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- ItemMicrobial regulation of barrier function in the gut-brain axis(University College Cork, 2023) Sánchez Díaz, Paula; Clarke, Gerard; Cryan, John; Leigh, Sarah-Jane; Advancement in Neurosciences (Geneva, Switzerland)The gut microbiome plays a critical role in host health through modulation of gut and blood-brain barrier integrity, responding to factors such as diet, stress, and medication. A key pathway by which the gut microbiota affects gut and bloodbrain barrier integrity is through the production of bioactive metabolites. This thesis explores the role of barriers in the microbiota-gut-brain axis, which are essential for the proper functioning of body systems and homeostasis. Chapter 2 investigates how fermenting infant nutrient formulations with different bifidobacteria strains isolated from infant gut microbiome can influence the integrity of gut and blood-brain barriers in vitro. The study found that the presence of bifidobacteria strains, in some cases, had protective effects on the barriers, and these effects sometimes differed depending on the barrier studied. Chapter 3 explores the effects of indole and two of its derivates, indole-3-acetate and indole- 3-propionate, on gut barrier function in vitro. The results indicate that indole has a protective effect on barrier function, particularly at higher concentrations, and indole-3-acetate has a protective effect at the lowest concentrations tested. Surprisingly, indole-3-propionate was not protective and at higher concentrations exacerbated the effects of LPS-induced disruption. Finally, Chapter 4 focuses on the effect of cancer therapy, specifically cisplatin, on gut and blood-brain barrier structure in mice, to further explore the role of gut microbiome in cancer-related cognitive impairment. The study used mice treated with cisplatin to investigate the expression of genes involved in the structural function of barriers and inflammation, as well as gene expression of receptors activated by microbial ligands in the ileum, colon, and hippocampus. The results showed that cisplatin affected gene expression in a region- and dose-dependent manner, leading to changes in anxiety-like and fatigue behaviours in mice. Overall, this research highlights the critical role of the gut microbiome in gut barrier and blood-brain barrier function. Microbial metabolite supplementation may present a useful therapeutic option for disease processes involving disruption of the gut and/or blood-brain barriers.
- ItemAn investigation of factors influencing early life gut microbiome in the piglet model, and potential functional food ingredients(University College Cork, 2023) Skillington, Orla; Stanton, Catherine; Ross, R. Paul; Vistamilk; Science Foundation IrelandIncreasingly regarded as an “invisible organ”, the microbiological supersystem that is the gut microbiome, has become a central field of research in recent years. Thanks to multi-omics approaches and modern day bioinformatic techniques, characterization of the gut microbiome has advanced hugely in the last three decades, and continues to evolve. Characterization of the gut microbiome to-date, has concerned infanthood through to old-age, across a variety of species, from humans to aquatic life. Due to ethical considerations involving human-targeted therapeutics and interventions for the gut microbiome, animal models must be considered. The porcine pig model is an attractive model for putative human interventions and their associated pre-clinical trials, due to certain physiological analogies. Herein, a porcine model was developed, to mimic the early life gut microbiota and to investigate the effects of prenatal stress exposure on the seeding of this microbial consortia. Piglets and neonatal infants share an early life gut microbiota rich in Bacteroides, Escherichia, Lactobacillus and Clostridium¸ however, whilst Lactobacillus dominate the early life porcine gut, the nascent infant gut may be characterized by high abundances of bifidobacterial species. Analysis by 16s rRNA sequencing revealed that, at two weeks of age, piglets exposed to low stress levels in utero harboured higher levels of Prevotella, Fusobacterium and Bacteroides, whilst high stress exposure influenced increased levels of Lactobacillus. The effects of stress on the gut microbiome projected into later life, following weaning, whereby piglets of the low stress group were seen to have significantly higher levels of Prevotella NK3B31 and Helicobacter and piglets of the high stress group harboured higher levels of Faecalbacterium and Megasphaera.. Taxa driving microbial clustering as the piglet aged were seen to be stress-related, with high stress piglets becoming enriched in Deferribacterota, Treponema and Pyramidobacter at the post-weaning stage. Conversely, increased populations of Ruminococcus were observed in the low stress groups at two weeks of age, and remained higher than that of their counterpart post- weaning. Commonalities between the stress groups existed at two weeks, with phyla such as Campilobacterota, Bacteroidota, Actinobacteria and Firmicutes being enriched and later declining with age. At post-weaning, both groups were found to have clusterings of Cyanobacterota, Fibrobacterota, Spirochaetota and Patescibacteria. Alpha and beta diversity metrics were unaffected by prenatal stress exposure, however differed significantly with respect to age, at two weeks of age versus four weeks (post-weaning). We also investigated the putative probiotic potential of a range of indigenous African fermented food that may serve to provide human health benefits. It is postulated that the succession of the development of probiotic strains from these foods, may be suited for further investigations within infant formula matrices, to help to equilibrate the microbial consortia of a dysbiotic infant gut which had been exposed to prenatal stresses. The bacterial isolates, obtained from seventeen different food sources, on a variety of growth media, were initially subjected to a preliminary subtractive process in order to reduce the 218 isolates to a smaller number. Following exopolysaccharide production screening, bile salt hydrolase production screening, and antimicrobial activity screening, seven isolates were chosen for further analyses. These were found to be dominated by lactic acid bacteria such as Enterococcus and Lactobacillus as identified by 16s rRNA Sanger Sequencing, with all isolates displaying average growth kinetics of between 8 and 10 log cfu mL-1. Analysis by whole genome sequencing revealed Enterococcus populations to be dominated by E. faecium and E. casselflavus, whilst Lactobacillus delbrueckii and Pediococcus pentosaceus were also identified in the isolated cultures. In silico mining and comparative genomic analysis revealed the presence of antimicrobial substances such as enterolysins, enterocins and helveticins in the samples, whilst predicted secondary biosynthetic metabolites revealed the presence of polyketide synthases and antimicrobial ribosomally synthesized and post-translationally modified peptides (RiPPs) such as lanthipeptides. Subjection of the seven isolates to simulated gastric environments revealed the ability of two strains, L. delbrueckii and P. pentosaceus, to survive gastric pH conditions after 4 hours. Furthermore, both of these strains were found to be capable of growing in simulated bile conditions. Haemolytic assays revealed all strains were non-hazardous to red blood cells, and produce no signs of lysis when grown on blood agar plates. African fermented foods present a largely untapped area of unidentified strains with potentially valuable technical properties, however may also serve as vehicles for antimicrobial resistant and pathogenic strains. There lies a substantial gap in knowledge surrounding these food products and their applications, which warrant investigation to benefit both the native consumers, and the market potential of these foods.
- ItemInvestigation of mechanisms underpinning cytokine-induced cell death of colon cancer cells(University College Cork, 2023) Linehan, Eva; Nally, Ken; Lee, CiaranInflammatory cytokines induce regulated cell death as part of a robust immune response. Many unique forms of regulated cell death have been described that are critically dependent on specific signal transduction molecules or cellular events. Often, molecules or events canonically associated with cell death signalling pathways, while contributing factors, are not essential for cell death. Given the extensive emerging crosstalk and plasticity between modes of cell death, determining dependency is crucial for categorising cell death and for downstream clinical applications. IFN-γ and TNF-α are inflammatory, Th1-type cytokines elevated in immune-mediated inflammatory diseases and infectious diseases such as COVID-19, that synergistically induce cell death in diverse cell types. Recently IFN-γ-induced JAK/STAT signalling has emerged as an important pathway for triggering cell death, but the downstream mechanisms underpinning JAK/STAT-mediated cell death are incompletely understood. Here, IFN-γ+TNF-α-induced synergistic cell death was confirmed to be dependent on JAK1, JAK2, STAT1, and partially dependent on CASP8, using knockout colon cancer cell lines. IFN-γ+TNF-α-treated cells displayed biochemical hallmarks of multiple cell death pathways. Use of JAK1/2 inhibitors highlighted the unique kinetics of IFN-γ+TNF-α-induced cell death, suggesting that an accumulation of intracellular event/s, reaching a threshold or point of no return, may be responsible for cell death. The inconsistent effects of p300/CBP inhibitors on IFN-γ+TNF-α-mediated cell death suggest that STAT1-dependent transcription is not the main effector of cell death downstream of JAK1 and JAK2. The STAT1-dependency observed may just be due to its effects on JAK2 expression in cytokine-treated cells over time. Flow cytometry assays were used to investigate the kinetics of several biomarkers of mitochondrial and cellular stress in IFN-γ+TNF-α-treated cells, compared to cells undergoing canonical cell death pathways. Loss of mitochondrial integrity coincided with the crucial turning point observed in JAK1/2 inhibitor chase experiments, followed by severe increase in superoxide levels. Though these events cannot be confirmed to be the main drivers of IFN-γ+TNF-α-induced cell death, they are hallmarks of death and may be contributing factors. IFN-γ+TNF-α-induced cell death occurs by a non-canonical and redundant cell death mechanism that is dependent on JAK activity and potentially involves induction of components of multiple cell death pathways.
- ItemNovel models for understanding traumatic stress(University College Cork, 2022-09-30) Lannon, Adam; Moloney, Rachel; Cryan, John; Brain and Behavior Research FoundationUndergoing trauma, be it physical, psychological, or observed, can induce pathological alterations leading to disorders such as Post-Traumatic Stress Disorder (PTSD). PTSD is highlighted by negative cognitive alterations, behavioural changes, and interruptions in arousal and sociability. PTSD is comorbid with disorders such as anxiety and depression, gastrointestinal disturbances, pain, and the gut microbiome is hypothesized to play a role in this trauma-related disorder. Direct experience of traumatic events is the most common method of generating traumatic-stress related pathologies, however indirect exposure through witnessing another endure a traumatic event can also lead to PTSD-like symptoms. This method of traumatic transference is called secondary traumatic stress (STS). While PTSD and STS are clinically relevant, and ever-growing in importance due to the recent COVID-19 pandemic, there is still a lot to be learned about their molecular underpinning, mechanisms, and biomarkers. In order to appropriately investigate these neurobiological features of traumatic stress, valid and effective animal models are absolutely essential. Utilizing the most appropriate animal models for the representation of neuropathologies is essential for extracting critical information in the process of developing novel therapeutic options. In chapter 2, we aim to develop the knowledge of secondary traumatic stress, we investigated whether a novel observational model, combining visceral pain, a common comorbidity of traumatic stress related disorders, and observed stress could result in a suitable phenotype. Utilizing colo-rectal distension (CRD) to induce visceral pain behaviours, we had rodents observe another rodent undergoing this procedure. These observer rodents then underwent the CRD themselves 24 hours later in order to assess whether they had visceral hypersensitivity. Indeed, it was seen that observer animals had hyperalgesia measured in visceral pain threshold and total behaviours, an impacted HPA axis, and altered neuronal activation in key brain regions. Our results suggest that this novel model was effective in producing secondary traumatic stress-like phenotypes, and would be well suited for further research into the social transference of pain and developing therapeutic options for traumatic-stress induced disorders and visceral pain comorbidity. In chapter 3, we look at Single prolonged stress (SPS), which is a well-validated and commonly used model however there are ethical concerns that limit its widespread use. The classical SPS model involves a 2-hour restraint, immediately followed by a 20-minute forced swim, a 15-minute rest and culminates with diethyl ether exposure until loss of consciousness. Recent focus on ethical standards and interests in refining animal models has led to concerns in the usage of diethyl ether, leading us to investigate whether the model would still be effective using isoflurane as a replacement for diethyl ether. Our findings suggest that this model is effective in recapitulating a key PTSD phenotype in the contextual fear conditioning paradigm. Impaired fear learning has been repeatedly found to be a key component of PTSD phenomenology, and our model induced significantly impaired fear learning in stress rats. Further to this, we found that SPS with isoflurane caused significant reduction in learned helplessness in rodents, paired with time specific changes in corticosterone concentration. Anxiety-like behaviours also appear to be implicated by this model, with Isoflurane exposure leading to reduced anxiety-like behaviour, suggesting its potential as an adequate PTSD model. The encouraging results from these two models of traumatic stress provide a significant interest in further studies using them. With the future intention of developing novel and effective therapeutics for undermedicated sufferers of these disorders, the hope is that these models can help provide valuable insights into the mechanisms of action behind the pathologies, illuminating potential therapeutic avenues.
- ItemNatural transformation in lactic acid bacteria(University College Cork, 2020-10-16) O'Connell, Laura Michelle; van Sinderen, Douwe; Mahony, Jennifer; Science Foundation IrelandFermentation has been commercially exploited to produce food products for millennia. The most intensely exploited microorganisms used in modern industry to commercially produce fermented products are yeasts and lactic acid bacteria (LAB). LAB are most widely employed in dairy processing, where they contribute to the biochemical changes that convert milk to cheese and yoghurt. To ensure consistency, large-scale, industrial fermentations use starter cultures of LAB, meaning a known strain or a sometimes-undefined combination of multiple strains with well-defined characteristics. Hypothetically, through natural genetic modification, which exploits DNA transfer systems occurring in nature, desirable traits could be introduced to strains to improve the efficacy of the starter culture. The focus of this thesis is natural competence. Competence refers to the specialised physiological state in which bacteria are able to undergo transformation through the internalisation of exogenous DNA. Recently an early com system was described for the industrially relevant LAB, Streptococcus thermophilus and a late com system homologous to that of S. pneumoniae was described in another LAB widely employed in cheese production, Lactococcus lactis. This thesis aimed to explore the functionality of the proven and predicted natural com systems encoded these LAB. In the case of S. thermophilus, this was achieved by defining the nutritional requirements of competence development, which involved defining a minimal cultivation medium. This medium was used in competence assays involving a model strain, S. thermophilus LMD-9, to confirm the functionality of the competence system in the minimal medium. The optimised medium and assay were then applied to a panel of industrial strains to determine the transferability of the transformation protocol. While it was observed that the minimal medium could support the growth of the industrially relevant strains, no transformation was detected in any industrial strains. A different approach was taken to study competence in L. lactis, as the system is far less defined compared to that of S. thermophilus. For this reason, an investigation of the gene activity of the lactococcal competence components was conducted, as opposed to competence assays. Specifically, the activity of the com-promoters was assessed via blue/white selection, using a promoter-probe vector constructed in this study. Several conditions were trialled to identify those optimal for competence development. This included the nisin-induction of the master competence regulator, ComX, which was the only condition that proved capable of activating the com-promoters. Chemical mutagenesis was employed in an attempt to generate random mutants that did not require comX-induction; however, this method did not prove effective, and no such mutants were isolated and the mechanism of lactococcal competence remains elusive.