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The role of myomiRs oxidation on muscle wasting in ageing and cancer cachexia
No full textProgressive loss of muscle mass and strength is a hallmark of ageing (sarcopenia) and disease, such as cancer (cachexia). Muscle atrophy and weakness are associated with increased risk of falls, frailty, worse cancer patient outcomes, and increased morbidity and mortality. The mechanisms of muscle loss are not fully understood due to complexity. Various cellular mechanisms have been demonstrated to underlie muscle loss, including changes in the expression of multiple genes. MicroRNAs (miRs), post-transcriptional gene expression regulators, are emerging as important regulators of skeletal muscle homeostasis, particularly due to their simultaneous targeting of multiple gene targets, supporting their role in regulating complex disorders like muscle loss.
One of the hallmarks of ageing and cachexia is oxidative stress. Whilst DNA, protein, and lipid oxidation have been characterised in muscle ageing and disease, much less is understood about RNA oxidation, and in particular microRNA oxidation. RNA is more sensitive to oxidative damage than DNA, and products of RNA oxidation (8-oxo-guanine) have been detected in different conditions associated with muscle loss. microRNA oxidation could affect how microRNAs regulate their targets, as 8-oxo-guanine can bind to adenosine as well as uracil—this could dysregulate microRNA ability to bind to its targets or even acquire new targets. To date, oxidation of miRs has been demonstrated during cardiac disease. Oxidised miR-184 was implicated in promoting apoptosis in cardiomyocytes through targeting genes that the native miR-184 does not target, while oxidised miR-1 was associated with the development of cardiomyopathy through target misregulation. However, the oxidation of microRNAs, or the functional consequences of this process, has not been investigated in muscle before.
This research examined whether microRNAs are oxidised in muscle during ageing and cancer cachexia and whether oxidised miRs contribute to muscle loss.
I hypothesised that oxidation of miRs contributes to muscle loss during ageing and cachexia through dysregulation of microRNA gene targeting. This hypothesis was first tested in muscle samples from older people and mouse models of ageing and lung cancer cachexia. In muscle from older people and old and cachectic mice, differential expression and oxidation of miRs were demonstrated, and the functional consequences were further validated using C2C12 mouse muscle cells and immortalised human muscle cells. Small RNA sequencing identified several oxidised miRs, including miR-1, miR-133, and miR-206. These oxidised myomiRs, muscle-enriched miRs, had pronounced effects on myotube size: exposure of C2C12 myotubes to oxidised myomiRs resulted in changes in myotube size and altered the expression of multiple genes, including those associated with oxidative stress, protein degradation, and autophagy. In addition to human myotubes, myotube size and the protein levels associated with oxidised RNA were decreased.
This supports the hypothesis that oxidised miRs, particularly myomiRs: miR-1, miR-133, and miR-206, may partially contribute to muscle atrophy through their regulation of key cellular processes: autophagy and oxidative stress response
Towards a system of rules: Charisius’ De Analogia as a witness of continuity and re-functionalisation in Latin grammatical discourse between Antiquity and the Middle Ages. A linguistic and philological commentary, with English translation
No full textIn my doctoral dissertation, I provide an introduction, a philological and linguistic commentary, and the first English translation of Julius Romanus’ De Analogia (approx. third century). This text is uniquely preserved in Charisius’s Ars Grammatica, a fundamental work on Latin grammar produced in the eastern part of the Roman Empire around 360 AD. De analogia, an independent section, is structured as an alphabetical list of 243 entries discussing issues of nominal morphology. In doing so, it often contrasts forms attested in use with standard ones (usus vs. ratio), quoting a remarkable variety of both literary and learned authors (364 quotations, from 71 different authors and 134 titles). The chapter builds on works such as Pliny’s Dubius Sermo and Caesar’s De Analogia, of which it represents our best source.
My study situates this text within its context of production and undertakes an in-depth analysis, revealing a complex stratification of sources and the reuse of materials resulting from its layered composition. Furthermore, it confirms the essential role of De Analogia as a key witness to the enduring vitality of learned discussion on the Latin language throughout the centuries.2019-2020 Digital Arts and Humanities Scholarship (DAH)
2020-2023 Irish Research Council - Government of Ireland Postgraduate Scholarship Programme (2020; Project ID: GOIPG/2020/1490)
A developmental glycobiology approach to formulate an enhanced hyaluronic acid-based hydrogel for intervertebral disc regeneration
No full textIntervertebral disc degeneration (IDD) is the leading cause of lower back pain, which affects the majority of the world population at some point in their lives. IDD is a progressive event marked by inflammation, loss of cellularity, extracellular matrix degradation, loss of water, and structural deterioration. Standard care includes symptom management, pain relief, and invasive surgeries in advanced cases. Addressing the need for regenerative therapeutic approaches, scientists have shifted their focus on the properties of a unique population of cells in the nucleus pulposus (NP) region of the intervertebral disc (IVD). Notochordal cells (NC) developmentally descend from the notochord before transitioning into what is identified as NP cells after maturity and age. Some animals, such as pigs and non-chondrodystrophic dogs, retain this population throughout their lives, while humans lose it after NP maturation. The changes that occur in the disc, including the loss of NCs, coincide with the pattern leading to degeneration, which suggests that the notochordal cells and their niche have a role in keeping IVD healthy. To understand these mechanisms better, molecular aspects of degeneration and maturation must be studied.
One overlooked subject in this regard is glycosylation. It is known that heavily glycosylated molecules are involved in disc degeneration. Considering the loss of NCs and the inflammatory changes that occur during the degeneration of the disc, it is hypothesised that introducing the healthy and young NP glycome within a supportive anti-inflammatory hydrogel network could induce changes in the disc that are indicative of tissue remodelling and regeneration. This study identified the glycosylation patterns in young and mature NPs that are rich in notochordal cells. A thorough analysis of the ultra-performance liquid chromatography (UPLC) and mass spectrometry (MS) data revealed over 300 N-glycan species in the NP, more than 45% of which are sialylated and/or fucosylated. In addition, 5% increase α(1,2/3) fucosylation and 9% decrease in α(2,3) sialylation was detected at maturation. With these properties in mind, a hyaluronic acid (HA) hydrogel network was formulated by crosslinking the carboxylic groups of HA with 8-arm polyethylene glycol (PEG), which was enhanced with the notochordal cell-derived matrix (NCM) to mimic the notochordal niche. This HA-NCM formulation formed a hydrogel within 15 minutes. The fabricated hydrogel showed high swelling capacity (up to 150% in weight), and supported cells such as NPs, NCs and stem cells for at least 14 days. The hydrogel also showed signs of mitigating the pro-inflammatory cytokines in the long-term. To test the regenerative potential of the HA-NCM hydrogel system, the last phase of the project investigated an in vivo study utilising a rat-rail model after introducing an acute injury to the NP. Even though the injury created is more than what is expected in the natural course of IDD, the HA-NCM hydrogel can improve the disc height by 36% after injury.
The work described in this thesis introduces a significant potential of a glycomic approach to a clinical problem. Future studies include more robust and efficient methods to analyse the glycosylation patterns and further charcaterisation of NCM to validate its potential while focusing on the mechanistic approaches to induce regeneration and remodelling of the IVD
Tracing a tyrant’s paper trail: A comparative assessment of the uses of archives in domestic proceedings addressing international crimes
No full text[No abstract available
Against the tide: Immigration into Ireland from continental Europe 1945-1990
No full textImmigration to Ireland in the second half of the Twentieth Century has been largely overlooked, particularly immigration from continental Europe. This examination of the motivation, experiences and impact of Europeans in the period before Maastrict and Nice opened the borders in Europe sheds new light on received historiograpy of Ireland in the late twentieth century
Modelling motion tracking data in elite soccer to classify and quantify collision intensity
No full textMotion tracking data is collected across an increasing number of sports. Some sports can be quite physical and can involve frequent collisions during both training sessions and matches. Depending on the motion tracking devices available for each sport, particularly in matches, it may limit the ability to accurately measure the severity of these collisions. Accelerometers can be used to measure the impact of collisions but they are not used in most sports. The development of a Collision Severity Index (CSI) using motion tracking data would allow analysts to measure collision severity without the use of accelerometers, which is currently absent from any literature. Therefore, the primary aim of this thesis is to present a novel approach to quantify collision severity in soccer by developing a CSI (Chapter 6) using Principal Component Analysis (PCA). In-game motion tracking data is used, which was collected from the English Premier League (EPL), the top soccer league in England. Loading scores can then be developed using the CSI, which can then be used to monitor the players during matches. The loading scores developed as part of this thesis were solely based on the accumulation of the collision severity scores for each player within each game. The loading scores could provide useful information for when players should be rested, which may lead to a reduction in the overall injury incidence in soccer and enhance the overall welfare for players in the sport.
Using video footage, collision-related events were manually labelled across 20 matches in the EPL and all of the studies which involved using motion tracking data were based on 9 of these matches as this was sufficient for the studies. In the first study, all of the collision-related events in a typical soccer game were labelled in order to develop a Soccer Match Collision Dataset (SMCD) (Chapter 3), which proved to be essential for the remainder of the studies in the thesis, mainly for validation purposes. The next two studies involved feature extraction and event detection (for collision-based events) (Chapter 4) and developing tackle and collision classification models (Chapter 5). The aim of the feature extraction and event detection study (Chapter 4) was to firstly develop features that could be used for later studies in the thesis, including the development of classification models and the CSI. Secondly, the aim was to develop event detection algorithms for collision-based events in soccer matches using motion tracking data which included tackles, headers, set-pieces (corners, goal-kicks, free-kicks and throw-ins) and collision detection algorithms. Headers, corners, goal-kicks, free-kicks and throw-ins could be detected with the highest level of accuracy. However, there were many false positives for the tackles and collisions. Developing event detection algorithms for tackles and collisions proved to be the most challenging. Given the diverse nature of tackles and collisions in the sport and the limitations with the data (one point per player, no player elevation, etc.), it made it extremely difficult to detect tackles and collisions. There was also a lot of borderline cases for both tackles and collisions which added to the complexity of the study.
The aim of the tackle and collision classification study (Chapter 5) was to develop classification models for the various tackle and collision types in soccer. Features were developed before and after the midpoints of the events in the hope to capture the motion of the players (and ball) in order to successfully develop the classification models. The study played a key part for a streamlined approach for the development of Collision-Specific Severity Indices (CSSI) as well as a general Collision Severity Index (CSI) for the collisions in soccer. Accurate tackle classification models could be developed. The accuracy was not high for the collision classification models due to the fact that a lot of the sub-classes were included in the body to body contact class, making it harder to make accurate predictions. There were not many observations for some of the collision classes also i.e., push and pull. Including all of the tackles and collisions that should really be classified in an 'other' class, also added to the complexity of the study.
The fourth study (Chapter 6) was the main goal of the thesis; the development of a Collision Severity Index (CSI) in soccer using in-game motion tracking data. Many features were developed around the point of contact of the collisions and Principal Component Analysis (PCA) was used on different combinations of features (and variations of frames) in order to develop a CSI. A general CSI was developed as well as Collision-Specific Severity Indices (CSSI) for different collision types in soccer. The accumulation of all of the collision severity scores from the collisions could then be incorporated into a loading score for each player. The loading scores could potentially be used by coaching staff to guide them in the decision-making process on when to rest (or substitute) players during matches as well as whether to start or rest players before a match has commenced. A dashboard was then developed using R Shiny (Chapter 7), where a collision-hurt map (CHM) was the main feature. Such a tool can be used by analysts, researchers and the medical team in order to inform post game rehab, identify players at increased risk of injury and ultimately improve player safety and welfare.
The general CSI (and CSSI) were proven to be very effective when the severity quantifications were analysed against the collision severity levels (heavy contact, medium contact, light contact and barely contact) using box plots (and violin plots) (Chapter 8).
The results of this thesis have shown that an accurate Collision Severity Index (CSI) (and Collision-Specific Severity Indices (CSSI)) can be developed in soccer using in-game motion tracking data. These indices coupled with loading scores could potentially be used to guide coaching staff on when players should be rested, which could minimise the risk of injuries and improve the player welfare in the sport. Future studies are warranted in order to use this novel approach to quantify collision severity to aid with any collision-based tools which are currently absent from any literature using motion tracking data
Towards non-invasive optical reconstruction of the human eye’s crystalline lens
No full textLight propagation in non-homogenous media with varying refractive index is challenging to analyse, even with knowledge of the gradient index (GRIN) structure n(x,y,z). Differential equations can be used to describe the path light rays take when travelling through a medium. In this project, a differential ray equation is used to calculate the effective focal length (EFL) and back focal distance (BFD) of a GRIN lens, with an emphasis on quadratic refractive index profiles commonly used to describe the crystalline lens of the human eye. The estimated values for EFL and BFD are then compared to the output of an optical design software, Zemax OpticStudio, which performs numerical ray-tracing through the lens. Ray-tracing in GRIN media is very difficult, and usually not possible without numerical methods, however for several types of GRIN lenses, the analytical solution exists.
By extending the ray-tracing from the paraxial region (near the axis) to a broader region, a general differential ray equation has been derived, enabling the analysis of rays propagating through the GRIN lens at finite heights. From this, the back focal distances for rays entering the lens at varying initial heights can be calculated, and thus, the longitudinal spherical aberration can be estimated. The solution to the general differential equation enables one to calculate the refractive indices along the ray. The BFDs obtained using the paraxial method, the along-ray method, and numerical ray-tracing in Zemax OpticStudio software have all been compared, showing consistent results. The EFL of the lens was estimated using the derived ray equation and then compared with the numerical solution in Zemax OpticStudio; the discrepancy in the values of the EFL did not exceed one-tenth of the wavelength of light.
The proposed ray-tracing methods have been applied to models of crystalline lenses found in nature, with specific interest on the lens in an octopus eye and Liou and Brennan model of a human eye. Reconstructing the refractive index profile of the crystalline lens presents a major challenge in representing the lens as a GRIN medium with a specific profile defined by polynomial functions of radial and axial distances. Without prior knowledge of the polynomial functions for a given lens, reconstructing the refractive index profile depends on predicting the path of light within the GRIN medium. This project explores a systematic approach to solving the GRIN reconstruction problem for symmetric and asymmetric lenses by utilizing the geometry of the lens's outer surface and the solution from the proposed ray equation based on the Frobenius method
The effects of inflammatory microenvironments on MSC viability, phenotype, secretome and function
No full textIntroduction: Acute respiratory distress syndrome (ARDS) is a severe clinical syndrome which is characterised by acute hypoxaemia and diffuse lung inflammation. The estimated incidence of ARDS (Berlin definition) is 10.4 % among intensive care unit (ICU) admissions and the estimated hospital mortality rate is around 40 % across mild, moderate and severe ARDS. Despite the high incidence and mortality, no efficient therapeutics have been identified for ARDS treatment. The pathogenesis of ARDS involves infectious and non-infectious aetiology, dysfunction of alveolar-capillary barrier and dysregulation of immune response. Mesenchymal stromal cell (MSC)-based therapies have shown therapeutic benefits in the restoration of alveolar-capillary barriers and immunomodulatory effects on neutrophils, macrophages, T cells and other immune cells in ex vivo and in vivo preclinical ARDS models. Clinical trials using different types of MSCs have shown the safety of MSC-based therapies, yet significant efficacy remains elusive. The variable clinical outcomes of MSC-therapies in ARDS are attributed to the tissue sources, donor variability, doses, administration route of MSCs and host microenvironment. A growing body of evidence suggests that inflammatory microenvironments play pivotal roles in evoking MSC therapeutic benefits in ARDS. In this project, inflammatory cytokines and lung homogenate were applied to investigate the effects of inflammatory microenvironments in ARDS on MSC phenotype, secretome and functions in vitro. In addition, healthy and Escherichia coli (E. coli)-induced ARDS models were used to investigate the interaction between MSCs and lung microenvironment in vivo.
Methods: 1) A cocktail of inflammatory cytokines including interleukin (IL)-1β, interferon (IFN)-γ and tumour necrosis factor (TNF)-α (cytomix) was used to mimic the inflammatory microenvironment. Three types of MSCs including bone marrow-derived MSC (BM-MSC), human wild type induced pluripotent stem cell-derived MSC (iMSC WT) and beta-2 microglobulin knockout iMSC (iMSC B2M KO) were cultured with or without cytomix. Naïve and cytomix-primed MSCs and conditioned media (CM) were harvested to analyse the therapeutic effects of MSCs, including their expression of immunomodulatory surface markers, their release of cytokines, their anti-microbial properties and their anti-inflammatory effects on pulmonary epithelia, macrophages and T cells. 2) Lung homogenates from sham and E. coli-induced ARDS animals were used to mimic healthy and ARDS lung microenvironments. BM-MSCs were exposed to lung homogenate for 24 h, and BM-MSCs and CM were harvested to analyse the apoptosis, immunophenotype, secretome, anti-microbial properties and anti-inflammatory effects on T cells. 3) BM-MSCs were administered to healthy and E. coli-induced ARDS animals and retrieved at 0.5 h, 1 h, 3 h, 6 h and 24 h post cell administration. The morphology, apoptosis and surface biomarkers of the retrieved BM-MSCs were analysed. The composition of immune cell populations and cytokines in healthy and ARDS lung microenvironment were analysed at the time points post cell administration.
Results: 1) Compared to naïve BM-MSC, iMSC WT and iMSC B2M KO, cytomix increased the expression of HLA-ABC on BM-MSC and iMSC WT, CD54 and CD200 on BM-MSC, iMSC WT, iMSC B2M KO, CD120b on BM-MSC and CD119 on two types of iMSC, respectively; cytomix increased the release of IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 from three types of MSCs while decreasing the release of TNF receptor 1 (TNFR1), transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF) from iMSC WT; cytomix enhanced three types of MSC inhibition on E. coli proliferation and BM-MSC inhibition on Klebsiella pneumoniae (K. pneumoniae); cytomix enhanced three types of MSC promotion on macrophage phagocytosis and three types of CM inhibition on T cell proliferation. 2) Compared to sham lung homogenate, E. coli-instilled ARDS lung homogenate induced lower levels of apoptosis in BM-MSCs, higher expression levels of CD54, CD200 and CD119 on BM-MSCs and higher levels of IL-6, IL-8, MCP-1 and VEGF in CM. Both sham and ARDS lung homogenate retained BM-MSC inhibition on E. coli, K. pneumoniae and Staphylococcus aureus (S. aureus) and enhanced BM-MSC inhibition on T cell expansion. 3) BM-MSCs were retrievable at 0.5 h and 1 h from healthy lungs and until 6 h from E. coli-induced ARDS lungs post cell administration. BM-MSCs underwent apoptosis in ARDS lung microenvironment. The expression levels of CD105, CD90, CD73, HLA-ABC, CD54 and CD200 on BM-MSCs were decreased in both healthy and ARDS lung microenvironment. On the other hand, BM-MSCs altered both healthy and ARDS lung microenvironment by increasing the percentage of non-classical monocytes in mononuclear phagocytes (MNPs) and decreasing the percentage of B cells in lymphocytes. In addition, BM-MSCs transiently increased the levels of cytokine-induced neutrophil chemoattractant 1 (CINC1), IL-6, MCP-1 and matrix metalloproteinase (MMP)-9 in ARDS lung proteome at 3 h post cell administration.
Conclusion: 1) Inflammatory cytokines enhance MSC therapeutic effects by increasing the immunomodulatory surface markers on MSCs, promoting MSC anti-microbial effects and anti-inflammatory effects on macrophages and T cells. 2) Both iMSC WT and iMSC B2M KO show similar therapeutic benefits as BM-MSCs, and iMSC B2M KO has no immunogenicity, providing promising alternatives to MSC-based therapies in ARDS. 3) BM-MSCs underwent apoptosis in ex vivo lung microenvironment. The inflammatory ARDS microenvironment improves BM-MSC therapeutic benefits by inducing higher expression levels of immunomodulatory biomarkers on BM-MSCs and more release of inflammatory cytokines and VEGF in CM, compared to healthy lung microenvironment. 4) BM-MSCs live a longer lifespan in the inflammatory ARDS lung microenvironment than in the healthy lung microenvironment. BM-MSCs undergo apoptosis and phenotype shifting in both healthy and ARDS lung microenvironment in vivo. BM-MSCs modulate the composition of immune cells and cytokines in inflammatory ARDS lung microenvironments. These findings provide new insights into MSC behaviours in healthy and ARDS microenvironment, implying inflammatory microenvironment as a key mediator for MSC therapeutic effects. Further research is required to identify different subtypes of ARDS microenvironment and to optimise the source, dose, functional characterisation and pre-activation strategies of MSC-based therapies in ARDS treatment
A feasibility study of the Redesigning Daily Occupations (ReDO®-10) programme for adults living with obesity attending specialist clinical services in Ireland
No full textIntroduction: Obesity is a complex, chronic, multifactorial condition requiring holistic treatment approaches. In Ireland, clinical specialist occupational therapists have been integrated into obesity management services, marking a new practice area with emerging evidence-based interventions. The Redesigning Daily Occupations (ReDO®-10) programme is a ten-week, group-based time-use intervention developed in Sweden, designed to explore the relationships between daily activities, time-use and health. This study is the first known evaluation of ReDO®-10 with adults living with obesity.
Methods: This was a sequential mixed-method feasibility study involving two non- randomised groups recruited from the Centre of Obesity Management services in Dublin and Galway. Participants were adults (18+) living with obesity and clinical specialist occupational therapists trained in ReDO® facilitated the programme. Quantitative data were collected at baseline, post-intervention and at 3-month follow-up using EQ-5D-5L, WHODAS 2.0, OVal-pd and MYCaW. Qualitative data were gathered using semi-structured interviews with ReDO® participants, occupational therapists and key interest-holders, along with weekly therapist reflections to monitor programme fidelity.
Results: Ten female participants living with obesity completed the 10-week ReDO® programme, with full retention and an average weekly attendance rate of 80%. Participants reported a positive experience, valuing the group format and the insightful content. Quantitative measures showed trends towards improvements in health outcomes. Occupational therapists reported professional satisfaction and identity, noting the manualised structure supported delivery in busy service settings, while remaining aligned with professional values.
Conclusions: The findings suggest that ReDO®-10 is a feasible, acceptable and well- received programme for adults living with obesity. The promising findings of this novel approach demonstrate preliminary benefits for health, wellbeing and occupational value. However, a future randomised controlled trial is needed to test the programme's efficacy
Targeting proteoglycan synthesis enzymes for spinal cord injury repair: Insights into ex vivo injury models, lentiviral vector-short hairpin RNA efficacy and hydrogel delivery systems
No full textThe obstacles to regeneration after spinal cord injury (SCI) are complex and persistent. Glial scar formation and proteoglycan (PG) dysregulation are key obstacles. Modifying PG levels after SCI can improve regeneration. However, due to extensive and persistent PG dysregulation, an intervention with widespread and long-lasting effects is required. Reducing enzymes involved in PG synthesis can beneficially alter the production of entire PG subgroups to have maximum benefit. This thesis focused on three PG synthesis enzymes; Xylosyltransferase I and II (XT1/XT2) which initiate the production of all PGs, and Chondroitin Sulphate N-Acetylgalactosaminyltransferase-I (Csgal1) that initiates the synthesis of chondroitin sulphate proteoglycans (CSPGs).
This project aimed to evaluate XT1, XT2, and Csgal1 as targets to enhance regeneration after SCI. The first research chapter examined these enzymes in the rat spinal cord and investigated the best age and sex from which organotypic ex vivo SCI models should be derived for glial scar and PG synthesis enzymes investigations. These enzymes were evaluated in mixed glial culture (MGC) models to gain insights into their regulation under different types of inflammation. Additionally, lentiviral vectors carrying short hairpin RNAs (LV-shRNAs) were assessed to knock down these enzymes in cell lines and two hydrogels were evaluated as carriers for LV delivery.
This thesis found that the mRNA levels of XT1, XT2, and Csgal1 increased in the spinal cords of rats as they aged, with adult rats (>6 months) having significantly more of these enzymes' mRNA than rat pups, from which ex vivo cultures are commonly derived. However, adult derived ex vivo cultures were not viable. Ex vivo SCI models derived from postnatal day 11 rats were viable and showed preliminary evidence of prominent glial scar production after transection injury, suggesting they may be favourable for studying PG synthesis enzymes and glial scar modifying therapies.
Only XT1 mRNA expression increased following lipopolysaccharide-induced inflammation of MGCs, but it was not increased following a physical scratch injury to MGC. CSPG levels increased after all inflammatory stimuli, regardless of changes in PG enzyme mRNA. This indicates PG synthesis enzymes can be regulated differently depending on the type of inflammation, highlighting the complexity of CSPG regulation.
LV-shRNA vectors targeting XT1, XT2, and Csgal1, successfully reduced their respective mRNA in at least one cell line. The XT2 targeting LV showed the most promise, consistently lowering XT2 mRNA, significantly reducing XT2 protein compared to control non targeting LV-shRNA and altering sugars related to CSPG and heparan sulphate PGs. Preliminary tests indicate this LV improved neurite outgrowth, but on average neurites were not significantly longer than controls. Further evaluation of all LV-shRNAs is needed to assess if lack in significant improvements in neurite outgrowth is due to inefficiency in LV-shRNAs or XT2 as a target.
This thesis evaluated LV delivery using tyramine-modified hyaluronic acid (HATA) and oligopoly-ethylene glycol fumarate (OPF/OPF+) hydrogels, both of which indicated beneficial properties for LV delivery and SCI treatment. The formulation of HATA gels tested was ineffective, potentially reducing LV stability. OPF and OPF+ hydrogels released 26% and 14% of the total LV loaded into them respectively, mostly within the first 24 hours, which is not an efficient total release or desirable release pattern. The inefficient release from both HATA and OPF based hydrogels were hypothesised to result from unexpected LV-biomaterial interactions. Future research should focus on optimizing both LV and hydrogel formulations to improve interactions and LV release.
This work provides insights into ex vivo SCI models, the expression of PG synthesis enzymes in the rat spinal cord and glial inflammation models. It demonstrates challenges with LV-shRNA-mediated enzyme reduction and hydrogel-based LV delivery. The thesis supports further research into PG synthesis enzymes in SCI pathology, alternative approaches to reduce PG for SCI regeneration, and optimisation of hydrogel systems for LV delivery in SCI treatment