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Impact of B cell secreted factors on myelin in progressive multiple sclerosis
No full textEx vivo organotypic slice models present several advantages over conventional cell culture systems by enabling the investigation of multiple cell types within their native 3D architecture, while preserving intrinsic cell-cell interactions and signalling pathways. In approximately 40% of individuals with progressive multiple sclerosis (PMS), B cell niches, referred to as ectopic lymphoid follicles (ELF), are observed in cortical sulci and are associated with cortical demyelination. This PhD thesis focused on developing a novel hybrid human-rat ex vivo platform to assess the cytotoxicity of B cells from individuals with PMS, hypothesizing that co-culture with brain slices may induce PMS-like cortical demyelination.
Coronal organotypic brain slice (OBS) cultures were optimized using both neonatal and adult rat brains. Initially, evaluation of the metabolic activity of slice cultures from neonatal rats (P10/11) was investigated. The data indicated that caudal slices (Bregma -1.10mm to -3.50mm) exhibited 29.6% higher metabolic activity than rostral slices (p<0.001, n=50). Furthermore, cortical slices without structural connections exhibited 38.5% lower metabolic activity than those with intact connections (p<0.001, n=26). Severing these connections resulted in a 25.5% reduction in metabolic activity (p<0.02, n=20), emphasizing the necessity of maintaining anatomical integrity and connectivity for ex vivo cortical studies. The caudal brain regions provided optimal metabolic activity and extended viability, making them ideal for mimicking cortical physiology under healthy and diseased conditions.
Adult rat brain slices typically exhibit limited viability in ex vivo cultures (often not exceeding 7 days, so fetal bovine serum (FBS) concentrations (0-10%) in growth media were tested. Cortical slices cultured in 10% FBS demonstrated significantly higher metabolic activity (p<0.001, N=4) compared to those cultured with reduced FBS concentrations, prolonging viability to 21 days. Caudal slices (~Bregma -1.33mm) remained more metabolically active (p<0.05) than rostral slices.
Since hybrid human-rat models, using LPC to induce demyelination, were being developed, culture conditions compatible with both neonatal rat OBS and human B cells had to be optimised. In the absence of FBS, slices treated with 0.5mg/ml LPC exhibited an 88% reduction in metabolic activity at T2 (p<0.001), and a 67% reduction at T4 (p<0.01) compared to untreated controls. The addition of 10% FBS improved metabolic activity, reducing cytotoxicity at T2 and T4, but tissue integrity was lost. The optimal FBS concentration for
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culturing both neonatal OBS and B cells was found to be 2%, which preserved tissue integrity and prevented cytotoxic effects.
Coronal OBS cultures were then treated with B cell supernatant, from cultures of peripheral blood from PMS+ and PMS- individuals. Proteome Profiler Rat XL Cytokine Array analyses demonstrated that OBS cultures treated with PMS+ supernatant exhibited cytokine expression patterns consistent with clinical PMS, including upregulation of IL-1, IL-17, and IL-22. PMS+ samples also showed the highest cytotoxicity at T2 (p<0.001, N=5) and significantly reduced myelin thickness (quantified on Image J using anti-MOG staining) at T2 (p<0.05) and T4 (p<0.01), disappearing by day T7 (N=5, n= 360). These findings suggest that factors released by PMS+ B cells are cytotoxic to myelin, inducing cortical demyelination in the OBS model.
Next, a series of pilot studies were carried out to further examine the potential of rodent models of MS. Firstly, a study of activated mouse B cell migration in the brain demonstrated that a cuprizone-induced inflammatory environment promotes B cell retention in brain sections, particularly when treated with chemokine ligand 13 (CXCL13), compared to LPC or untreated controls (~30.6% and ~57.4% cells less, respectively). This highlights the necessity of developing an ex vivo model of PMS-like pathology and reinforces the role of B cells in MS-associated cortical myelin toxicity. Injecting B cells into an inflamed brain milieu facilitates their retention despite active glymphatic and cerebrospinal fluid flow. Secondly, further understanding of an inflamed brain milieu was sought by experimenting with biomaterials that could potentially be used to model ELF-like structures. For that purpose, hydrogel (HG) formulations were tested to obtain the storage modulus most closely resembling the malignant human lymph node, as that of ELFs has not yet been documented, and a non-adherent cell line (THP1) was successfully embedded within the hydrogel's 3D structure, showing proof-of-concept. Thirdly, attempts were made to mimic ELFs by promoting extracellular matrix (ECM) deposition in meningeal cultures, using the macromolecular crowder (MMC) carrageenan (CG). Unfortunately, at higher concentrations, cells treated with CG exhibited significant cell death, and no acceleration of collagen deposition was observed.
Finally, the established ex vivo model was used to test the effect on remyelination of a peptide previously shown to be immunomodulatory in vivo. Peptide FhHDM, and its truncated version, were tested on demyelinated cerebellar slices (0.5mg/ml LPC). Treatment with full-length FhHDM resulted in improved metabolic activity and reduced cytotoxicity, suggesting a
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protective effect on myelin. However, further tests and quantification of the myelin area are required to confirm the peptide’s myelin-rescuing potential.
Overall, this model has been extensively optimised and tested and introduces a novel platform for using adult OBS cultures to study neurodegenerative diseases, including drug screening and cortical pathology analysis, particularly in conditions like MS
A pharmacological investigation of cholinergic modulation of emotion-related brain connectivity and cognition in bipolar disorder
No full textIntroduction: In bipolar disorder (BD), alterations in functional and effective connectivity and involvement of an impaired muscarinic-cholinergic system contribute to substantial emotional and cognitive disturbances which significantly negatively impact quality of life. Compared with controls, functional neural network connectivity alterations are correlated with compromised executive function, social cognition, and memory ability in BD, while effective connectivity alterations between prefrontal and limbic regions may underlie emotion processing deficits in the disorder. Based upon emotion-related task-based fMRI and muscarinic-cholinergic positron emission tomography research, the muscarinic-cholinergic system is also implicated in emotional and cognitive processing, is altered in BD, and may further impact the emotional and cognitive disturbances experienced across BD states. However, there is little research concerning how intrinsic, resting-state network and prefrontal cingulate effective connectivity alterations, together with underlying muscarinic- cholinergic dysfunction, underpins the poorer cognitive and emotional processing experienced in BD. Yet, this information will elucidate both the intrinsic, neuroanatomical composition and mechanistic neurochemical basis of these widespread symptoms, which may inform novel, more efficient treatment options for those with BD. Therefore, in the current thesis, we aim to use resting-state functional magnetic resonance imaging methods, effective connectivity techniques, and muscarinic-cholinergic drug challenges to investigate how connectivity differences and modulated muscarinic-cholinergic transmission may underlie the persistent emotional and cognitive deficits in BD.
Methods: The manuscripts herein comprise data from individuals with euthymic or depressed BD and psychiatrically healthy controls (HC). In the first manuscript (Chapter 3), subjects with euthymic BD and HC underwent structural and resting-state fMRI scans and cognitive testing. Independent component analysis of resting-state timeseries extracted signal components organized into the following intrinsic networks: occipital, pericentral, midcingulo-insular, and dorsal, lateral, and medial frontoparietal networks. Intra- and inter- network connectivity and related relationships with cognitive performance was assessed between groups through the use of a generalized linear model. In the second manuscript (Chapter 4), individuals who are euthymic and have a diagnosis of BD or are HC underwent fMRI scanning during an emotion inhibition task at baseline and during intravenous physostigmine or placebo challenge. Effective connectivity between the anterior cingulate, posterior cingulate, and middle frontal gyrus was investigated at baseline and during the drug-challenge using bilinear dynamic causal modeling and Bayesian statistics to elucidate the optimal model which explains the observed neuroimaging data during emotion inhibition of the negative valence and underlying involvement of the muscarinic cholinergic system in BD compared with HC. In manuscript three (Chapter 5), muscarinic-cholinergic antagonism (through scopolamine, which has an opposing mechanism of action to physostigmine) and related cognitive impairments in depressed BD were investigated using the administration of intravenous scopolamine or placebo and the Cambridge Neuropsychological Test Automated Battery (CANTAB) to measure cognition. Cognitive testing for emotion recognition, executive function, memory, and attention was conducted three times: first at baseline following placebo run-in, then at an acute timepoint 40-50 minutes following scopolamine or placebo (between 2 and 7 days following baseline), and thirdly, approximately one week following the third of three doses to assess the long-term effects of scopolamine on cognition. Acute and long-term effects of scopolamine on cognitive accuracy and latency were examined using linear regression, accounting for baseline performance.
Results: In manuscript one (Chapter 3), we observed altered resting-state functional connectivity within the lateral frontoparietal network, related to executive functioning, altered pericentral, lateral and dorsal frontoparietal, and occipital network connectivity, correlated with social cognition, and altered midcingulo-insular, lateral and dorsal frontoparietal, and occipital network features related with memory performance in BD compared with HC. In manuscript two (Chapter 4), in effective connectivity, we also observed that a modulation on the connection from the posterior cingulate to the middle frontal gyrus best explained the emotion inhibition of negatively valenced stimuli. After the administration of physostigmine, analyses revealed an increase in modulatory connection strength, at this connection, in HC that was absent in BD. Furthermore, positive and negative connection strength values indicated increased and decreased effective connectivity in HC and BD, respectively. In manuscript three (Chapter 5), there were no significant effects of scopolamine on emotion recognition accuracy during averaged anger and disgust trials at acute or long-term timepoints. Additionally, at acute and long-term timepoints, there were no significant effects of scopolamine on accuracy or latency during emotion recognition (total score and six emotions separately), visual memory, sustained attention, or executive function.
Discussion: The findings in manuscript one (Chapter 3) suggest that intrinsic, network connectivity disruptions contribute to persistent cognitive features in BD. Cognitive impairment may be further underpinned by altered effective connectivity between prefrontal and cingulate cortex regions, and the results of manuscript two (Chapter 4) suggest a compromised muscarinic-cholinergic system in BD, with diminished influence of the posterior cingulate cortex on the middle frontal gyrus during emotion inhibition of the negative valence. In the BOLD fMRI study which formed the basis for the effective connectivity analyses (Chapter 4), physostigmine, an agent that increases cholinergic transmission, improved accuracy in BD on emotion inhibition task performance (Nabulsi et al., 2022). However, a novel cholinergic drug, scopolamine (used in Chapter 5), acts in the opposite direction to physostigmine, and has been proposed to provide antidepressant relief in BD depression. Therefore, as a muscarinic acetylcholine receptor antagonist which decreases cholinergic transmission, it was crucial to investigate whether scopolamine may impair cognition in a depressed BD sample, where cognitive deficits already exist. However, our results in manuscript three (Chapter 5) suggest that, at the hypothesized therapeutic dose of intravenous scopolamine (4 μg/kg), emotion recognition, memory, attention and executive function are not further impaired at acute or long-term timepoints. If intravenous scopolamine is utilized as an antidepressant in the future, it may not exacerbate the cognitive deficits associated with bipolar depression. Overall, cognitive impairment is a core feature of BD across states, is sustained during clinical remission, and greatly impacts quality of life of impacted individuals. This is depicted through alterations in resting-state functional connectivity in intrinsic, cognitive-related networks and differential, cholinergic-based effective connectivity patterns between prefrontal and cingulate regions. Current treatments for BD depression remain suboptimal; taken together, the three manuscripts observe how functional and effective connectivity differences and related muscarinic-cholinergic alterations underpin cognitive impairment in BD, which may inform the development of targeted, effective treatments (such as scopolamine) which preserve or enhance cognitive function with the aim of improving the quality of life of individuals with bipolar disorder
Regulation of cancer testis antigens by unfolded protein response and their function in breast cancer
No full textStressful conditions of the tumour microenvironment, which overwhelm the folding capacity of the endoplasmic reticulum (EnR), activate an evolutionarily conserved signalling pathway known as the unfolded protein response (UPR). UPR is regulated by three EnR transmembrane sensors—ATF6, PERK and IRE1—each playing a distinct role in restoring EnR homeostasis. Through the coordinated actions of these sensors, the UPR aims to restore EnR homeostasis by reducing the influx of client proteins, enhancing the cell’s ability to fold misfolded proteins and removing unfolded proteins via the EnR-associated degradation (ERAD) pathway. The degradation of unfolded proteins and the enhancement of protein folding capacity, mediated by the coordinated efforts of the UPR, attempts to restore protein homeostasis. UPR is an adaptive reaction that reduces unfolded protein load to maintain cell viability and function. UPR can exert both pro-survival and deleterious effects on the survival of cancer cells. In addition to promoting cellular survival, UPR can initiate apoptosis under conditions of chronic stress. Chronic EnR stress has been linked to the occurrence of many diseases, including cancer, neurodegeneration, ischemia and diabetes. Accordingly, a comprehensive mechanistic understanding of UPR signalling is very critical for the evaluation of its biological effects in normal and/or disease conditions, as well as for developing preventive and therapeutic measures against UPR-associated diseases.
Cancer/testis antigens (CTAs) are a group of proteins that are restrictively expressed in testes but are either absent or expressed at very low levels in somatic cells and normal tissues. CTAs are highly expressed during embryonic development to promote cell proliferation, migration and survival, playing a crucial role in embryonic development. After birth, most CTAs, especially the CTA genes located at the X-chromosome, are restricted to expression only in male testis, with no expression in somatic cells, mainly due to the hyper-methylation of genomic DNA in the promoter and enhancer regions of CTAs. Their expression is tightly controlled by DNA methylation and histone modifications, involving epigenetic modulatory proteins such as the germ-cell specific CCCTC-binding factor (CTCF) and the brother of regulator of imprinted sites (BORIS). While the expression of CTAs is a recurrent observation in tumours, the extent of their expression differs between cancers and even among tumours of the same cancer type. CTAs promote tumour growth, metastasis and drug resistance, and are closely associated with poor prognoses in various types of cancer patients. Given their highly tumour specific expression and pro-tumourigenic functions, CTA-based therapy provides novel opportunities for potent treatment responses with minimal adverse effects. Despite the wealth of information about their function in cancer, the regulation of their expression in cancer remains poorly understood.
In this study, we performed transcriptomic analysis using total RNA from parental T47D breast cancer cells treated with the UPR activators, thapsigargin (TG) and brefeldin A (BFA). Through bioinformatic analysis of the RNA-Seq data, combined with a comprehensive literature review, we identified eight candidate CTA genes, including MAGEA11, DMRT1, SPATA17, LYPD4, MAGEB18, Calmegin (CLGN), PLAC1 and ZNF165. The RNA-Seq and qPCR results showed that CLGN expression was significantly upregulated in breast cancer cells under UPR conditions. CLGN protein is localized within the EnR membrane, where it plays an important role in maintaining EnR homeostasis. Pan-cancer analysis suggests that CLGN expression was significantly upregulated across various cancers. Further bioinformatic analysis revealed that CLGN expression was significantly upregulated in primary tumour samples of breast cancer, and the higher expression of CLGN indicated poorer survival. Our results show that CLGN expression was upregulated in a PERK-dependent manner during UPR. This study also demonstrated that CLGN knockdown reduced the proliferation, migration and colony formation abilities of breast cancer cells. Furthermore, CLGN knockdown sensitized 293T cells to UPR-induced cell death and the chemotherapeutic drug cisplatin.
EnR is the major cellular compartment where the folding and maturation of secretory and membrane proteins take place. When protein folding demands exceed EnR capacity, the UPR pathway reduces the client protein load in the EnR by inhibiting protein translation and increases protein handling capacity by upregulating genes encoding EnR-resident molecular chaperones. The main pathway for translational repression in response to EnR stress has been the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by PERK. In addition, PERK-dependent phosphorylation of eIF2α plays a critical role in the inactivation of RRN3/TIF-IA, leading to the downregulation of ribosomal RNA (rRNA) synthesis. Downregulation of rRNA transcription occurs simultaneously or slightly prior to eIF2α phosphorylation-induced translation repression. The EnR transmembrane kinase/ribonuclease IRE1β induces 28S rRNA cleavage and represses protein synthesis in response to EnR stress. It is an efficient strategy that IRE1β directly cleaves 28S rRNA on the rough EnR. rRNA methylation and pseudouridylation can fine-tune protein synthesis. Small nucleolar RNAs (SnoRNAs) are a family of small non-coding RNAs located in the nucleolus and guide the post-transcriptional modifications of rRNAs. SnoRNPs are mainly separated into two subtypes: C/D box and H/ACA box. C/D box SnoRNP is a stable assembly of C/D box SnoRNA and core proteins (NHP2L1, NOP56, NOP58 and FBL) and guides rRNA 2'-O-methylation. H/ACA SnoRNP consists of H/ACA box SnoRNA and core proteins (NHP2, NOP10, GAR1 and DKC1) and catalyses the pseudouridylation modifications of rRNAs. We hypothesize that the UPR may regulate translational fidelity by regulating the expression of core proteins of C/D and H/ACA box SnoRNPs.
The bioinformatics results suggest that these core proteins were upregulated, except for FBL, in breast cancer, and higher expression levels of NOP56, NOP58, NHP2, NOP10 and DKC1 indicated poorer survival. Here, we evaluated the expression of core proteins belonging to C/D and H/ACA box SnoRNPs during EnR stress. This study also suggests that NHP2L1, NOP56 and FBL were significantly downregulated by UPR. In addition, FBL knockdown reduced cell proliferation and colony formation. Furthermore, we show the effects of UPR and FBL knockdown on rRNA methylation and translation fidelity, including alterations in nonsense suppression, frameshifts, ribosome pausing and translation initiation by internal ribosome entry site (IRES).
In conclusion, this study reveals the regulation of CLGN expression by UPR and its function in breast cancer, as well as the regulation of translational fidelity during UPR by mediating the downregulation of C/D box SnoRNP, leading to change in rRNA methylation.China Scholarship Council 20200637006
The mechanics of biological growth: A study through the vertex model
No full textThis doctoral thesis investigates the mechanics of growth and remodeling in biological tissues through a discrete framework known as the vertex model. While this model has been extensively used for numerical simulations in biological contexts, the present work focuses on a fundamental aspect: the generation of elastic stresses in vertex-based systems. This study introduces the concept of incompatibility in such tissues, a well-established source of residual stresses in continuous mechanics in the absence of external loads.
A key contribution of this thesis is the identification and characterization of two distinct types of incompatibility in the vertex model. The first, termed internal incompatibility, arises when the target area and perimeter of individual cells violate the isometric inequality. Internal incompatibility is recognized as a regulator of the critical transition between fluid-like and solid-like cell behavior, which plays a crucial role in processes such as cancer cell migration. The second, termed external incompatibility, pertains to the manner in which cells are interconnected to form specific tissue morphologies. Both types of incompatibility act as sources of residual stresses in tissues described through the vertex model.
The second part of this work explores the consequences of elastic stress accumulation on the possibility of inelastic tissue evolution. Specifically, the study examines phase transitions, including T1, T2, and T3 transitions, as well as cell division, analyzing how such non-elastic processes enable the system to evolve toward a minimal energy configuration. This evolution represents a potential pathway for growth in biological tissues.
The analysis, implemented using a MATLAB code, further investigates the influence of parameters such as area stiffness, perimeter stiffness, and line tension on growth progression, to single out the conditions leading to the instabilization of initially flat or regular surfaces. Preliminary results suggest that incompatibilities and stiffness parameters significantly contribute to the onset of corrugations at the interface between domains with differing control parameters. These findings open the door to explore connections with tumor growth, particularly in the context of metastasis spreading into healthy tissues
Exploring novel antibiotic and enzyme combinations to improve the treatment options for biofilm-associated diabetic foot infections
No full textStaphylococcus aureus isolates collected from the wounds of a cohort of patients presenting at University Hospital Galway (UHG) with diabetic foot infections were shown to produce a mix of polysaccharide and protein type biofilms in vitro. Several of the MSSA isolates also demonstrated the ability to switch from a polysaccharide to protein mechanism of biofilm formation. This latter phenotype is more commonly associated with MRSA isolates following growth in culture media supplemented with glucose. Hyperglycaemia is common in patients with diabetes mellitus and may be accompanied by increased levels of glucose in the wound milieu, which in turn would promote protein-adhesin type biofilm formation. Several coagulase-negative staphylococci including two methicillin-resistant Staphylococcus epidermidis isolates, which are often commonly overlooked in chronic wounds, also formed robust biofilms in vitro suggesting they were clinically significant in the pathogenesis of the wound infections from which they were isolated.
In a related collaborative study with colleagues at the University of Liverpool on chronic infections in patients with cystic fibrosis, twelve MRSA isolates were collected sequentially over a period of four years from the lungs of a single patient. All of these isolates formed robust protein-type biofilms in vitro. Isolate LCF269, which was the fourth sequential strain isolated from the patient produced approximately 4X more biofilm than the other isolates. Whole genome sequencing analysis revealed that LCF269 and subsequent isolates were genetically different to the first three isolates. Furthermore LCF269 was found to contain 6 synonymous mutations in sdrC, which encodes a cell wall anchored protein previously implicated in the biofilm phenotype.
Interestingly, two of the clinical DFI-associated MSSA isolates were shown to be very similar to one another. Both demonstrated the ability to switch between the protein and polysaccharide type biofilm production and both possessed similar antimicrobial susceptibility profiles. Upon taxonomic classification these were the only two isolates collected from the patients at UHG shown to share a top common hit during sequence similarity searches using NCBI BLAST. These isolates were collected from two separate patients presenting at UHG within one week of each other raising the possibility that similar MSSA strains are circulating among patients attending this clinic.
Antibiotic doses up to 1000X the minimum inhibitory concentration were required to inactivate biofilm bacteria, even when the drugs were used in combination, reflecting the high tolerance of biofilms to antimicrobial drugs. The most effective combination therapy identified in this study comprised rifampicin which targets bacterial transcription, gentamicin which targets translation and the clinically used enzymatic debridement agent trypsin. Exposure of biofilms to this triple combination for 48 hours resulted in a > 3 log (99.9%) reduction in the number of colony forming units for all of the DFD staphylococcal isolates tested, surpassing the proposed threshold for clinical significance. Moreover, this triple combination was also highly effective against S. aureus and Pseudomonas aeruginosa mixed species biofilms using a chronic wound biofilm model at clinically achievable drug and enzyme concentrations. The rifampicin/gentamicin/trypsin combination represents a promising new therapeutic strategy for the treatment of biofilm-associated diabetic foot infections
Merging psychology and technology: Understanding, quantifying and predicting driver fatigue during conditionally automated driving
No full textBackground. Conditionally automated driving, which constitutes the continuous, operational design domain-specific performance of the driving task by an automated driving system, will soon become commonplace in everyday driving. However, the monotony, extended periods of exposure, and increase in automation which characterises the change in the driver’s role from active operator to passive supervisor incurs a risk of the driver developing dangerous levels of fatigue. Driver fatigue, which is distinct from drowsiness and is already a leading cause of road traffic accidents, is thought to imperil safe transitions of control between automated system and human driver by decreasing attention, alertness and vigilance, rendering the driver ill-equipped to respond to time-sensitive situational demands. This has prompted researchers, vehicle manufacturers and legislators alike to recommend the inclusion of driver monitoring systems, which are capable of assessing the driver’s mental state, within future conditionally automated vehicles. Current driver monitoring technology, however, largely relies on behavioural measures which do not necessarily permit the detection of more subtle changes in the driver’s mental state, such as an increase in fatigue. Physiological measures therefore represent a promising means of overcoming the limitations of traditional measures of fatigue whilst also affording a higher temporal resolution.
Aims. This thesis aimed to: (1) Understand the presentation and impact of driver fatigue during conditionally automated driving, as well as drivers’ subjective experiences and perceptions of the interactions between behaviour, vehicle automation and in-vehicle monitoring; (2) Quantify driver fatigue using physiological, subjective and behavioural measures, to understand the effect of automation on physiological activity, as well as how activity and arousal vary due to task-related factors in automated driving; (3) Predict the progression of driver fatigue during conditionally automated driving by identifying the point at which fatigue reaches its highest level during prolonged automation.
Methodology. A sequential, mixed-methods approach was taken to addressing the aims of this thesis, whereby four studies were completed. Study 1 constituted a systematic review and meta-analysis concerning the effect of non-driving related tasks on drivers’ physiological responses during conditionally automated driving. Study 2 employed qualitative focus group interviews to examine drivers’ perspectives and attitudes towards the use of driver monitoring systems during automated driving. Study 3 involved a driving simulator study which sought to investigate the effect of prolonged conditionally automated driving on the development of fatigue, drivers’ physiological responses, and their takeover performance. Lastly, in study 4, supervised machine learning methods were applied to understanding the progression and peak of driver fatigue, as captured by physiological activity, during prolonged conditionally automated driving. Studies 1 and 2 contributed to the design of studies 3 and 4 through refining their conceptual and methodological underpinnings, and by including the driver’s perspective in the design of the research. The physiological markers of fatigue that were used in study 4 were also selected on the basis of study 3’s findings.
Findings. The systematic review and meta-analysis reported that, when drivers engaged with a non-driving-related task during conditionally automated driving, an increase in physiological arousal was observed, supporting the use of secondary tasks as a means of increasing physiological activity during automated driving. Study 2 found that drivers perceived driver monitoring and automated driving as a secondary layer of support with the potential for maladaptive consequences for the user, citing a perceived risk of overreliance on automation forming over time. The results of study 3 demonstrated that a prolonged period of conditionally automated driving led to a significant increase in subjective fatigue, as well as significant differences in several physiological measures compared with manual driving which were indicative of heightened parasympathetic activity. Finally, study 4’s findings revealed that the progression of driver fatigue was characterised by a series of peaks and brief recovery periods, supporting the use of self-regulatory strategies to mitigate fatigue during conditionally automated driving.
Conclusions. The findings of this thesis make several important contributions to the literature concerning the psychological and cognitive processes underpinning drivers’ interactions with automated driving systems. Namely, the findings have quantified the evidence for the physiological effect of driver fatigue, as well as vehicle- and task-related factors during conditionally automated driving, providing strong justification for the use of physiological measures to assess driver mental states. They have clearly outlined the relationship between driver fatigue and conditionally automated driving by documenting changes over time, thus providing a clear profile of the temporality of fatigue. An understanding of drivers’ perceptions and representations of this technology has also yielded several recommendations for the development of user-centred driver safety assistance features. Lastly, predicting the progression and peak of driver fatigue using physiological measures has implications for emergent driver monitoring systems that can detect fatigue in an accurate and timely manner.This work was conducted with the financial support of the Research Ireland Centre for Research Training in Digitally-Enhanced Reality (d-real) under Grant No. 18/CRT/6224
Peptide-based targeted covalent inhibitors of cysteine proteases
No full textTargeted covalent inhibitors (TCIs) can modulate dysregulated enzymes by covalently engaging with nucleophilic residues in active sites. Cysteine cathepsins, implicated in diseases such as cancer, osteoporosis, and viral infections, are promising targets for TCI development. The nucleophilic cysteine residue in the active site reacts with inhibitory electrophilic molecules, but structural similarity between cathepsins has resulted in a lack of specificity of inhibition. This work explores the synthesis of peptide-based isothiazolones, a class of reactive heterocycles, as selective covalent inhibitors of cathepsins. These compounds exhibit a reactive sulfur–nitrogen bond that can be targeted by thiol nucleophiles, forming labile disulfide linkages and have previously been shown to interact with biological thiols.
Cysteine-containing peptides were synthesised via solid-phase peptide synthesis and modified to incorporate isothiazolone moieties by using a Norrish Type II transformation of a phenacylsulfide derivative. Optimisation of reaction conditions led to successful isothiazolone formation, alongside other interesting side products, for several peptide candidates. The scaffold of a known peptidic inhibitor of cathepsin B, a protease strongly implicated in tumour progression, was selected for TCI design and showed promising isothiazolone conversion rates. One candidate was evaluated in vitro and showed potential inhibitory activity under non-reducing conditions, supporting the scaffold’s suitability for further biological investigation. C-terminal carboxylate analogues were also shown to undergo isothiazolone formation, providing constructs relevant for interactions with the occluding loop hidtidine residues of cathepsin B. Ongoing work is directed toward the improved design, isolation and purification of these peptide isothiazolones in sufficient quantities to permit expanded biological evaluation as TCIs
The management of the scaling process from the perspective of serial scalers
No full textVenture scaling has garnered significant attention from practitioners, policymakers, and academics within the fields of entrepreneurship and management. The focus has been on the inputs and outputs of scaling ventures, with limited exploration into the management of the scaling process itself. This thesis addresses this gap by investigating how serial scalers—professional managers with extensive scaling experience—navigate the complexities of the scaling process. The central research question is: How do serial scalers manage the scaling process?
Given the exploratory nature of this inquiry, I conducted an inductive, qualitative study involving semi-structured interviews with 30 serial scalers. This novel approach provides fresh insights into the field of scaling, particularly as our understanding of how to manage scaling ventures successfully is still nascent. The study incorporates diverse perspectives from serial scalers in senior roles across various functions in scaling enterprises located in Ireland and Portugal.
By examining the experiences of serial scalers, this research reveals new perspectives and practices for managing the "growing-up" and "scaling-up" stages of venture development. Utilizing organizational culture theory, the study elucidates the intricate dynamics of managing scaling ventures. The findings reveal emergent scaling practices, highlighting how scalability is first achieved and then enhanced throughout the scaling process. Specifically, the research demonstrates how practices generated in the "growing-up" stage evolve during the "scaling-up" phase, underscoring the necessity of adaptive management strategies to sustain and enhance scalability. Furthermore, the study emphasizes the critical role of a high-performance organizational culture as the scaffolding for the successful managing of scaling.
This research contributes to the entrepreneurship and management literature by identifying key practices and their evolution, offering valuable insights for both theorists and practitioners. Finally, the thesis discusses limitations and implications for future research, paving the way for continued exploration in this area
Democratising adaptation - The role and place of public participation in climate change adaptation policies in Ireland
No full textAdapting to future socio-environmental changes induced by climate change represents one of the greatest challenges of our time. Climate change adaptation (CCA) policies are crucial, as these long-term strategies enable local communities to anticipate, prepare for, and build capacity to cope with potentially devastating extreme weather impacts. Despite significant challenges arising from long-term planning requirements and high uncertainties, adaptation policies also present a unique opportunity for public participation. The context-specific nature of local adaptation strategies and their cross-sectoral scope provides an avenue to rethink traditional decision-making processes, facilitating the active involvement of diverse actors in the formulation of these comprehensive, long-term roadmaps.
Reconsidering and redefining our perceptions of democracy is equally essential for addressing future environmental changes and socio-economic uncertainties. Participatory decision-making processes can generate multiple benefits, from the formulation of locally responsive policies to the promotion of social learning and the strengthening of social capital. Beyond these functional outcomes, public participation embodies a democratic ideal: fair, inclusive, and egalitarian engagement.
However, public participation rarely achieves its normative and democratic promises, despite being frequently cited as a pillar of climate policy. In practice, participation is institutionalised within complex governance arrangements, often reduced to consultative exercises on pre-formulated policies. Furthermore, the concept of participation is often vaguely defined in policy, rendering it discursively malleable and open to diverse interpretations (e.g., what constitutes participation and who should be engaged).
This thesis situates itself within this context, aiming to provide new interpretive insights to better understand the role and place of public participation in contemporary decision-making processes. Drawing on Ireland’s experience in formulating and implementing national and local CCA policies, with a particular focus on the North-West region, the research adopts three complementary perspectives to examine public participation. The study deliberately spans from a post-structuralist critique of public participation to an exploration of individual motivations for engagement.
The findings reveal profound challenges to public participation at both institutional and individual levels. Beyond structural and practical barriers, public participation discourse and practice is shaped by systematic governmental rationalities, which are reinforced through research. Building on this critique, the thesis goes beyond policy framing to critically examine the experiences of those implementing policy and the perspectives of local residents, highlighting practical-institutional barriers and individuals’ preferences for participation. By integrating both institutional “top-down” and individual “bottom-up” perspectives, the research delineates the constraining space for public participation and highlights two areas requiring particular vigilance in future research and practice on public participation in CCA policies
Iron sulfide–driven denitrification: Mechanistic insights into chemical and biological pathways and microbial regulation effects
No full textIron sulfide–driven autotrophic denitrification has emerged as a promising carbon–free strategy for nitrate (NO3–) removal in anoxic environments. While biological denitrification using iron sulfides as electron donors has been extensively studied, the role and mechanism of abiotic NO3– chemodenitrification remain largely unresolved. This research systematically investigated the transformation of NO3–, product selectivity, and coupled elemental cycling of N, S and Fe in chemical and biological denitrification systems mediated by three representative iron sulfides, FeS, FeS2, and pyrrhotite. The specific objectives of this PhD research were: (1) study of the chemical denitrification of NO3− by iron sulfide under mild conditions and its transformation mechanism; (2) investigation of the effect of the surface structure of iron sulfide on autotrophic denitrification and its role in nitrogen cycle; (3) Elucidation of microbial–induced mechanisms driving the shift in nitrogen reduction products from ammonium (NH4+) to nitrogen gas (N2) in FeS–mediated autotrophic denitrification.
FeS was found to chemically reduce NO3– to NH4+ with a high NO3– reduction efficiency of 97.5% and NH4+ product selectivity of 82.6%, whereas FeS2 and pyrrhotite showed negligible reactivity. Electrochemical analyses revealed that FeS exhibited a superior electron release rate. Quenching experiments and density functional theory (DFT) calculations confirmed that surface Fe(II) and sulfur vacancies on FeS played key roles in facilitating NO3– reduction through selective oxygen adsorption and water dissociation, leading to NH4+ formation.
In microbial systems, FeS and pyrrhotite supported effective autotrophic denitrification, while FeS2 remained biologically inactive. The differences in reactivity were attributed to their mineral–specific surface structures and Fe–S bond energies: pyrrhotite, with iron vacancies and a lower bond energy (1.35 eV), facilitated microbial electron transfer, whereas FeS2, with strong bonding (1.63 eV), hindered microbial colonization and electron mobility. FeS, with intermediate bond energy (1.39 eV) and sulfur vacancies, supported both abiotic and biotic NO3– transformations.
Furthermore, microbial colonization of FeS altered its surface redox structure, including Fe(II) oxidation and sulfur speciation changes, enhancing electron transfer capacity and shifting NO3– reduction from NH4+ production toward N2 formation. This demonstrated the critical role of mineral–microbe interactions in modulating reaction pathways and product outcomes.
This PhD research provides new mechanistic understanding of iron sulfide–driven NO3– reduction, highlighting the importance of surface defects and electron transfer dynamics in determining chemical and biological reactivity. Beyond advancing knowledge of nitrogen, sulfur, and iron biogeochemistry, these findings carry practical implications for engineering design and operation of wastewater treatment systems. FeS, with its strong chemical activity and high product selectivity toward NH4+, offers promise as a redox–active medium or cathode material in engineered systems where abiotic NO3– reduction is desirable. In contrast, pyrrhotite demonstrated the highest efficiency in supporting microbial autotrophic denitrification toward N2, making it a suitable candidate for biofilter media or long–term bioreactor operation. The demonstrated influence of microbial colonization on FeS further provides insights into managing reaction selectivity and long–term stability. Overall, this research offers strategies for tailoring reactive media selection and process design to achieve targeted NO3– removal and sustainable nutrient management in next–generation carbon–neutral wastewater treatment technologies