Pharmacology and Therapeutics - Doctoral Theses
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Item Characterisation of the role of mitochondrial dysfunction and meta-inflammation as a shared pathogenic network in gestational diabetes mellitus(University College Cork, 2023-05-02) McElwain, Colm; McCarthy, Cathal; McCarthy, Fergus; Health Research BoardBackground Gestational diabetes mellitus (GDM) is one of the most prevalent obstetric complications, with a growing incidence resulting from upward trends in global obesity and metabolic syndrome. GDM pathology is considered a state of exaggerated insulin resistance in pregnancy, mediated by pancreatic beta cell insufficiency, adipose dysregulation and systemic meta-inflammation. Although the pathophysiology of GDM is complex, adipose tissue dysfunction is an established cause of metabolic dysfunction and systemic insulin resistance. Furthermore, the placental microenvironment is highly sensitive to GDM physiology and there is significant evidence that the hyperglycaemic state of GDM drives placental dysfunction, in part mediated by exaggerated oxidative stress and inflammation. This thesis aims to characterise the role of systemic and local tissue mitochondrial dysfunction and meta-inflammation in promoting GDM pathology. Methods In this study, the inflammatory and metabolic profiles of omental visceral adipose tissue (VAT), placental tissue and fasting maternal blood were extensively studied to identifiy pathways which may drive insulin resistance in GDM and contribute to both acute and chronic adverse maternal and fetal outcomes. Samples were collected at term pregnancy from nulliparous women with normal glucose tolerant pregnancies and women diagnosed with GDM. Various immune-endocrine mediators were investigated in VAT, placental tissue and the maternal circulation including markers of mitochondrial dysfunction, monocyte/macrophage population frequency, hormones, inflammatory cytokines and insulin signalling transduction. Results Firstly, distinct phenotypes of GDM were confirmed, relating to both insulin-deficient and insulin-resistant pathologies. Insulin-deficient GDM participants had lower body mass indexes (BMIs) and were significantly hypoinsulinaemic compared to insulin-resistant GDM participants, who had elevated BMIs with substantial VAT dysfunction, including adipocyte hypoplasia and dysregulated insulin signalling capacity. The placental microenvironment was also negatively affected in GDM, with a potent pro-inflammatory secretome including increased release of IL-6, TNF-α and IL-18. This inflammatory phenotype was mirrored in the maternal circulation, confirming that systemic low-grade inflammation is present in term GDM physiology and is, in part, facilitated by placental signalling. Mitochondrial dysfunction was also confirmed in GDM participants, characterised by exaggerated mitochondrial superoxide production in placental macrophages and elevated circulating levels of cell-free mitochondrial DNA. Ex vivo mitochondrial antioxidant therapy attenuated excessive placental inflammation in GDM, proposing a promising therapeutic avenue for alleviating systemic inflammation in GDM patients. Conclusion In this thesis, we have provided substantial evidence of metabolic dysfunction and inflammation in the maternal circulation, omental VAT and placental tissue of women with GDM. This includes evidence of defects in adipocyte expansion and impaired insulin signal transduction, in addition to placental and systemic mitochondrial dysfunction and inflammation, which may ultimately orchestrate GDM pathophysiology. These findings identify novel pathological pathways in GDM, which may be further delineated to establish their potential as therapeutic targets to alleviate adverse maternal outcomes.Item The impact of host-microbe interactions on murine colonic secretomotor function(University College Cork, 2014) Lomasney, Kevin W.; Hyland, Niall P.; Cryan, John F.; Science Foundation IrelandThe overall objective of this thesis was to gain further insight into the mechanisms underlying commensal microbial influences on intestinal ion transport. In this regard, I examined the impact of commensal host-microbe interactions on colonic secretomotor function in mouse. I first examined the influence of two different probiotic (microorganisms which, when given in adequate amounts, can confer health benefits upon the host) strains, Bifidobacterium infantis 35624 and L. salivarius UCC118 on active colonic ion transport in the mouse, using the Ussing Chamber. I found that both probiotics appear to have converging effects on ion transport at a functional level. However, L. salivarius UCC118 may preferentially inhibit neurally-evoked ion transport. Next I examined the impact of the host microbiota itself on both baseline and stimulated colonic secretomotor function as well as probiotic induced changes in ion transport. I provide further evidence that the microbiota is capable of mediating alterations in colonic ion transport, and specifically suggests that it can influence cAMP-mediated responses. Finally, it has been well documented that many probiotics elicit their effects via secreted bioactives, therefore, I studied the effects of microbially produced GABA, contained in supernatants from the commensal microbe Lactobacillus brevis DPC6108, on colonic secretomotor function. In conclusion, I believe that commensal microbes have an important and strain specific functional influence on colonic ion transport and secretomotor function and these effects can be mediated via extracellular bioactives. Moreover, I believe that functional ex-vivo studies such as those carried out in this thesis have a critical role to play in our future understanding of host-microbe interactions in the gut.Item Investigation of the genotoxic potential of the marine biotoxins okadaic acid and azaspiracids(University College Cork, 2014) Dörr, Barbara Valentina; Van Pelt, Frank; O'Halloran, John; Higher Education AuthorityThe present study investigated the genotoxic potential of the marine biotoxins okadaic acid (OA) and azaspiracids (AZAs). Harmful algae blooms (HABs) are an increasing global problem with implications for the ecosystem, economy and human health. Most data available on human intoxication are based on acute toxicity. To date, limited data has been published on possible long term effects, carcinogenicity and genotoxicity. To investigate genotoxicity in the present study, DNA fragmentation was detected using the COMET assay. In contrast to most other available studies, two further endpoints were included. The Trypan Blue Exclusion assay was used to provide information on possible cytotoxicity and assess the right concentration range. Flow cytometer analysis was included to detect the possible involvement of apoptotic processes. In house background data for all endpoints were established using positive controls. Three different cell lines, Jurkat T cells, CaCo-2 cells and HepG-2 cells, representing the main target organs, were exposed to OA and AZA1-3 at different concentrations and exposure times. Data obtained from the COMET assay showed an increase in DNA fragmentation for all phycotoxins, indicating a modest genotoxic effect. However, the data obtained from the Trypan Blue Exclusion assay showed a clear reduction in cell viability and cell number, indicating the involvement of cytotoxic and/or apoptotic processes. This is supported by data obtained by flow cytometer analysis. All phycotoxins investigated showed signs of early/late apoptosis. Therefore, the combined observations made in the present study indicate that OA and AZA1-3 are not genotoxic per se. Apoptotic processes appear to make a major contribution to the observed DNA fragmentation. The information obtained in this study stresses the importance of inclusion of additional endpoints and appropriate positive controls in genotoxicity studies. Furthermore, these data can assist in future considerations on risk assessment, especially regarding repeated exposure and exposure at sub-clinical doses.