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RNA sequencing of non-coding RNAs in ischaemic heart disease
Get PDFIschaemic heart disease is a major cause of death worldwide and a leading cause of mortality and morbidity in New Zealand. Older adults and those of Māori and Pacific ancestry are particularly affected. Ischaemic heart disease accounts for over half of all cardiovascular disease mortality and, again, rates are more than twice as high among Māori than non-Māori. Ischaemic heart disease can lead to myocardial infarction (heart attack) which, if not fatal, can then lead to heart failure, a complex, multifactorial disease characterised by neurohormonal signalling and remodelling of the heart. Currently the natriuretic peptides are the international gold standard for diganosing heart failure and are also excellent prognostic markers in patients with heart failure. However, there is still a clinical need for early biomarkers of myocardial ischaemia (to identify people at risk of myocardial infarction) and to identify patients at risk of developing heart failure before detrimental remodelling has occurred.
As sequencing technologies have evolved there has been intense research in the fields of circulating cell free DNA and RNA, especially non-coding RNA. As RNA is actively transcribed, it has the advantage of providing a ‘real time’ insight into the disease status of an individual. Recent discoveries have highlighted the regulatory roles and diseases associated with non-coding RNAs, including long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). LncRNAs have been demonstrated to have mutliple functional roles both within the nucleus and cytoplasm such as chromatin remodelling, histone modification, transcription factor recruitment, formation of subnuclear structures and control of mRNA translation and decay. CircRNA, a relative newcomer, has also been demonstrated to have functional roles such as sequestering miRNAs, binding proteins and even coding for peptides. There is great excitment for the potential utility of circRNAs as biomarkers as, due to their circular structure, they are more resistant to degradation in the circulation than their linear RNA counterparts.
The overall aim of this thesis was to identify non-coding RNAs associated with ischaemic heart disease. To address this aim, a bioinformatics pipeline was developed to identify mRNAs, lncRNAs including putative novel lncRNAs, and circRNAs using short-read RNA Sequencing (RNA-Seq) data. This pipeline was tested and validated with publicly available data and used to screen for candidate mRNA and lncRNA biomarkers associated with ischaemic heart disease in human heart tissue. A whole genome network correlation approach identified several promising candidate biomarkers for myocardial ischaemia including several novel lncRNAs, which were validated with long-read Nanopore sequencing in independent samples. The sub-cellular localisation of three promising lncRNAs candidates (two annotated lncRNAs, one novel lncRNA) was identified using the in-situ hybridisation assay, RNAscope®. Next, an RNA-Seq protocol was developed to detect mRNAs, lncRNAs and circRNAs in human plasma. This protocol was applied to plasma from patients with ischaemic heart disease and healthy controls to screen for candidate mRNA, lncRNA and circRNA biomarkers for progression from ischaemic heart disease to heart failure. Although candidate biomarkers for disease progression could not be detected in these patients several additional lncRNA candidates for the presence of ischaemic heart disease were identified.
In summary, this study has established a bioinformatics pipeline and methodology for identifying and validating putative novel lncRNAs and circRNAs in human tissue and plasma. This work has identified several promising candidate lncRNA biomarkers for ischaemic heart disease, which, if validated, may provide early diagnostic information in high-risk patients.
The pipeline is freely available to download at https://github.com/zoeward-nz/Ph
N-Alkylation of imidazoles
Get PDFThe regioselectivity of N-alkylation in unsymmetrical imidazoles was studied.
The ambident nature of imidazole was scrutinised in an examination of substrate (ie. substituent(s)), alkylating agent, and medium effects on the ratios of isomeric N-alkylated products obtained under standardised alkylation conditions. To this end various 4-mono- and 4-nitro-5-substituted-imidazoles were synthesised and subsequently treated with alkylating reagents in both "neutral" (ethanolic) and basic (aqueous sodium hydroxide) reaction media to produce isomeric products in proportions rigorously determined by 1H nmr analysis.
A number of factors were shown to affect the product ratios. It is generally accepted that the free base is methylated by an SE2' process while the imidazole anion obeys SE2cB kinetics. The ratios of 1-alkyl-4- : 1-alkyl-5-substituted products determined from reaction in basic medium depend largely on polar and steric factors. Electron-withdrawing groups in the 4(5)-position render the more remote nitrogen the least deactivated to electrophilic attack. Correlation of the results obtained is much better with ơ1 or ơm than with ơp substituent constants, indicating that the major effect of the substituent is inductive. Steric effects play a very significant role in the overall regiochemistry of alkylation. An increased preference for the less-hindered nitrogen is demonstrated as both the size of the substituent and the size of the incoming electrophile increases. In "neutral" conditions matters are more complex. Certainly steric effects can be seen, but the expected electronic effects are overcome by a dominant tautomeric effect when the substituent is an electron-withdrawing group. KT values were determined for a number of substrates and these demonstrate that the 4-substituted tautomer is dominant when the substituent is electron-withdrawing, and even though this is the less-reactive tautomer in an SE2' reaction, it still controls the product ratio,
leading to high proportions of 1-alkyl-5-substituted imidazole in the product
mixture.
Diazomethane methylation was also briefly examined. Substitution is predominantly at the nitrogen proximate to an electron-withdrawing group, and reaction yields are seen to be dependent on substrate acidity
Using non-coding RNAs and proteins to predict acute kidney injury in heart failure
Get PDFAcute decompensated heart failure (ADHF) is associated with a high incidence of acute kidney injury (AKI), an abrupt loss of kidney function associated with a near doubling of mortality at one year. In addition to the direct threat ADHF itself poses to kidney function, several drugs commonly used to treat HF can exacerbate kidney dysfunction and the beneficial effects of these treatments must be weighed against their adverse effects on glomerular perfusion. Serum creatinine (SCr), the gold-standard marker for glomerular filtration, is a delayed and insensitive marker of kidney function that can delay the diagnosis of AKI by 24-72 hours. During this time, irreversible kidney damage can occur. Consequently, there is an urgent need to identify early markers for AKI and long-term renal impairment in ADHF to facilitate timely implementation of supportive measures to minimise kidney damage and improve patient outcomes.
This project performed a search for novel markers of AKI in ADHF across two classes of potential biomarkers, circulating microRNAs (miRNAs) in patients with ADHF and kidney proteins in an ovine model of renal impairment in ADHF. First, a large-scale screen of 375 miRNAs was performed in a well-phenotyped discovery cohort consisting of ADHF patients who incurred AKI (n=19), and age- and gender-matched ADHF patients who did not incur AKI (n=20), to identify a panel of candidate miRNA markers for AKI in ADHF. Subsequently, circulating concentrations of the 10 most promising candidate miRNAs were measured in 200 consecutively-recruited ADHF patients (with a spectrum of kidney dysfunction) to determine whether any of the candidate miRNAs had potential to provide independent prognostic information beyond established markers such as SCr and markers of ADHF severity including amino-terminal pro b-type natriuretic peptide (NT-proBNP).
Second, kidney tissue samples from an ovine model of ADHF were used to characterise the changes in renal protein expression that occur in response to the development of, and recovery from, renal impairment as a result of ADHF, and identify candidate protein markers of AKI in ADHF. Relative protein quantification was performed in kidney tissue from healthy control sheep (n=5), sheep with established ADHF (n=8) and sheep recovered from ADHF (n=7) using Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS). By investigating associations between candidate miRNA concentrations and AKI, and characterising alterations in renal protein expression in response to development of renal impairment following ADHF, this work aimed to identify clinically useful biomarkers which could be used to identify ADHF patients at risk of AKI.
Key findings from this research included identification of 26 candidate miRNAs which were differentially expressed between patients with ADHF who incurred AKI, and patients with ADHF who did not. Of these candidates, only miR-454-3p validated in a larger, independent cohort. Higher concentrations of miR-454-3p prior to discharge from hospital were associated with lower risk of AKI or intermediate kidney dysfunction in ADHF patients and may add value beyond established risk factors including age in predicting AKI (Odds Ratio 0.58, 95% CI 0.34-1.01, adjusted for ethnicity, age, gender and NT-proBNP). The association between miR-454-3p and AKI was consistent across all major ethnic groups in the validation study (European, Māori and Pasifika), but the study was only sufficiently powered to detect this effect in patients of European ancestry.
Through protein expression profiling, this work identified 189 proteins that were altered > 1.2-fold during the development of and recovery from ADHF in an ovine model. Of these, 11 proteins were detectable in serum, plasma or urine and had a fold-change > 1.2 with an adjusted p-value < 0.05. These included seven candidate protein markers of kidney injury (Filamin A, T-complex protein 1 subunit gamma, Talin 1, Chaperonin containing TCP1 subunit 8, T-complex 1, Crystallin zeta, Peroxiredoxin), one potential protein marker of kidney recovery (Apolipoprotein A4) and three potential protein markers of long-term renal impairment (Apolipoprotein E, Tripeptidyl peptidase 1, Chaperonin containing TCP1 subunit 6A). Of these candidates, eight had never previously been associated with AKI. Differentially expressed proteins were enriched in two pro-inflammatory signalling pathways: the Glycoprotein VI signalling pathway was activated during ADHF development (Z-score 2.65, p< 0.01), and the acute phase response signalling pathway was repressed during recovery from ADHF (Z-score -2.25, p< 0.01).
In conclusion, these findings suggest that miR-454-3p and candidate protein biomarkers, potentially together and in combination with existing clinical risk factors, have the potential to improve detection of AKI, kidney recovery and long-term renal impairment in ADHF. New biomarkers for AKI in ADHF may facilitate targeted treatment and monitoring of patients with ADHF
High and increasing rates of skin infection hospitalisation for Pacific children in Aotearoa
Get PDFBackground:
Skin and subcutaneous tissue infections are common among children, and are typically bacterial in origin (e.g. impetigo, cellulitis, and skin abscesses which can be caused by Staphylococcus aureus or Streptococcus pyogenes). Hospitalisations are potentially avoidable through good primary care.
Rates of childhood skin infections in Aotearoa are the highest amongst Western countries , and high risk of hospitalisation for Māori and Pacific children and children living in areas of high socioeconomic deprivation. The hospitalisation rates of skin infections in Aotearoa has been gradually increasing, most notably for Pacific children. The study outlines the epidemiology of Pacific children hospitalised with skin infections.
Methods:
This study undertook a retrospective analysis of acute and arranged hospitalisations of children aged 0–14 years with a primary diagnosis of serious skin infection (ICD-10-AM L00–L08, H00.0, H01.0, J34.0, L98.0) from 2000 to 2018.
Results:
The hospitalisation rate for children aged under-15 years with a skin infection increased by 19% from 3.0 per 1,000 in 2000 to 3.5 in 2018. For Pacific children, rates increased significantly (by 36%) from 6.7 per 1,000 in 2000 to 9.2 in 2018. Within the Pacific children hospitalised for skin infections, under-5 year olds have had rates consistently higher than those aged 5–9 and 10–14 years, however, hospitalisation rates have increased significantly for these older age groups. Over 80% of skin infection hospitalisations of Pacific children were for ‘cutaneous abscess, furuncle and carbuncle’ or for cellulitis. Hospitalisation rates for these conditions have increased since 2000, particularly for cellulitis.
Conclusion:
Given the high hospitalisation rate for Pacific children with skin infections, and the increasing rates of hospitalisation for 5–14 year olds, an effective strategy to promote targeted community-level prevention of skin infections and early access to treatment in primary care is recommended for reducing skin infection-associated hospitalisations.Peer Reviewe
New Zealand Deprivation Index 2018 - TA41: Tararua District
No full textFor further information about data sources, interpretation of the graphs, and cautions, please see the separate Introduction Chapter
All data relating to the 2018 census is provided by Stats NZ, https://www.stats.govt.nz/
New Zealand Deprivation Index 2018 - TA65: Mackenzie District
No full textFor further information about data sources, interpretation of the graphs, and cautions, please see the separate Introduction Chapter
All data relating to the 2018 census is provided by Stats NZ, https://www.stats.govt.nz/
New Zealand Deprivation Index 2018 - TA71: Dunedin City
No full textFor further information about data sources, interpretation of the graphs, and cautions, please see the separate Introduction Chapter
All data relating to the 2018 census is provided by Stats NZ, https://www.stats.govt.nz/
New Zealand Deprivation Index 2018 - TA45: Upper Hutt City
No full textFor further information about data sources, interpretation of the graphs, and cautions, please see the separate Introduction Chapter
All data relating to the 2018 census is provided by Stats NZ, https://www.stats.govt.nz/
"Folauga" - Pacific health, well-being and success in higher education
Get PDFBackground
Pacific peoples are migrants who have a unique and close relationship with Aotearoa New Zealand. The people of the realm countries, Cook Islands, Niue and Tokelau, have the right to New Zealand citizenship, and Samoa has a Treaty of Friendship with New Zealand. People from many neighbouring Pacific Island countries travelled to New Zealand since the 1800s related to trades and employment. New Zealand also had a colonial relationship with some Pacific countries, and utilised the Pacific migrant labour workforce to support its agricultural and manufacture industries. Many Pacific peoples chose to remain in New Zealand for employment and education opportunities.
Many have, however, not realised their hopes for better employment and education opportunities. Pacific peoples are disproportionately represented in poor health and education outcomes in New Zealand. The New Zealand Government and tertiary institutions have attempted to address this disparity over the years. Education is one of the determinants of health. Increasing the Pacific health workforce and improving education outcomes is likely to contribute to better employment opportunities and health outcomes for Pacific peoples in New Zealand. There is very little research on Pacific students studying towards a career in health in tertiary institutions. Understanding how best to support students in this environment will assist in enabling them to achieve their goals and aspirations.
The specific objectives of this research are:
1. What are the enablers and barriers for Health Sciences Pacific students' transition in the first two years at university?
2. To what extent can the enablers and barriers for Pacific students in this study be understood in the context of Vincent Tinto’s theoretical model(s) with regards to first-year integration and retention?
3. What conceptual holistic theoretical model could be proposed that would be relevant for the experiences of Pacific students' success in higher education?
Methods
The Kakala Pacific research methodology and Talanoa method were utilised as the overarching Pacific research frameworks. Twenty students purposively sampled from all students who identified with at least one Pacific ethnicity enrolled at the University of Otago Health Sciences First Year Programme in 2015, were eligible for inclusion as participants. Eligible participants were stratified by ethnicity and gender and randomly selected to be invited for the research. Of the twenty participants, fifteen were recruited during the University Early Orientation Programme (EOP), and five were recruited from those who did not attend the EOP. Where a potential participant declined to be involved, the next person on the randomised list was approached. Participants signed a consent form which included the consent to participate and access to their academic performance and progress. Each participant was interviewed three times in their first year and once in their second year at university (80 planned interviews in total). All interviews were conducted at a time and place that suited the participants. Seventy-six of the total 80 interviews were completed, with an average time of 50 minutes for each interview. Two students left the University after their first year and were interviewed by telephone. Thematic analysis of the data was conducted utilising both inductive and deductive approaches, with information from the four interview periods, and for each participant across their four interviews. Ethical approval was obtained from the University of Otago Ethics Committee.
Results
The enablers and barriers were explored specifically for the areas outlined in Tinto’s Model. These were family background, individual attributes (skills/abilities), pre-college schooling (prior schooling), goal and institutional commitments, institutions academic and social systems integration, interactions with Faculty (staff) and the interactions between areas that can impact on students’ decision to drop out or persist at the University. The results showed that all components of Tinto’s Model were important for Pacific students, as they transitioned into the tertiary environment. There were, however, additional components identified inductively from the data that were very important for Pacific students, but not included in Tinto’s Model. These were culture, identity, and health and well-being. The components that were important for the participants’ health and well-being were: spiritual well-being, psychological well-being, physical well-being and social well-being.
Discussion
Tinto’s models were relevant for Pacific students in all their aspects, but could not explain in full, what was important for the integration and retention of Pacific students in higher education. These additional areas were related to their culture and identity as well as their health and well-being. Pacific Health is viewed in a holistic way and there were four components of their well-being that were identified from the data as important. These were their spiritual well-being, psychological well-being, physical well-being, and their social well-being. These aspects are aligned with Pacific models of health such as the Fonofale Model as well as the Hauora Māori Model of Health Te Whare Tapa Whā. These additional components will be important for institutions in New Zealand to consider, for effective support for Pacific students’ transitioning into the tertiary environment. To encompass all of these, this research proposes a revised Tinto model which includes these key components for the success of Pacific students in higher education. The new model is called Folauga. Folau or Folauga means The Journey in the Samoan language. Pacific peoples are navigators travelling long distances to populate the many islands in the Pacific using double-hull canoes (vaa or waka). The Folauga model is presented as an outrigger/canoe. The body of the vaa consists of the Tinto model. The additional components of the vaa consists of culture, identity and the four aspects of health and well-being. These additional components provide stability for the waka’s journey of success, for Pacific peoples in higher education.
Conclusion/Recommendations
This research provides recommendations that education institutions and the government ought to consider, to ensure effective support for the transition into, and the success of Pacific students in higher education:
1. Holistic Approach – a holistic approach is required for the effective support of Pacific students’ transition and success in higher education.
2. Cultural Capital – embracing the cultural capital Pacific students bring to the tertiary environment is critical to their success.
3. Identity – create an environment that is supportive of students’ diverse backgrounds and identities.
• Health and Well-being – provide support for Pacific students’ spiritual, mental, physical and social well-being within the tertiary environment, ensuring both preventative interventions, and easy access to well-being and relevant pastoral care support.
4. Capacity Building – strategic investment in a national Pacific-coordinated pipeline encouraging Science, Technology, Engineering and Maths (STEM) subjects in schools (e.g. Health Sciences academies) to support students’ preparedness for tertiary studies.
5. Targeted support:
• Academic support – increase investment in targeted academic support for Pacific students in tertiary studies, as many enter higher education lacking the required academic preparation from high schools.
• Mentoring support – increase investment in mentoring programmes to support the transition of Pacific students into the tertiary environment, and where possible utilise senior Pacific students as mentors.
6. Racism – institutional leaders to take an active approach to address issues of institutional racism, and to show leadership through modelling positive behaviour.
7. Other – these findings and recommendations are relevant for all students. Much can be learned from this research project that can benefit students from all ethnicities
Biological characterisation of morphological alterations of the cornea following collagen cross-linking (CXL) treatment
Get PDFIntroduction: Keratoconus (KC) is a disease included in a broader range of conditions called corneal ectasia which leads to spontaneous, non-inflammatory, self-limiting ectasia of the cornea that results in localized corneal thinning with protrusion of the thinned cornea. In recent years, corneal crosslinking (CXL) treatment with ultraviolet light (UVA) and riboflavin has been introduced as a minimally invasive treatment option to arrest the progression of keratectasia and it has revolutionized the treatment of KC. Several animal models, including rabbits, have been used to study the morphological alterations of the cornea following CXL treatment. Few studies have investigated the corneal morphological alterations following CXL treatment in porcine and rabbit corneas due to their structural similarity to the human cornea. Currently, no studies that have compared the normal corneal collagen fibrillar (CF) diameter, interfibrillar distance and interlamellar distance between human and these animal models. In addition, none have compared the morphological alterations of collagen fibrils (CFs) in both porcine and rabbit corneas with human, to identify an animal model that reacts in a similar manner to the human cornea, following CXL treatment.
Aims: In the present study, we have compared the normal morphology of CF’s in porcine and rabbits to find an animal model comparable to human. These models (rabbit and porcine) were also investigated to find the optimal animal model that mimics reactions similar to human, to CXL treatment. Finally, we have investigated the effects of crosslinked corneas maintained in vitro for two weeks. We have also explored the possibility of extending the time a crosslinked cornea that can be kept in vitro beyond 2 weeks.
Study 1:
Materials and methods: The normal morphology of porcine (n=5), rabbit (n=5) and human (n=5) corneas were analysed using light and Transmission Electron microscopy (TEM).
Results & conclusion: Histologically, all five layers (epithelium, Bowman's layer, stroma, Descemet membrane and endothelium) of the cornea were visible in all the three species. The CF diameter, interfibrillar distance and interlamellar distance were compared between porcine, rabbit and human corneas for any statistically significant difference. It was evident that the CF diameter and interfibrillar distance of porcine and rabbit corneas were significantly different (p<0.001) from the human corneal values, but not from each other. While neither of animal models were structurally identical to the human cornea, they are both relatively similar to being used as models to study the biomechanical effects of external insults/treatments to be extrapolated to the human cornea.
Study 2:
Materials & methods: Porcine (n=10), rabbit (n=10) and human (n=10) corneas were treated with CXL treatment. The central corneal thickness (CCT) was measured before and after CXL treatment. Each treated and control corneas were bisected, and one half was used for histological examination; the other half imaged by Transmission Electron Microscope (TEM) to investigate the morphological changes of corneal CF’s. These parameters were compared to identify the optimal animal model that mimics reactions of human following CXL treatment.
Results & conclusion: The CXL treatment leads to an increase in CF diameter, a decrease in the interfibrillar distance, and a decrease in interlamellar distance. The effect of the CXL treatment was greatest in the anterior region of the corneal stroma, followed by the intermediate region, followed by the posterior region. All the species showed statistically significant keratocyte apoptosis and a significant reduction in the CCT following CXL treatment. When the magnitude of the change post-CXL treatment is taken into consideration, it was evident that porcine corneas were more suitable as an animal model to study reactions of CXL treatment.
Study 3:
Materials & methods: The effects/changes to the morphology of the CF’s in crosslinked corneas in vitro for two weeks were investigated, by maintaining 15 human corneas in a culture medium.
Results & conclusion: The results revealed that the effect of crosslinking (increase in collagen fibre diameter, decrease in interfibrillar and interlamellar distance) does not significantly change during the 2 week post-treatment. In the next part of the in vitro study, porcine (n=12) and human (n=3) corneas were investigated for the possibility of extending the viability of the crosslinked cornea in vitro beyond 2 weeks. However, we were unable to maintain the crosslinked corneas for more than two weeks, and we postulate that epithelial damage due to de-epithelialization before crosslinking treatment has an adverse effect on maintaining a healthy endothelium in vitro, hindering the de-epithelialized cornea from being maintained in a culture medium for a longer period of time