intervention subjects reached step 12 (ie drinking unheated cow’s milk) compared to 2/20 (10%) of the controls (p=0.049). 12 months post randomisation 31/36(86%) of the intervention group and 15/19(79%) of the control group were on step 6 or above.However 23/36(64%) of the intervention group were at step 12 compared to 7/19(37%) of the control group. Maternal state and trait anxiety were significantly associated with their infants’ response/progress on the milk ladder and with changes in skin prick test and spIgE levels at 6 and 12 months. Conclusion Using the 12 step IMAP milk ladder accelerates natural tolerance induction in infants under 12 months with CMPA. A supervised single dose at the ED05 significantly accelerates this further, probably by giving parents the confidence to proceed. Maternal anxiety generally reflects infants’ progress towards tolerance but preexisting high levels of maternal anxiety are associated with poorer progress.
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    Sensitising pancreatic cancer cell lines to electrochemotherapy by modulation of the cell cycle
    (University College Cork, 2021-03) Cooke, Katie Deniese; Forde, Patrick; Collins, Dearbhaile; Breakthrough Cancer Research
    We chose to research pancreatic cancer as pancreatic adenocarcinoma (PAC) is quite the aggressive disease, which even with the vast improvements in cancer treatments over recent years still has limited treatment options. Monotherapy treatment of pancreatic cancer with chemotherapy drugs initially show hopeful results, decreased disease symptoms and can lead to a slight improvement in survival but unfortunately does often ends in recurrence (1–3). Surgery has shown to be successful for respectable tumours followed by chemotherapy. Chemotherapy alone has proven to be associated with chemoresistance and can be quite intense, often patients are not able to continue with strong treatment regimes. FOLFIRINOX is classed as the gold standard for treating PAC, however, patient fitness ultimately determines suitability. Electrochemotherapy (ECT) is a new and emerging cancer therapy which allows for decreased doses of chemotherapy drugs to be used. We have evaluated pancreatic cancer cell lines response to low dose chemotherapy and Electrochemotherapy. The human pancreatic cell lines BxPC-3, PANC-1, and MIAPaCa-2 were used for all experiments in this thesis. Based on previous studies we predicted that PANC-1 cells would be most resistant to drug treatment followed by BxPC-3 and MIAPaCa-2 cells. All cell lines survived treatment with Gemcitabine (GEM) and Nab-paclitaxel (Nab), however, they had decreased survival when treated with 5’Fluorouracil (5’FU). When Electrochemotherapy (ECT) was applied to the cell line using GEM, Nab and 5’FU, cells survived and recovered but again had decreased survival and recovery with 5’FU ECT. Cells were also treated with Oxaliplatin (Ox) ECT, this had more of an impact to cell survival than with GEM/Nab/5’FU ECT. Bleomycin and cisplatin are the conventional drugs used with ECT due to their effectiveness (4,5), but we chose Ox as it is part of the FOLFIRONOX cocktail which is the gold standard for treating patients with PAC. Electrochemotherapy (ECT) has emerged as a promising treatment strategy for patients with pancreatic cancer (PC). ECT is safe for patients and has reduced side effects of systemic chemotherapy as drugs are concentrated to treatment site(6). ECT has shown to be very effective in treating melanoma (7). ECT has recently been used to treat locally advanced PC (8). Development of preclinical and clinical agents that inhibit cell cycle progression have proven effective in the treatment of cancer. Targeting cyclin dependent kinases (CDKs) and cell cycle checkpoint proteins can induce cell cycle arrest and apoptosis in cancer cells. Cell cycle deregulation is regularly seen in pancreatic cancer. Cells grow out of control and can lead to mortality. Controlling the deregulation with cell cycle inhibitors has shown to be beneficial for survival. Our study aimed to investigate CDK4/6 inhibitors and their ability to inhibit cell cycle progression. We specifically chose CDK4/6 inhibitors as they have proven to be quite effective in previous studies of breast cancer(9). Our hypothesis was that using a cell cycle inhibitor as a pre-treatment to ECT would make cells more susceptible to treatment. We used Palbociclib (PAL), a CDK4 inhibitor which causes cell cycle arrest at G1 and prevents cells progressing to S phase. We carried out experiments to investigate CDK4 levels in cells treated with PAL and cell cycle profiles of these cells post treatment. PAL prevents cell from passing to S phase of the cell cycle. We could not finish our planned work due to time, ceased funding and the COVID-19 global pandemic. We theorise that this combination treatment could enhance the efficiency of ECT and could potentially be translated into a clinical study as a new treatment option and improve patient quality of life.
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    Epitope mapping of the E2 glycoprotein, including the hypervariable region 1, of the hepatitis C virus genotype 3a, in the context of humoral immune pressure
    (University College Cork, 2020-12) Walsh, Nicole Ellen; Fanning, Liam J.; O'Farrelly, Cliona; Bristol-Myers Squibb
    The hepatitis C virus (HCV) is an enveloped +ssRNA virus, belonging to the family Flaviviridae. HCV is notable for displaying extraordinary genetic diversity and variability, having seven recognised genotypes and over sixty subtypes. HCV is responsible for the disease known as hepatitis C, which is associated with cirrhosis and hepatocellular carcinoma (HCC). The Global Hepatitis Report released by the World Health Organisation (WHO) in 2017 estimated that viral hepatitis was responsible for 1,340,000 deaths in 2015. The report also estimated that 71,000,000 people have ongoing HCV infections. HCV is largely transmitted via exposure to infected blood, with intravenous drug use accounting for approximately 55% of cases. HCV infections can be categorised as acute or chronic. During chronic HCV infections, antibodies (Abs) are produced against HCV - however, the host Abs are unable to neutralise HCV and only accelerate the evolution of circulating HCV variants. HCV variants resistant to the current generation of host Abs become the dominant variant through selective pressure. The variants of HCV within a host are known as quasispecies. Although the host Ab response is not able to resolve the chronic HCV infection, some Abs can bind to particular HCV variants. These Abs form complexes with virus particles and are known as AAVs (antibody-associated virus). AAVs are detectable in the blood of patients with chronic HCV infections and examination of these AAVs could reveal conserved viral structures and vulnerable HCV epitopes. Twenty genotype 3a serum and plasma samples from patients with chronic HCV infections were obtained from the National Virus Registry Laboratory (NVRL) and from the Molecular Virology Research and Diagnostic Laboratory (MVDRL). HCV genotype 3a was chosen for this research project given its prevalence (estimated to account for 17.9% of chronic HCV infections), resistance to treatment, and increased risk of causing severe steatosis and HCC when compared to other genotypes. Building on previous research carried out by the MVDRL, the patient samples were screened for the presence of AAVs. Initially, AAV+ samples were going to be processed and used to generate HCV pseudoparticles (HCVpp). The HCVpp system is a model system that incorporates the E1E2 glycoprotein from HCV into a plasmid. The E1E2 glycoprotein is responsible for HCV entry and infection, meaning the HCVpp can be used to infectivity and Ab neutralisation assays. However, the E1E2 glycoproteins could not be extracted from the AAV+ patient samples. Instead, the IgG from the AAV+ samples was extracted and used for a series of neutralisation experiments on HCV pseudoparticles generated using the HCV H77 isolate. H77 (GenBank: AAB67037.1) is an infectious genotype 1a isolate that has undergone complete genome sequencing. The Abs that showed the greatest neutralisation potential against the H77 pseudoparticles were selected for epitope mapping. The epitope mapping procedure tested the selected Ab samples against a synthesised H77 E2 glycoprotein structure, and characterised the sites where the patient Abs bound to the synthesised E2. This revealed vulnerable epitopes on the HCV E2 glycoprotein. The epitope mapping also revealed a large number of glycosylation sites around the vulnerable epitopes – a phenomenon known as glycan shielding. Glycan shielding is used by a number of viruses (including HCV and HIV) to protect conserved and vulnerable epitopes from Abs. However, strategies are being developed to counter viral glycosylation, including modifications to glycosylation sites and the use of polysaccharides derived from non-mammalian sources as therapeutic agents against glycosylated viruses.
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    Preparation for clinical practice in radiology: a survey of interns and radiologists
    (University College Cork, 2019-11-05) Simelane, Thabisile; O'Connor, Owen; Ó Tuathaigh, Colm
    Objective: The purpose of this study was to evaluate interns’ preparedness to deal with radiology departments, and to identify knowledge gaps which will facilitate the design of simplified evidence-based radiology orientation course for students or newly qualified doctors, to ease the transition from theory to practice. Methods: A cross-sectional, mixed-methods approach was employed. A sample of Irish interns and practicing radiologists were asked to complete a quantitative survey regarding perceptions of intern readiness to interact with the radiology department. A mixed quantitative/qualitative analysis using group concept mapping was also conducted involving perspectives of doctors in various categories, including NCHDs, and consultants, to evaluate what topics should be covered in a related preparatory course. Pearson’s Chi Square analysis was employed to examine the association between socio-demographic and educational variables and selected categorical item responses. Kruskal-Wallis analysis of variance (ANOVA) and/or Mann–Whitney U tests were employed to carry out univariate comparisons. Results: The majority of interns 66%( N=66) and radiologists 52%(N=26) felt that undergraduate medical training did not prepare the interns to interact with radiology department. More than half of the intern respondents 52%( N=52) were frequently uncertain about radiology exam indication when completing a request form. Most interns identified challenges in choosing appropriate examinations and communicating with the radiology department. A significant number also regarded the radiology department as unapproachable. Most radiologists 92%( N=92) felt that intern understanding of indications for imaging modalities is inadequate, reflecting intern uncertainty regarding exam indications. Most radiologists 86%(N=43) perceived that interns’ understanding of contrast medium was inadequate, and 78%( N=39) perceived that interns were lacking in the knowledge of (N=39) radiation protection. Results of the group concept analysis suggested the following topics to be included in the intern radiology preparatory module: ordering investigations; clinical decision support; radiology department IT and communication; adverse reactions and risks; urgent imaging; interpretation of radiology results. Conclusion: This study has highlighted vital topics to be included if one were to design a preparatory module in radiology which would promote smooth transition from theory to practice. Future research would be to design, implement, and evaluate an appropriate preparatory module.