Pharmacology and Therapeutics - Doctoral Theseshttps://hdl.handle.net/10468/17892024-03-29T00:11:23Z2024-03-29T00:11:23Z121A study of magnesium stearate behaviour in pharmaceutical blends and tablets employing Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS)Peddapatla, Raghu V. G.https://hdl.handle.net/10468/96992023-04-04T07:27:23Z2019-01-01T00:00:00Zdc.title: A study of magnesium stearate behaviour in pharmaceutical blends and tablets employing Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS)
dc.contributor.author: Peddapatla, Raghu V. G.
dc.description.abstract: Magnesium Stearate (MgSt) is the most commonly used lubricant in pharmaceutical industries. Effects of MgSt on the final product have been extensively studied in batch processing. In recent times pharmaceutical companies have been increasingly interested in continuous processing, where the relative effects of material properties and process parameters on blend behaviour during continuous processing has gained significant attention. It is important to assess the behaviour of materials and it is a challenging feat to monitor behaviour of very cohesive materials like MgSt in continuous processing. The main aims of this thesis were, to investigate the role of MgSt supplier variability during continuous feeding through a loss in weight feeder (LIW) and to investigate the capability of Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS) to discriminate between blends and tablets with variable MgSt distribution. Initially, the variability among four different grades of MgSt samples from two different suppliers was studied. The variability among the samples was evident (chapter-3) and the effect of this variability on continuous feeding performance of MgSt samples was studied (Chapter-4). Bulk density of the samples dictated the feed factor achieved for the MgSt samples, when fed through K-Tron MT12 feeder (Chapter-4). Higher variability in the feed rate RSD was noticed for the MgSt samples, when fed at lower feed rate of 0.15 kg/hr and for samples (Ligamed MF-2- V and Ligamed MF-2-V-BI) with similar properties. Post feeding characterisation of MgSt samples was performed to identify any effect of feeding on particulate properties. A reduction in particle size due to feeding of the samples was noted and these samples when included in tablet blends, showed a delayed drug release, which was more prominent in tablets with fed MgSt of Alfa Aesar and Ligamed MF2-V samples. Ligamed MF-3-V was least effected by feeding and when fed samples were included in formulations a very slight delay in drug release was noted compared to other tablets with other MgSt samples (Chapter-4). A novel technology, BARDS was employed for the first time to analyse the excipients, tablet blends and tablets (Chapter-5 and Chapter-6). Analysis of unlubricated and lubricated blends using BARDS, clearly discriminated between the blends, resulting inan extended acoustic response for lubricated blends. K-Tron MT12 feeder, was used to feed the unlubricated and lubricated blends at three different feed rates (0.2238 kg/hr, 0.5594 kg/hr and 1.006 kg/hr), anticipating lubricated blend with varied degrees of blend lubrication. When analysed using BARDS, unlubricated blend fed at increasing feed rates showed similar acoustic response, whereas lubricated blend showed extended acoustic response, which was dependent on the feed rate (Chapter-5). Gas elimination rate constant was used to determine the degree of lubrication within blends. The degree of overlubrication was further confirmed by the standard wetting techniques and tabletability of the blends (Chapter-5). Tablets were produced from unlubricated and lubricated blends at a compression pressure range of 30 MPa to 234 MPa. The influence of compression pressure and tablet properties, on the BARDS acoustic response was investigated in Chapter-6. The yield pressures calculated from the Heckel analysis was 98 MPa and 102 MPa for unlubricated and lubricated tablets. A significant change in the BARDS acoustic response was noticed for tablets produced above and below yield pressure for both blends. Lubricated tablets produced at higher compression pressures, showed delamination, which was identified by BARDS. Slow gas elimination rate constant was observed for lubricated tablets compared to unlubricated tablets.
2019-01-01T00:00:00ZCharacterisation of the role of mitochondrial dysfunction and meta-inflammation as a shared pathogenic network in gestational diabetes mellitusMcElwain, Colmhttps://hdl.handle.net/10468/145832023-11-06T11:14:46Z2023-05-02T00:00:00Zdc.title: Characterisation of the role of mitochondrial dysfunction and meta-inflammation as a shared pathogenic network in gestational diabetes mellitus
dc.contributor.author: McElwain, Colm
dc.description.abstract: Background
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.
2023-05-02T00:00:00ZDiscovery and pharmacological characterisation of angiotensin-(1-7) receptors and identification of their importance in diabetes mellitusTetzner, Anjahttps://hdl.handle.net/10468/73332023-04-04T07:13:11Z2018-01-01T00:00:00Zdc.title: Discovery and pharmacological characterisation of angiotensin-(1-7) receptors and identification of their importance in diabetes mellitus
dc.contributor.author: Tetzner, Anja
dc.description.abstract: The renin-angiotensin system (RAS) is known to be the main regulator of blood pressure and fluid balance. Within the RAS, angiotensin (Ang)-(1-7) is known to have cardiovascular protective effects. It represents the opponent of the often detrimental Ang II, which is known to stimulate the angiotensin receptor type 1 (AT1), causing negative effects such as a pathological rise in blood pressure. Beside the well accepted fact that the G-protein coupled receptor Mas is a receptor for the heptapeptide, it was not possible to characterise the ligand/receptor pharmacology or to identify further receptors, since there was a lack in the understanding of the initial intracellular signalling pathways stimulated by Ang-(1-7). In this study, cyclic adenosine monophosphate (cAMP) was identified as a second messenger stimulated by Ang-(1-7). The heptapeptide elevates cAMP concentration in various cell lines, as well as in Mas transfected HEK293 cells, confirming that Mas is a functional receptor for Ang-(1-7). Even more important, MrgD was identified as a second receptor for the peptide, while AT2 could be excluded to be targeted by the heptapeptide. It was also examined, if there are any changes in the intracellular signalling if the first amino acid of the peptide is decarboxylated. The receptor fingerprint for Ala1 -Ang-(1-7) was discovered, and the consequences for pharmacodynamics characterised. The dose-response curves were clearly different from the curves generated with Ang-(1-7). They showed a much lower EC50 and a bell-shaped curve for Ala1 -Ang-(1-7). Furthermore, pharmacological proof was provided that both, Mas and MrgD, are functional receptors for Ala1 -Ang-(1-7). Interestingly, it was also discovered that the AT2 receptor blocker PD123319 is not AT2 specific, but can also block the effects of Ang-(1-7) and Ala1 -Ang(-1-7) in Mas and MrgDtransfected and in primary cells. This raised the question whether the selective nonpeptidic AT2 receptor agonist, Compound 21 (C21), is also unspecific and stimulates Mas and MrgD too. This hypothesis was supported by the fact that the chemical structure of C21 is similar to the Mas receptor specific, non-peptidic agonist AVE0991. Using cAMP and downstream molecules as readouts, pharmacological proof that Mas and MrgD are functional receptors for C21 was generated. The last part of the study examined the role of Ang-(1-7) and its receptors in diabetes mellitus (DM). Previous studies demonstrated that the ACE2/ Ang-(1–7)/ Mas axis has beneficial effects on glucose homeostasis, but the underlying mechanisms remained unknown. The effects of Ang-(1–7) and its receptor Mas on the function of β-cells were investigated. Islets isolated from Mas-deficient and wild-type mice were stimulated with Ang-(1–7) or its antagonists and effects on insulin secretion were determined. It was found that Ang-(1–7) was able to increase the insulin secretion from wild-type islets, but not from islets derived from Mas deficient animals. Interestingly, Ang-(1-7) antagonist DPro, but not A779 could block the Ang-(1-7) mediated effects indicating the involvement of another Ang-(1-7) receptor. However, the heptapeptide did not affect the insulin gene expression or the excitation-secretion coupling, but increased intracellular cAMP involving exchange protein activated directly by cAMP (EPAC), leading to a higher insulin secretion by the β-cells. Ang-(1–7) was also applied to normo-glycaemic mice for 14 days using osmotic pumps. The effects of the heptapeptide in vivo had only marginal effects on glucose tolerance in wild-type mice. However, Ang-(1-7) had improved the insulin secretion in islets isolated from these mice. Interestingly, although less pronounced than in wild-types, Ang-(1–7) still affected insulin secretion in islets derived from Mas deficient mice. The effect of Ang- (1-7) in mice with STZ-induced diabetes was marginal, as in normo-glycaemic mice. Taken together, these results lead to an expansion and partial revision of the reninangiotensin system, by identifying a second receptor for Ang-(1-7), and by excluding AT2 as a receptor for the heptapeptide. Furthermore, the identification of Ala1 -Ang-(1-7) as a peptide with specific pharmacodynamic properties can be used as a basis for the design of more potent and efficient Ang-(1-7) analogues, which can be useful in therapeutic interventions in a rapidly growing number of diseases. The proof that C21 and PD123319 are not AT2 receptor specific as generally assumed, but also interact with the two Ang-(1- 7) receptors, Mas and MrgD, might be an explanation for the partial overlap in beneficial effects of both compounds. Thus, the better understanding of the interaction of small molecules like C21 with their receptors, lays the foundation for the development of small molecules which stimulate all or just one of the Ang-(1-7) receptors, which may be beneficial in diseases like diabetes mellitus. Since it could be shown that Ang-(1–7) plays a significant role in the regulation of insulin secretion from mouse islets in vitro and in vivo, mainly, but not exclusively, by Mas-dependent signalling, modulating the accessory pathway of insulin secretion via increase in cAMP, makes clear that Ang-(1-7) and its receptors are very promising therapeutic targets.
2018-01-01T00:00:00ZExamining potential causes of neuronal dysfunction in Alzheimer’s diseaseJaisimha, Anirudh Vinayhttps://hdl.handle.net/10468/86522023-04-04T07:30:11Z2019-01-01T00:00:00Zdc.title: Examining potential causes of neuronal dysfunction in Alzheimer’s disease
dc.contributor.author: Jaisimha, Anirudh Vinay
dc.description.abstract: Alzheimer’s disease (AD) is a debilitating neurodegenerative disease that is characterised by a number of intraneuronal hallmarks, which include the accumulation of autophagic vacuoles (AVs) within dystrophic neurites, and neurofibrillary tangles (NFTs) composed of both truncated and full-length forms of tau protein. Work presented in this thesis, outlines findings from three lines of investigation that set out to determine potential causative factors that contribute to AD-related neurodegeneration. For Aim 1, I investigated the role of impaired lysosomal digestion as a cause of AV accumulation in AD. Having developed a novel assay that utilised the detection of specific truncated forms of amyloid precursor protein C-terminal fragments (APP-CTFs), which preferentially accumulate when lysosomal digestion is impaired, findings from post-mortem human brain tissue at different Braak stages of AD (0 – VI), indicate that the accumulation of AVs in the AD brain is not caused by an impairment in lysosomal digestion. For Aim 2, I investigated the role of altered glucose availability as a cause of tau hyperphosphorylation in AD. To determine if excessive or insufficient amounts of glucose availability to neurons is a direct cause of tau hyperphosphorylation in the AD brain, I utilised a primary rat neuron culture system, to determine if hyperglycaemic or hypoglycaemic stress could lead to tau hyperphosphorylation. Despite finding high basal amounts of the AD-related tau phospho-epitope (PHF1), in both primary neurons and mouse brain, I did not report any change in levels of phospho-tau under glucose altering conditions, suggesting these changes are not directly responsible for inducing tau hyperphosphorylation in AD. For Aim 3, I investigated the role of dysfunctional neuron-glial interactions as a cause of truncation tau in AD. Having identified truncated forms of tau as early as Braak stage II in post-mortem human brain tissue, I subsequently found that neurons grown in co-cultures with glial cells, develop truncated forms of tau after two weeks in culture, which correlated with the progressive proliferation of astrocytes and microglia. I also found that certain excitatory stimuli, in particular glutamate and zinc, produced a rapid but transient increase in truncated tau, which was prevented by kynurenic acid (KynA). Concluding thoughts from all three investigations suggest that dysfunctional neuron-glial interactions are likely to occur early in AD pathogenesis and the therapeutic targeting of autonomous (neuronal) or non-autonomous (glial-mediated) factors that contribute to dysregulated neuronal excitation may prove to be beneficial in treating AD.
2019-01-01T00:00:00ZHIF-1alpha role in oxygen-dependent radio- and chemosensitivityGebolys, Kingahttps://hdl.handle.net/10468/19052023-04-04T07:10:36Z2014-01-01T00:00:00Zdc.title: HIF-1alpha role in oxygen-dependent radio- and chemosensitivity
dc.contributor.author: Gebolys, Kinga
dc.description.abstract: Poor oxygenation (hypoxia) is a common characteristic of human solid tumours, and is associated with cell survival, metastasis and resistance to radio- and chemotherapies. Hypoxia-induced stabilisation of hypoxia-inducible factor-1α (HIF-1α) leads to changes in expression of various genes associated with growth, vascularisation and metabolism. However whether HIF-1α plays a causal role in promoting hypoxic resistance to antitumour therapies remains unclear. In this study we used pharmacological and genetic methods to investigate the HIF-1α contribution to radio- and chemoresistance in four cancer cell lines derived from cervical, breast, prostate and melanoma human tumours. Under normoxia or hypoxia (<0.2% or 0.5% oxygen) the cells were exposed to either a standard irradiation dose (6.2 Gy) or chemotherapeutic drug (cisplatin), and subsequent cell proliferation (after 7 days) was measured in terms of resazurin reduction. Oxygen-dependent radio- and chemosensitivity was evident in all wild type whereas it was reduced or abolished in HIF-1α (siRNA) knockdown cells. The effects of HIF-1α-modulating drugs (EDHB, CoCl2, deferoxamine to stabilise and R59949 to destabilise it) reflected both HIF-1α-dependent and independent mechanisms. Collectively the data show that HIF-1α played a causal role in our in vitro model of hypoxia-induced radioresistance whereas its contribution to oxygendependent sensitivity to cisplatin was less clear-cut. Although this behavior is likely to be conditioned by further biological and physical factors operating in vivo, it is consistent with the hypothesis that interventions directed at HIF-1α may improve the clinical effectiveness of tumour treatments.
2014-01-01T00:00:00ZInvestigation of the genotoxic potential of the marine biotoxins okadaic acid and azaspiracidsDörr, Barbara Valentinahttps://hdl.handle.net/10468/17882023-03-31T07:10:14Z2014-01-01T00:00:00Zdc.title: Investigation of the genotoxic potential of the marine biotoxins okadaic acid and azaspiracids
dc.contributor.author: Dörr, Barbara Valentina
dc.description.abstract: The 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.
2014-01-01T00:00:00ZNovel insights into the expression and function of p38δ MAPK in oesophageal squamous cell carcinomaO'Callaghan, Carolhttps://hdl.handle.net/10468/20822023-04-04T07:39:23Z2015-01-01T00:00:00Zdc.title: Novel insights into the expression and function of p38δ MAPK in oesophageal squamous cell carcinoma
dc.contributor.author: O'Callaghan, Carol
dc.description.abstract: Oesophageal cancer is an aggressive malignancy which is resistant to conventional therapy and has a poor prognosis. A greater understanding of the underlying molecular biology of oesophageal cancer and the identification of novel targets is necessary for the future treatment of this disease. This thesis focuses specifically on the ill-defined and understudied p38δ mitogen-activated protein kinase (MAPK) and its function(s) in oesophageal squamous cell carcinoma (OESCC). In contrast to the three other p38 isoforms (p38α, -β and –γ which have to-date been relatively well-studied), p38δ MAPK signalling is poorly understood. Thus, this research elucidates some of the role(s) played by p38δ MAPK in cancer progression. This work outlines how loss of p38δ MAPK expression confers greater tumourigenicity in oesophageal cancer. Restoration of p38δ MAPK expression, however, has anti-proliferative and anti-migratory effects and decreases OESCC capacity for anchorageindependent growth. Using a novel application of an enzyme-substrate fusion approach, the effect of phosphorylated p38δ (p-p38δ) MAPK expression is also considered. The work goes onto describe the effect(s) of p38δ MAPK status on the chemosensitivity of OESCC to conventional cisplatin and 5-fluorouracil (CF) versus the effectiveness of doxorubicin, cisplatin and 5-fluorouracil (ACF). ACF treatment of p38δ MAPK-negative OESCC results in decreased proliferation, migration and recovery, and increased apoptosis when compared with CF treatment. This thesis examines the potential mechanisms by which p38δ MAPK expression is lost in OESCC and identifies epigenetic regulation as the probable cause of differential p38δ MAPK expression. Also analysed is the role p38δ MAPK and p-p38δ MAPK play in the cell cycle. In summary, this research identifies p38δ MAPK as a possible molecular target and a potential predictor of response to chemotherapy in OESCC patients.
2015-01-01T00:00:00ZPreclinical characterisation of fingolimod as a potential therapeutic agent for strokeDiaz Diaz, Andrea C.https://hdl.handle.net/10468/140662023-04-04T11:05:50Z2022-12-01T00:00:00Zdc.title: Preclinical characterisation of fingolimod as a potential therapeutic agent for stroke
dc.contributor.author: Diaz Diaz, Andrea C.
dc.description.abstract: Stroke is the leading cause for death and disability worldwide, and the search for novel drug treatments has been affected by repeated clinical trial failures. One novel drug that has garnered promising results in preclinical and clinical stroke studies is fingolimod, an FDA approved drug for the treatment of multiple sclerosis. Even though there are several studies supporting the effectiveness of fingolimod for the treatment of stroke, the recommended characterisation based on the Stroke Treatment Academic Industry Roundtable (STAIR) guidelines is incomplete. Furthermore, the quality of the preclinical studies supporting fingolimod has been poor, thus rigorous studies are required to validate the effectiveness of fingolimod prior to evaluation in clinical trials. This thesis aimed to inform whether fingolimod is effective for the treatment of stroke in intracerebral haemorrhage and ischaemic stroke, and to inform whether fingolimod is a good candidate for evaluation in large randomised clinical trials. This goal was achieved by first using a model of intracerebral haemorrhage to evaluate the effect of administering fingolimod at 30 min, 24 and 48 h after stroke on lesion size and behaviour in a 14-day study on male and female mice. This was followed up by a series of studies using middle cerebral artery occlusion to cause a focal ischaemia. First an optimal dose of fingolimod was determined in a dose response study; then we evaluated the optimal drug dose in two animal model of common comorbidities associated with stroke, age and hyperlipidaemia; and the last study evaluated the effect of an extended treatment duration on stroke. For the ischaemic stroke studies we focused on lesion and behavioural measurements as primary outcomes, and secondary data was collected from daily scores, plus additional measures where necessary. The intracerebral haemorrhage study fingolimod treatment had no measurable effect on either lesion size or behavioural outcomes irrespective of sex, the only finding was that fingolimod treatment reduced mortality in female mice. The dose response study showed no difference in lesion size or behavioural outcomes between the two fingolimod doses (0.5 and 1.0 mg/kg) and control mice, the study did show that saline treated mice had a significantly larger atrophy compared to the lower dose of fingolimod. The lower dose was selected as optimal for further studies. The study evaluating the effect of 0.5 mg/kg fingolimod on stroke in aged mice showed that fingolimod-treated mice had a significantly larger atrophy and a significant improvement in the grid score 7 d after stroke compared to saline-treated mice. The study evaluating the effect of fingolimod on stroke in hyperlipidaemic mice showed that fingolimod-treated mice had a significantly reduced lesion size, without an effect on any other outcome measures. The final study evaluated two fingolimod treatment durations compared to saline controls, the study showed no difference between any of the outcome measures, with a trend towards improved behaviour outcomes in mice receiving 10 d of fingolimod treatment. Lastly, considering the fact that the results of these studies were inconclusive, we decided to pool the data of the ischaemic studies and evaluate whether fingolimod had an effect on the primary outcome measures in a heterogenous animal population. The pooled data showed that fingolimod treatment improved behaviour 7 d after stroke without an effect on lesion size or atrophy. The results of this thesis cast a doubt on the effectiveness of fingolimod and its suitability for translation into larger clinical trials. Furthermore, they highlight the need for thorough preclinical studies for promising drugs as well as the need for studies to meet the proposed STAIR guidelines prior to translation. Confirmatory studies, like those presented here, performed with measures intended to control for internal and external biases are all good measures to be implemented for future studies of novel and highly promising drugs for stroke treatment.
2022-12-01T00:00:00ZThe impact of host-microbe interactions on murine colonic secretomotor functionLomasney, Kevin W.https://hdl.handle.net/10468/20022023-04-04T06:56:58Z2014-01-01T00:00:00Zdc.title: The impact of host-microbe interactions on murine colonic secretomotor function
dc.contributor.author: Lomasney, Kevin W.
dc.description.abstract: The 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.
2014-01-01T00:00:00ZThe impact of whey protein isolate on energy balance regulationMcAllan, Liamhttps://hdl.handle.net/10468/20042023-04-04T07:06:31Z2014-01-01T00:00:00Zdc.title: The impact of whey protein isolate on energy balance regulation
dc.contributor.author: McAllan, Liam
dc.description.abstract: Using C57BL/6J mice fed whey protein isolate (WPI) enriched high fat (HFD) or low-fat diets (LFD), this study tested the hypothesis that WPI directly impacts on adiposity by influencing lipid metabolism. WPI suppressed HFD-induced body fat and increased lean mass at 8 weeks of dietary challenge despite elevated plasma triacylglycerol (TAG) levels, suggesting reduced TAG storage. WPI reduced HFD-associated hypothalamic leptin and insulin receptor (IR) mRNA expression, and prevented HFD-associated reductions in adipose tissue IR and glucose transporter 4 expression. These effects were largely absent at 21 weeks of HFD feeding, however WPI increased lean mass and cause a trend towards decreased fat mass, with notable increased Lactobacillus and decreased Clostridium gut bacterial species. Increasing the protein to carbohydrate ratio enhanced the above effects, and shifted the gut microbiota composition away from the HFD group. Seven weeks of WPI intake with a LFD decreased insulin signalling gene expression in the adipose tissue in association with an increased fat accumulation. WPI reduced intestinal weight and length, suggesting a potential functional relationship between WPI, gastro-intestinal morphology and insulin related signalling in the adipose. Extending the study to 15 weeks, did not affect adipose fat weight, but decreased energy intake, weight gain and intestinal length. The functionality of protein sensing lysophosphatidic acid receptor 5 (LPA5) in 3T3-L1 pre-adipocytes was assessed. Over-expression of the receptor in 3T3-L1 pre-adipocytes provided a growth advantage to the cells and suppressed cellular differentiation into mature fat cells. In conclusion, the data demonstrates WPI impacts on adiposity by influencing lipid metabolism in a temporal manner, resulting possibly due to changes in lean mass, hypothalamic and adipose gene expression, gut microbiota and gastrointestinal morphology. The data also showed LPA5 is a novel candidate in regulating of preadipocyte growth and differentiation, and may mediate dietary protein effects on adipose tissue.
2014-01-01T00:00:00Z