ItemPiRAMiD: predicting early onset autism through maternal immune activation and proteomic discovery(University College Cork, 2023) Carter, Michael; Murray, Deirdre M.; Gibson, Louise; O'Keeffe, Gerard W.; English, Jane; National Children's Research CentreAutism spectrum disorder (ASD) is a heterogeneous developmental disorder arising early in life. ASD is composed of a wide variety of clinical characteristics, neuropsychological impairments and complex phenotypes. The classical triad of ASD symptoms includes disrupted social function, atypical verbal and non-verbal communication skills, and restricted interests with repetitive behaviours. These core symptoms often coexist with other psychiatric and neurological comorbidities. Attention Deficit Hyperactivity Disorder (ADHD), epilepsy, migraine, and anxiety are much commoner in children with ASD. Children and adults with ASD often encounter difficulties with emotional and behavioural problems (EBPs) such as emotional reactivity, aggression, and depression. Up to 50% of those affected can have intellectual disability (ID) and limited verbal communication. Social, emotional and behavioural deficits in children with ASD are also important modifiers of outcome and are linked to elevated stress, mental and physical health problems, and lower overall family and caregiver well-being. We know that early intervention can be effective, and may be parent or therapist delivered. Pharmacological treatment of ASD can be successful insofar as it is useful for symptomatic management of some ASD comorbidities such as ADHD, and depression. Although genetic susceptibilities are increasingly recognised, the mechanism of disease development in ASD remains unknown. We are aware of both common and rare genetic risk factors with more than four hundred diverse high confidence genes now linked to ASD (https://www.sfari.org/resource/sfari-gene/). Singly, these genetic factors each convey only a modest increase in ASD risk (~1%), however collectively they can contribute to a far greater risk. Both de novo and inherited genetic defects are recognised but ASD risk in progeny does not follow a clear pattern of inheritance. Estimates of heritability of ASD in twin pairs vary widely between 50 – 90%. The apparent male preponderance in ASD persists with a clear bias towards males. Rates of ASD among males exceed those of females by three or fourfold hinting at a possible sex differential genetic foundation. Up to 20% of individuals with ASD may possess copy number variants (CNV) and de novo loss of function single nucleotide variants (SNV) that are individually rare but in combination, increase an individual’s overall ASD risk. While newer methods of genetic analysis (such as whole genome sequencing) are uncovering new candidate genes with regularity, the heterogeneity of the clinical and phenotypic groups within ASD strongly suggest that in those with a genetic predisposition, environmental factors may act in concert to bring about a multisystem dysfunction leading to ASD. Despite recent advances in gene analysis, we are yet to discover a single gene determinant that can account for more than a small percent of ASD cases. The current ASD literature suggests that mutations occurring in genes involved in synapse formation, cell adhesion molecule production (Cadherin), scaffolding proteins (SHANK proteins), ion channels (sodium, calcium, and potassium channels), and signaling molecules can disrupt regulatory or coding regions and affect synapse formation, plasticity and synaptic transmission. All this suggests that we cannot explain many cases of ASD by genetic factors alone, or at least we cannot explain them using our current understanding of ASD genetics or our current techniques of genetic analysis. The flawed picture of ASD genetics has led some to investigate the role of environmental exposures in the aetiology of ASD. Researchers have identified many environmental risks in ASD. Advanced parental age, foetal environmental exposures, perinatal and obstetric events, maternal medication use, smoking and alcohol use, psychosocial hardship, nutrition and toxic exposures have all been implicated as risks in the pathogenesis of ASD. While authors attribute between 17 - 41% of ASD risk to non-genetic or environmental exposures, the exact balance between genetic and environmental determinants and their roles in aetiology remains disputed. Multiple mechanisms have been proposed through which each of these exposures may exert an influence on genetic and epigenetic risk in ASD , but there are only a handful that are likely to effect abnormal neurodevelopment. Animal models of inflammation and maternal immune activation are particularly well characterised, and have successfully modelled ASD type behaviours and social difficulties in mice, rats and non-human primates. Maternal immune activation (MIA) is defined as an increase in measured levels of inflammatory markers in mothers during pregnancy. Through this process, a cytokine cascade transmits to the foetus, resulting in adverse neurodevelopmental phenotypes and even remodelling of the immature foetal brain. Many studies have profiled cytokine, chemokine, immune cell and inflammatory signatures in ASD affected individuals. Only a much smaller number have characterised cytokine profiles in expectant mothers who progressed to birth children who develop ASD. The few previous studies, which have examined gestational serum, have indicated mid-gestational upregulation in specific pro-inflammatory cytokines or indeed down-regulation in anti-inflammatory cytokines. Metabolomic analysis refers to the systematic identification and quantitation of all metabolites in a given biological sample. This collection of metabolites, known as the metabolome, is thought to directly reflect the biochemical activity of the studied system at a specific point in time. The metabolome has become an area of interest, as some inborn errors of metabolism (IEM) are clearly linked to ASD phenotypes. Phenylketonuria (PKU) and Smith-Lemli-Opitz syndrome (SLOS) are disorders of amino acid and cholesterol metabolism respectively. Untreated PKU is associated with strongly autistic phenotypes, while SLOS is phenotypically heterogeneous, but autism remains a common feature in these children. Similarly, proteomics is defined as the study of the complete protein profile in a given tissue, cell or biological sample. Proteomic studies of human sera have so far noted altered levels of proteins involved in inflammation or immune system regulation, including acute phase reactants and interleukins. Abnormalities of the complement system have also been found in ASD and other psychopathologies such as schizophrenia. Recent works demonstrate that the complement pathway can affect synaptic remodelling and has roles in neurodevelopmental processes. The initial focus of ASD research on genomics has largely failed to result in the much-hoped-for silver bullet of ASD aetiology, i.e. a common genetic cause. Instead, the genetic landscape has proven to be exceedingly complex and interdependent on a multitude of factors, including environmental exposures and other modifiers of genetic risk. Research examining the aetiology of ASD has shifted focus from genetics to a multimodal approach. In recent years, funding has become available for a far wider variety of ASD aligned research topics, beyond those with a focus on genetics. Opportunities now exist to adopt a multifaceted approach to ASD aetiology, shifting the focus from a narrow genetic base, to a broader multimodal approach to examine other potential mechanistic players. While this adds further complexity to what is already a complicated picture, the strived for parsimonious answer is simply never likely to materialise. Newer fields and modalities such as proteomics, metabolomics and machine learning will help to further refine and untangle the complicated web of ASD, and this variety of granular detail is what is likely to result in a practicable biomarker or effective therapy in the future. In this thesis using a multimodal approach (ELISA, metabolome and proteome analysis) we aim to explore further the role of MIA and alterations of the proteome and metabolome in the pathophysiology of ASD. We hope that our findings may ultimately help to identify a potential gestational biomarker of ASD, which will improve access to early diagnosis and treatment. We also aim to characterise co-morbid emotional and behavioural problems, which arise early in children with ASD and are pervasive throughout all spheres of life. Early recognition and intervention with these co-morbidities can improve treatment outcomes, patient, and family wellbeing. ItemStudies in the synthesis and impurity profiling of pharmacologically active benzodifurans(University College Cork, 2022) O'Connor, Richard Eric; Keating, J J; Higher Education AuthoritySubstituted 2,3-dihydrobenzofuran derivatives as well as their benzofuran and benzodifuranyl analogues are versatile heterocycles that are increasing in prominence as key building blocks in organic chemistry. In particular, benzodifuran-containing structures are gaining significant interest in medicinal chemistry as pharmacophores and in industrial chemistry as scaffolds for organic electroluminescent devices, organic field-effect transistors, solar cell sensitizers and semiconducting polymers. The synthesis of pharmacologically active benzodifurans are complex multi-step processes and each individual step has the potential for considerable side-product/impurity formation. This project focused on synthetic strategies to benzofuran and benzodifuran derivatives in addition to their hydrogenated and substituted analogues. The chosen synthetic routes to target molecules further focused on the propensity of synthetic steps to produce impurities, modifiable variables to modulate impurity formation and the impurity profile of each synthetic route explored. Organic process impurities identified were isolated and fully characterised where possible, in addition to intended products. ItemPrimary care reform: international evidence and primary care doctors’ perspectives within the context of health system reform in Saudi Arabia(University College Cork, 2023) Alyousef, Mohammed; Naughton , Corina; Bradley, Colin; Savage, EileenBackground The Kingdom of Saudi Arabia (KSA) has launched and ambitious reform programme Vision 2030 including health system reform (Government of Saudi Arabia, 2016a). The health system challenges include the increasing burden of chronic diseases, inadequate access to healthcare, and health service fragmentation. The health strategy articulates a new model of care (MOC), targeting financial reform, provider reform, eHealth development, and public private partnerships. A strategic priority for the KSA health system is to strengthen primary healthcare (PHC) and to position primary care (PC) as the first point of access to healthcare. To date there is limited evaluation of the health reforms with a particular evidence gap on reform implementation in PC. The research aims: 1) to examine the international evidence on the implementation of PHC reform for chronic conditions in countries with high or very high human development index (Review 1); 2) to examine the qualitative evidence on PC doctors’ perspectives on PC reform within the context of health system reforms (Review 2); 3) to explore PC doctors’ perspectives on PC reform within the context of the health system reform in the KSA using a qualitative study design. Methods Review 1 was guided by the PRISMA statement including academic articles and grey literature from 1 March 2008 to 1 September 2020. Descriptive analysis and narrative synthesis were applied. Review 2 used a thematic synthesis of qualitative studies guided by Thomas and Harden (2008) three stage framework to identify descriptive and analytical themes. The qualitative study used interpretive description (ID) methodology. Semi-structured interviews were conducted with a purposive sample of 14 PC doctors (general practitioners (GPs) & family physicians (FPs)), recruited from primary health care centers (PHCC) in the Makkah region of KSA. Constant comparative analysis and Thorne (2016) six-steps was used to analyze the data. Results Review 1 included eight articles from four countries. It revealed a shift in chronic disease management from hospital to PC services involving new organizational and funding models (PC networks, commissioning of services, and shared governance structures). The need for leadership and engagement from PC doctors to support PC reform was identified. Review 2 included sixteen qualitative studies from eight countries. Three themes were identified: (1) Health system reform: lacking integrated and co-ordinated services; (2) Funding primary care: competition versus collaboration; and (3) GP/FP engagement in shaping primary care reform. The qualitative study on PC doctors’ perspectives on KSA PC reform identified five major themes: Changes in daily practice; Changes in the infrastructure of PC; Changes in Relationships, Gradation of awareness to understanding of the PC reform; and Barriers and enablers to engaging in PC reform. Participants observed changes in their daily practice with reference to chronic disease prevention and early intervention with a shift from hospital to PC management model. This corresponded to changes in infrastructure especially diagnostic services, access to medication, and information systems. Changes in relationships with patients were perceived as more patient/person-centred wile relationships between PHCCs were described as more collaborative which was attributed to the formation of health clusters as a collective of PHCC and acute hospitals serving a defined population. In contrast, participants’ viewed relationships with the hospital sector as largely hierarchical with no real collaboration. Participants’ understandings and engagement with the PC reforms as stakeholders varied. While there was some understanding of the MOC especially among FP participants, their understanding of financial reform and provider payments was limited. The GP/FP participants believed they had an important contribution to make to the reform agenda as frontline providers. Barriers and enablers to engagement related to trusted sources of information, education and leadership opportunities, and communication with policy decision making. Conclusion This thesis provides important insights from the literature reviews and the qualitative study into the crucial role that PC doctors as frontline health service providers can play in PC reform. PC doctors’ experiences need to be considered by policy makers to ensure successful implementation of PC reform that meets individual and population needs, and that are adequately resourced financially. There is a need for a deliberate process to engage with PC doctors to help them appreciate how observed changes link to policy and to support them as change agents rather than passive actors in the reorientation of the KSA PHC and wider health system. ItemA trilogy of stressors in the neonatal intensive care unit: towards therapy for preterm adversity(University College Cork, 2023) Dias Casacao, Maria Luis; McDonald, Fiona; Carolyn Sifton Foundation; Science Foundation IrelandPremature infants are born with immature breathing network and an innate immune system that responds differently to that of infants born full-term. All infants born less than 28 weeks of gestation develop apnoea of prematurity-related symptoms, which decreases to about 20% of infants born at 34 weeks of gestation and to less than 10% of those infants born beyond 34 weeks of gestation. Premature infants are not only at risk of developing breathing disorders but also at increased risk of infection due to early life vulnerability (the earlier a baby is born, the more likely is to have health problems due to immature organ development). Gram-positive (GP) infections are the most common type of late onset infection in preterm infants. Activation of specific toll-like receptors (TLRs) is reported to modulate cardiorespiratory responses during infection and may play a key role in driving homeostatic instability observed during sepsis. Firstly, we sought to characterise the expression of TLRs in the brainstem, adrenal gland and in the diaphragm respiratory muscle in naïve rats during our developmental period of interest (postnatal day (PND) 3 and PND13). These studies demonstrated mRNA expression of these receptors at PND3. TLR expression fluctuated in early life depending on the subtype and tissue examined with a relative decrease in some of the mRNA expression at PND13; TLR1, 2, 4, 6, 9 and NOD2 in sternohyoid muscle, TLR1, 2, 4 and 6 in diaphragm muscle, TLR2 in adrenal glands and NOD2 in brainstem and spinal cord, but a relative increase in mRNA expression of CX3CR1 in brain and brainstem, TLR2 and 9, IL1R1, in brain and TLR2 and TLR9 in spinal cord. Sex differences were revealed in mRNA expression of TLR9 in brain and NOD2 and IL1R1 in brainstem with upregulation of expression in males. These results relating TLRs and postnatal development suggest a developmental regulation of the immune system. Given these results, we reasoned that oxygen dysregulation coupled with GP bacterial immune stimulation would modulate redox sensitive genes and TLR expression that could alter hormonal expression and impinge on respiratory function in a sex-specific manner. To test this, we developed a novel neonatal rat model in which male and female neonatal rats were exposed to intermittent hypoxia, normoxia and hyperoxia from PND3 for 10 days, followed by combined administration of GP bacterial proteins lipoteichoic acid (LTA) and peptidoglycan (PGN). This model sought to mimic physiological challenges encountered by infants born preterm. Hypoxia challenges during the intermittent hypoxia and hyperoxia (IHH) protocol, induced a significant peripheral oxygen desaturation in treated animals. LTA and PGN (3mg and 5mg, respectively) evoked a significant immune response in PND13 rat pups when measured 3 hours post administration. Serum cytokine analysis revealed LTA&PGN triggered an increase in CCL2, IL-1α, IL-1ß, IL-5, G-CSF, IL-13, CCL3, Gro/KC, CXCL10, CX3CL1, CXCL2, IL-10, IFNy, leptin, VEGF, IL-17A and TNF-α serum concentration compared to vehicle. Interestingly, IL-1ß, Gro/KC, IL-10 and leptin expression were upregulated with combined IHH and LTA&PGN exposure. Respiratory function demonstrated an overall decrease in breathing frequency that was mainly impacted by LTA&PGN administration due to an increase in expiratory time. A decrease in minute ventilation was reported with LTA&PGN however, regarding the metabolic function, the ventilatory equivalent for carbon dioxide was similar across the groups, which is consistent with normal pH levels obtained. Additionally, a mild response to the GP challenge in the late periods of hypoxia were associated with decreased number of gasps with IHH. We analysed mRNA expression of TLRs and redox modulated genes using real-time polymerase chain reaction (RT-PCR) in brainstem, diaphragm muscle and adrenal glands. Brainstem gene expression was similar across groups. In adrenal glands tissue, there was an overall upregulation of TLRs mRNA expression with IHH exposure, except for TLR6. Moreover, TLR2 mRNA expression was upregulated with IHH compared to Sham groups, in males compared to females and in LTA&PGN compared to vehicle. An inverse trend from that of adrenal glands was reported in diaphragm muscle. We also analysed redox modulated proteins in serum using bioassays to detect 8-OHdG, 8-iso-PGF2α, AOPP and SOD in plasma. No differences were observed with IHH and LTA&PGN. However, sex differences were found in 8-iso-PGF2α and AOPP redox proteins, with upregulated expression in males compared to females. Finally, we sought to characterise the postnatal development in our animal model using a battery of developmental and motor assessments. These studies demonstrated a delay in pinnae detachment in IHH pups, a decrease in time to righting with IHH and an increase in motor and locomotion faults in IHH females. Tactile stimulation was decrease with IHH suggesting a delay in brainstem reflexes. IHH treated males presented with an increased expression of stress and anxiety-related behaviours illustrated by increased time spent in the corners of the open-field test with LTA&PGN and distance travelled with IHH, and decreased time spent in open arms in the elevated plus maze experiment in males compared to females. Altogether, these results suggest that early life stress can profoundly impact the expression of TLRs and redox genes in adrenal glands, impact the expression of cytokines such as leptin and alter development, motor coordination, stress levels in this novel neonatal rat model of early life stress. In conclusion, these studies specifically target the gaps in knowledge of the pathophysiological alterations experienced in prematurely born babies who present with impaired breathing function and contract GP infection as mimicked in this novel animal model. The results presented disclose novel insights on the physiology and hormonal alterations that these babies could face in similar conditions, with potential to positively contribute to the field of study by enlightening future targets of research. Future directions relying on the spatial characterisation of TLRs and leptin receptors and possible long-term influences on behavioural performance would also be helpful to better characterise the model. ItemDeprescribing for frail older adults residing in long-term care facilities: a mixed-methods evaluation(University College Cork, 2023) Heinrich, Clara H.; Donovan, Maria; Mccarthy, Suzanne; Mchugh, Sheena; School of Pharmacy, University of SydneyIntroduction Globally, the population is ageing and the demand for long-term care (LTC) is increasing. Older adults residing in LTC have a higher degree of physical frailty compared to their community-dwelling counterparts. The physiological changes associated with ageing and frailty can increase the risk of experiencing an adverse event from a medication. Polypharmacy and potentially inappropriate medications (PIMs) are even more of a concern in frail older adults as frailty increases the risk of adverse outcomes. Deprescribing has been shown to reduce PIMs for older adults residing in LTC however, deprescribing is not universally implemented. The aim of this thesis is to develop and test an evidence- and theory-based implementation strategy to support HCPs engage with deprescribing in routine practice for frail older adults residing in LTC. Methods A mixed-methods approach was utilised for this thesis, informed by the Medical Research Council’s framework for designing and evaluating complex interventions. Firstly, the ‘best-fit’ framework method was used to synthesise the qualitative evidence, using the Theoretical Domains Framework. The resulting ‘best-fit’ framework was used to inform a semi-structured interview study of HCPs working in LTC to identify context-specific deprescribing barriers/enablers. A retrospective chart review was conducted in two long-term care facilities (LTCFs) using Screening Tool of Older Persons Prescriptions in Frail adults with limited life expectancy version 2 (STOPPFrail v2) to identify PIMs. Results supported the design of a deprescribing implementation strategy. The implementation strategy design consisted of 3 phases; a mapping process to identify behaviour change techniques (BCTs) which could form a strategy, a modified online Delphi survey to select feasible BCTs and a roundtable discussion to prioritise barriers/enablers and tailor the proposed strategies for deprescribing in LTC. A feasibility study of the resulting strategy was conducted in two public LTCFs focusing on implementation outcomes whilst collecting intervention data. Results The qualitative evidence synthesis and interview study summarised the deprescribing barriers/enablers in LTC. Barriers included insufficient resources, lack of co-ordination between healthcare settings and negative social influences. Deprescribing enablers included interprofessional support and patient social influence. The retrospective chart review study identified that lack of a documented indication for prescribed medications was the most prevalent PIM criterion, with antihypertensives the largest drug class potentially inappropriately prescribed. The final deprescribing implementation strategy consisted of an education-enhanced 3-monthly multidisciplinary team deprescribing review, led by a nurse, conducted at the LTC site targeting antihypertensives, named the DEFERAL strategy. Of HCPs involved in the feasibility study, the DEFERAL strategy was unanimously considered acceptable and appropriate, with 90% agreeing that it was feasible. Qualitative feedback outlined site-specific modifications for future consideration. Conclusion This thesis has designed and feasibility tested a theory- and stakeholder-informed implementation strategy to support HCPs to engage with deprescribing PIMs prescribed to frail older adults residing in public LTCFs in Ireland. The DEFERAL strategy helped to address a gap reported in the deprescribing literature by incorporating concepts from behavioural and implementation science to target barriers and enablers inherent with the LTC setting and translate deprescribing into routine practice. The strategy was considered acceptable, appropriate and majorly feasible, with modifications reported for consideration in future piloting and effectiveness research.