Biology and Environmental Science Theses

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This collection is made up of doctoral and master theses by research, which have been received in accordance with university regulations.

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Now showing 1 - 5 of 21
  • Publication
    Investigating chemopreventive and chemotherapeutic applications of cannabinoids in prostate cancer
    (University College Dublin. School of Biology and Environmental Science, 2022) ;
    0000-0001-8301-5846
    Prostate cancer is the 5th leading cause of cancer death in men, responsible for more than 375,000 deaths worldwide in 2020. For patients with localised prostate cancer, the 5-year survival rate is close to 100%. However, metastatic prostate cancer remains a lethal disease, with a 5-year survival rate of just 30%. Therefore, novel therapeutic strategies are urgently needed to improve clinical outcomes for patients with advanced prostate cancer. Cannabinoids, chemical components of the cannabis plant, pose a possible solution. Substantial preclinical evidence demonstrates that cannabinoids can modulate key hallmarks of cancer, including cell death, proliferation, migration and invasion, and angiogenesis. However, few studies have investigated the anti-cancer potential of cannabinoids in prostate cancer. Our overall hypothesis was that plant-derived cannabinoids (phytocannabinoids) have chemotherapeutic and chemopreventive effects in prostate cancer. To test this hypothesis, our specific aims were to assess the effects of cannabinoids on various hallmarks of cancer, to investigate the mechanisms underlying the observed phenotypic effects, and to determine whether phytocannabinoids have antioxidant or chemopreventive effects in non-cancerous prostate cells. First, we used cell line models of prostate cancer to measure the effects of cannabinoid treatment on cell viability, survival, proliferation, apoptosis, and migration and invasion. The non-psychoactive phytocannabinoid GL1a inhibited prostate cancer cell viability and proliferation, with no significant increase in apoptosis. GL1a reduced the invasiveness of highly metastatic PC-3 cells. We also found some evidence that combinations of cannabinoid compounds produce enhanced anti-cancer activity. However, GL1a also reduced viability and induced apoptosis in non-cancerous prostate cells and further investigation into possible off-target effects is warranted. Having shown that GL1a reduced cell proliferation and invasion, our next goal was to investigate the underlying mechanisms. Specifically, we aimed to identify the receptor targets of GL1a in prostate cancer cells and to measure the effects of GL1a treatment on the expression of cell cycle proteins, the phosphorylation of protein kinases, and the expression and secretion of proteins involved in cell invasion. GL1a reduced the expression of the key cell cycle proteins cyclin D3, CDK2, CDK4, and CDK1, and reduced the phosphorylation and activation of the protein kinase AKT. Additionally, we found some evidence that GL1a may increase the expression of E-cadherin, an adhesion protein associated with a non-invasive phenotype. The effects of GL1a on cell viability were not blocked by CB1 or CB2 antagonists, TRPV ion channel blockers, or a GPR55 agonist, suggesting that GL1a acts independently of these targets in prostate cancer cells. Finally, we assessed the antioxidant and cytoprotective potential of low-dose cannabinoids in non-cancerous prostate cells. Hydrogen peroxide was used to induce oxidative stress. Phytocannabinoid treatment at the doses tested produced no significant cytoprotective or antioxidant effects. However, drug exposure times and doses may require further optimisation. This study provides novel insights into the phenotypic effects and mechanisms of action of cannabinoids in prostate cancer cells. GL1a reduces prostate cancer cell proliferation and invasion. The underlying mechanisms include altered expression of key cell cycle regulators and modulation of AKT phosphorylation. Future studies should aim to identify the receptor target(s) of GL1a and further investigate its mechanisms of action, as well as testing the effects of cannabinoid treatment in more biologically relevant experimental models. Ultimately, clinical trials will be needed to determine whether the observed phenotypic effects of GL1a in vitro can translate to therapeutic benefits and improved outcomes in patients with prostate cancer.
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  • Publication
    Urea transporters in the mammalian brain
    (University College Dublin. School of Biology and Environmental Science, 2022)
    Facilitative urea transporters encoded by two distinct genes: SLC14A2 (UT-A) and SLC14A1 (UT-B) facilitate movement of urea across plasma cell membranes and have been found in different locations within different tissues in various mammalian species – holding important physiological implications in the urine concentrating mechanism, urea nitrogen salvaging process, and increasing interest in the physiological role of urea in the aging brain and in neurodegenerative disease. Other studies have relatively recently reported increased brain urea levels in Alzheimer’s disease human brain tissue and increased levels of urea and urea transporter SLC14A1 transcript in a prodomal transgenic sheep model of Huntington disease. Comprehensive studies of UT-B transporters in the mammalian (rat, mouse, human, etc) brain investigating expression, abundance, and localization of UT-B in the brain, specific UT-B isoform(s) expressed in the brain, and regulation of UT-B in cell lines were undertaken. Such characterization of the precise nature of UT-B in the brain (i.e. aging and diseased state) is essential because very little is understood about UT-B in the brain and the important implications on brain health. Studies conducted in this research project using a range of techniques (i.e. endpoint PCR, Western blotting, and immunolocalization) have determined UT-B RNA expression increases in aging brain although likewise was not definitively shown for UT-B protein abundance, how UT-B is regulated in C6 astrocyte cell lines, and novel UT-B isoforms are present in human brain. Much more work remains to be done to determine the functional relevance of UT-B transporters and hence understand their physiological significance in the mammalian brain under physiological and diseased states.
      8
  • Publication
    Investigation into chemotherapeutic and chemopreventive properties of seaweeds against prostate cancer
    (University College Dublin. School of Biology and Environmental Science, 2022) ;
    0000-0002-3220-7036
    Background: Prostate cancer (PCa) is the second most common cancer and the fifth leading cause of cancer related deaths in men worldwide, with an estimated 1.4 million cases and 375,000 deaths in 2020. PCa is a disease of older age. The late age of onset provides ample opportunity for chemoprevention. PCa incidence rates range from 6.3 to 83.4 per 100,000 men in South Central Asia and Northern Europe, respectively. The low incidence among Asian men has been connected with multiple dietary elements. Brown seaweeds have been afforded numerous health benefits and are a staple constituent of the Asian diet. Anti-cancer properties of some seaweed compounds have been previously demonstrated across different cancer types. However, knowledge on the effects of seaweeds against PCa is lacking. The aim of this thesis was to investigate the anti-cancer properties of 10 seaweed species using cell line models of prostate cancer. Methods: Ten seaweed extracts were screened for their effects on the viability of three PCa cell lines (LNCaP, PC3 and DU145) using MTT assay. To determine cancer specificity, we tested the effective species on viability of prostate non-cancerous (BPH1 and PWR-1E) cells. The effect of Fucus vesiculosus, a brown seaweed, on migration and invasion of PCa cells was measured using the wound healing and Transwell invasion assays, respectively. Fucus vesiculosus impact on LNCaP cell cycle, apoptosis, and proliferation was measured by flow cytometry. Western blotting was used to measure the expression of cell cycle proteins (CDK1, CyclinD3 and CyclinE1), apoptotic proteins (cleaved caspase 3 and PARP) in PCa cells cultured in the presence of F. vesiculosus. Regulatory protein of migration and invasion (E cadherin, vimentin, survivin) were studied in PC3 and DU145 cells. The secretion of matrix metalloproteinases (MMP1, MMP3, and MMP9) in PC3 and DU145 cells were measured by multiplex ELISA. Cytoprotective effects of F. vesiculosus against H2O2 induced PWR-1E cytotoxicity was measured by MTT assay. Glutathione levels in PWR-1E cells was measured by GSH-GLO assay. Results: Three seaweed extracts demonstrated a reduction in viability of androgen dependent LNCaP cells. LNCaP cells were more sensitive to F. vesiculosus than either of the non cancerous cells. F. vesiculosus induced apoptosis in LNCaP cells by increased cleaved caspase 3 and cell cycle arrest at G1 phase by reducing expression of Cyclin D3. F. vesiculosus had no effect on the expression of the androgen receptor. None of the seaweed extracts had any effect on the viability of the androgen independent PC3 and DU145 cells. However, F. vesiculosus did reduce the migration and invasion of PC3 cells and invasion of DU145 cells, although no mechanism could be identified. Pre-treatment of non-cancerous prostate epithelial cells PWR1E with low-dose F. vesiculosus extracts were not found to have any cytoprotective or antioxidant effects against H2O2 induced cytotoxicity. Conclusion: The work presented in this thesis provide strong evidence for therapeutic potential of the Fucus vesiculosus brown seaweed against PCa. Further research is needed to decipher the identity of the bio-active compound(s) responsible for these effects as well as to further elucidate the mechanisms of action.
      16
  • Publication
    Tropical marine biodiversity through a molecular lens: eDNA metabarcoding across oceans, habitats, and time
    (University College Dublin. School of Biology and Environmental Science, 2022) ;
    0000-0001-6684-5194
    Environmental DNA (eDNA) metabarcoding is a relatively new tool in the field of marine ecology, yet in the last decade its use has grown exponentially. Encouraging metabarcoding efforts that support and/or augment conventional survey results have paved the way for implementation of molecular based monitoring, yet it is unclear whether the tools should be directly compared or validated with e.g., visual methods. Afterall, the sensitivity of molecular methods goes far beyond what can be seen, even with a microscope. Furthermore, the tools are also still held back by the paucity of reference sequences in online databases. In this thesis, I examined the use of eDNA metabarcoding in the context of highly diverse tropical marine habitats. In chapter two, the ability of metabarcoding to reflect currently accepted biodiversity patterns was assessed. I used standardised methods across two vastly different yet characteristically similar regions, namely the Caribbean Sea and the Western Indian Ocean, to be in a position to directly compare how metabarcoding performs in different environments. Despite quite a range of habitats being studied with metabarcoding to date, to my knowledge no one has done a comparison of this scale to evaluate metabarcoding performance. The results of this chapter show clearly how an understanding of underlying biodiversity levels is crucial for effective experimental and monitoring design, as well as bioinformatics processing when using metabarcoding approaches. Chapter three takes a closer look at one of the sites studied in chapter two, by analysing temporal data from a reef and a mangrove habitat in Tela, Honduras. Metabarcoding efforts often suffer from a lack of resolution as a result of gaps in reference databases, and therefore this chapter examined the differences in spatio-temporal patterns that could be found whether using taxonomically higher resolution data or data that purely passed through bioinformatics processes and was thus assumed to be of good quality. The taxonomically unassignable data was found to be more informative in terms of finding temporal changes, yet it is clear that taxonomic information of some level is required to draw conclusions and make predictions from the observed changes. With this in mind, chapter four presents a new tool for investigating the so-called “dark matter” of metabarcoding; the conventionally unassignable molecular operational taxonomic units (mOTUs) or actual sequence variants (ASVs). Instead of strict taxonomic assignments, this tool uses a phylogenetic placement approach of your query reads to a large reference tree of COI and COI-like (i.e., of bacterial and archaeal origin) sequences currently available in online databases. By applying this tool to metabarcoding data from various projects, we found further evidence that a large proportion of the resulting mOTUs/ASVs from marine eDNA samples originate from bacterial sources. In chapter five, to address this issue, I developed a capture probe protocol with the aim to isolate target metazoan DNA templates from eDNA samples, or alternatively remove the abundant bacterial DNA templates, prior to the amplification step of a conventional metabarcoding effort. In order to fully understand the effects of the protocol, all of the steps were evaluated for changes in the abundance and richness of COI and bacterial 16S, by amplifying and sequencing for both. The results clearly demonstrated the nonspecificity of popular COI primers, but also revealed the range of DNA that capture probes are able to pull out from eDNA extracts, providing a basis for suggesting an exciting new approach for isolation of target DNA.
      81
  • Publication
    Optimising macroalgal diversity and ecosystem function on coastal infrastructure - an eco-engineering approach
    (University College Dublin. School of Biology and Environmental Science, 2022)
    Artificial structures are becoming ubiquitous features of coastal landscapes. The habitat these provide differs from natural shorelines in several ways, and consequently there has been significant interest in eco-engineering, which combines ecological and engineering knowledge to design artificial structures to support more natural biological assemblages. A number of designs have been developed, but most have been tested in only one context. Research on how biota on artificial structures compares to natural shores has focussed primarily at the level of diversity and community structure, and we do not yet have a clear understanding of how ecological processes may differ between these two habitats. The biological responses of key habitat-formers on artificial structures are also not yet well understood. In terms of eco-engineering, stakeholders perceive a lack of evidence that such interventions contribute to biodiversity and ecosystem function on artificial structures, and thus there is a need to strengthen the existing evidence base. In Chapter 2, I cleared plots on seawalls and natural shores at two separate timepoints and monitored these over one year to determine the influence of timing of disturbance on recruitment and succession. Additionally, I cleared plots on one seawall at a single timepoint, installed exclusion cages and monitored these over 18 months in order to determine the influence of grazing pressure on colonisation. I found that timing of disturbance was significant in determining biofilm structure on both types of habitat after 3 months, and in determining species richness on both shore types after 6 months. Timing of disturbance also influenced the direction and trajectory of the subsequent successional sequence. Grazer exclusion increased biofilm concentrations on artificial structures, and influenced community composition in comparison to plots without exclusion. In Chapter 3, I characterised and compared populations of a key habitat-forming species on natural and artificial substrata in terms of abundance, growth rate, survival and aspects of reproduction. Data were collected from artificial structures and natural shores along the coasts of Ireland and Wales. Abundance and growth rate did not differ between artificial and natural shores. While there was considerable site-level variation, populations on natural shores produced a higher number of mature receptacles overall during the peak reproductive period in April, and had lower rates of dislodgement compared to artificial structures. In Chapter 4 I investigated the ecology and function of seaweed assemblages on artificial bolt-on rockpools on seawalls established in different environmental contexts along the Irish coastline. A total of 32 artificial rockpools were retrofitted on eight seawalls, and were monitored regularly for a period of two years. Data were collected in terms of productivity, community respiration, and community composition. Seaweed colonisation consisted of early dominance of ephemeral species followed by the appearance and establishment of perennial habitat-formers. Overall species richness differed both among sites and among structural sections of the artificial rockpools. By 24 months, all sites supported populations of large habitat-forming seaweeds. Productivity and community respiration differed among sites, but not among environmental contexts at any timepoint. This thesis serves to highlight the impacts of artificial structures on coastal biodiversity and ecosystem function, particularly in terms of ecosystem processes and the ecology of habitat-forming species. Knowledge of how these characteristics differ on artificial structures compared to natural shores, along with an understanding of how ecological processes are altered on these structures, will aid
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