53 results for Undergraduate, 2016

  • 24 – hour oxygen saturation recordings at discharge in preterm infants

    Wellington, Grace Charlotte (2016)

    Undergraduate thesis
    University of Otago

    Preterm infants have an immature respiratory system and therefore experience an increased number of respiratory pauses and oxygen desaturations. There is now advanced pulse oximetry technology that can record oxygen saturations every two seconds for extended periods of time. There has been insufficient literature that reports the incidence of intermittent hypoxia at time of discharge home from the neonatal unit in preterm infants using new generation oximeters. These respiratory events preterm infants experience have been shown to have some effect on neurodevelopment, but their effect on growth has not been investigated before. The primary aim of this study was to determine the prevalence of intermittent hypoxia in preterm infants at time of discharge home from the neonatal unit. The study also addresses the issue of artefact within oximetry recordings and compares results from automatically edited, manually edited and unedited oximetry data, as well as determining whether overnight 12-hour recordings are of equal value to full 24-hour recordings. The secondary aim of this study was to determine whether intermittent hypoxia at discharge has any effect on post discharge growth and to determine changes in amount of intermittent hypoxia from discharge oximetry to oximetry one-month post discharge. We recruited preterm infants from the Wellington neonatal intensive care unit. A 24-hour pulse oximetry recording was performed immediately prior to the infant’s discharge home. These oximetry recordings were analysed and median values for measures reported from oximetry recordings were determined. Rules to manually edit oximetry data were created and applied to oximetry recordings. These manually edited reports were then compared with automatically edited and unedited reports. Each recording was also edited to resemble a 12-hour overnight recording and this was compared to the full 24-hour recording. Infants born less than 32 weeks gestational age were further followed up with weekly growth measurements for one-month. A repeat 24-hour oximetry recording was performed at one-month post discharge for these infants and compared to their discharge recording. We report high rates of intermittent hypoxia in preterm infants at time of discharge home from the neonatal unit. For example the median 4% oxygen desaturation index (DSI 4%). was 57.9 events per hour. The incidence of these events decreased with advancing post-menstrual age. Rates of intermittent hypoxia one month post discharge were greatly decreased from discharge with improvements of 42% - 57% seen, with DSI 4% reducing to 25.5 events per hour. This study did not show a significant association between intermittent hypoxia and post-discharge growth, possibly because of the small sample size in this study subgroup. There were few clinically relevant differences on reports edited manually compared with automatically edited reports, with some difference when compared to unedited reports. We recommend automatically editing oximetry reports as this gives similar results to manual editing for the majority of measures, however the nadir of the fall in oxygen saturation is often artefact, and even after automatic editing is the one measure that may remain false. The 24-hour oximetry reports were clinically similar to 12-hour recordings and therefore we suggest 12-hour oximetry studies are sufficient.

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  • What’s the harm in waiting? Patient harms in the referral waiting gap

    Patel, Vimal (2016)

    Undergraduate thesis
    University of Otago

    Background Patient safety research seeks to improve the delivery of care, and ensure that patients’ risk of injury from healthcare itself is minimised. Referral between primary healthcare, specialist diagnostic agencies (such as community medical laboratories and radiological centres), and hospital based healthcare is common and important in primary care, yet patients have highly variable waiting times before receiving their care. However, there is almost no research exploring what happens to patients while they wait. Aims This study aims to investigate patient’s waiting periods between referral from their General Practitioner (GP) and receiving specialist healthcare. Specifically, this study aims to determine if patients come to any harm in this waiting gap, and if so, which patients are harmed and what types of harm happen. Methods I reviewed 5 years (2003-2007) of healthcare records of 201 general practice patient’s notes. Each consultation record was examined to identify the types of referral that were made and to find evidence of harms while the patient was waiting for referred healthcare. A subset of 101 of these patients also had the records reviewed for investigation types and evidence of harm while waiting for investigation. A broad definition of harm was used to capture a greater number of harms. Harms were categorised as related to referral for investigation, referral to medical specialty or referral to other non-medical specialty. Harms were also graded in severity (mild, moderate and severe) and were described under the following: ‘continued symptoms’, ‘delay in subsequent management’, ‘deterioration of condition’, ‘financial cost to patient’, ‘anxiety/mental harm’ or ‘other’. Comparisons were made between patients whose referrals had evidence of harm in the waiting gap with patients who did not. Comparisons included length of waiting gap, age, gender and specialty referred to and used t-tests or non-parametric tests, as appropriate. Results 5003 Consultation records were reviewed. A referral rate of 0.21 per person per year for medical and non-medical specialties was found, and a referral rate of 1.00 per person per year for investigations was found. 45 of 183 (25.5%) of referrals to medical or non-medical specialties had evidence of harm in the waiting gap, whereas 9 of 105 (1.8%) of referrals for investigation had harm in the waiting gap. Of the 58 total harms, 43 (74.1%) of harms were minor, 12 (20.5%) were moderate and 3 (5.2%) were severe. The largest broad classification of harm was “continued symptoms” with 38 harms (65.5%), followed by “delay in subsequent management” with 14 harms (24.1%) and “deterioration in condition” with 14 harms (24.1%). There were no statistically significant relationships between the age of patient nor sex of patient nor length of waiting time and the incidence of harm in the waiting gap. Conclusion This is the first study of harm in the referral waiting gap. The findings indicate that harm does happen while patients wait for referred care, and more research is needed to explore these harms. While the relatively small number of patients in this study limits the ability to draw robust implications for changed clinical practice, it is a strong starting point for larger, future research.

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  • Umbilical Cord Serum Chemokines and the Development of Atopic Dermatitis

    Townsley, Hermaleigh (2016)

    Undergraduate thesis
    University of Otago

    Background: Atopic dermatitis (AD) is a chronic skin condition characterised by the development of pruritic and inflamed lesions. One key component of AD pathogenesis is a cutaneous hyper-reactivity to allergens influenced by a Th2-polarised immune response. Macrophage-derived chemokine (MDC) and Thymus and activation-regulated chemokine (TARC) are two chemokines involved in this pathological immune response. Research has shown a strong association between MDC and TARC levels in blood and AD severity. Recently, some cohort studies have suggested that levels of MDC and TARC in umbilical cord blood (UCB) may be predictive of whether infants will develop AD during childhood. This cohort study aimed to further investigate the potential predictive value of UCB MDC and TARC for AD development in childhood. Methods: This project involved a retrospective analysis of data obtained from the NZA2CS population birth cohort study. Information about AD-related outcomes was gathered using questionnaires at various time points, along with physical examination of flexural dermatitis and measurement of total and specific IgE levels at age six. UCB MDC levels were measured using enzyme-linked immunosorbent assay (ELISA) techniques for a total of 647 participants. Haemolysis of UCB samples was found to affect TARC measurement; therefore fewer (n = 270) samples were analysed for TARC. Haemolysis of UCB samples did not affect measurement of MDC concentration. Logistic regression was used to calculate odds ratios to determine the association between UCB chemokine levels and development of AD- related outcomes in childhood. Results: UCB MDC and TARC levels were not predictive of development of AD at age six. Neither were they consistently significantly associated with the development of AD-related outcomes such as an itchy rash or atopy. Some statistically significant associations were found, although their value is difficult to interpret as these were isolated findings. UCB MDC levels were significantly associated with the development of an itchy rash at four years of age (p = 0.027) and with the level of specific IgE to cat allergen at age six (p = 0.05). When the data was segregated by sex, UCB MDC levels in males were consistently significantly associated with development of an itchy rash during childhood (p < 0.05). Conclusion: UCB MDC and TARC concentrations are unlikely to be clinically useful biomarkers for the development of AD in childhood. This was the largest cohort study so far to investigate cord MDC and TARC levels as predictors of future AD onset, and the findings are concordant one other large cohort study. Therefore, these results do not warrant further research into UCB MDC and TARC as predictive biomarkers of AD development.

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  • An analysis of vessel loading of export logs at four New Zealand ports.

    Duval, Alfred W. (2016)

    Undergraduate thesis
    University of Canterbury Library

    Over half of New Zealand’s annual harvest was exported as logs in 2015 (MPI, 2016). The large scale and economic importance of log exports highlights the importance of efficient port operations. Productive cycle elements for the log loading operation were defined. The vessel loading cycle was split into six elements: three ‘action elements’ (loading, tallying, and slinging), and three ‘carting elements’ between the ‘action elements’. Time study measurements were carried out at four New Zealand ports (Tauranga, Marsden Point, Gisborne, and Port Chalmers) to identify differences in productive time to load log export vessels. Port Chalmers wasn’t compared to the other ports as it was too different operationally. Loading had the longest productive element time, followed by slinging and tallying, and lastly the ‘carting elements’. Loading was uninfluenced by port but affected by log grade, length, operator skill, and the time of day. Tallying was significantly different between the three ports with Marsden Point fastest and Tauranga slowest. Slinging was quickest in Gisborne and faster whilst loading below-deck and during the daytime. Carting elements were heavily influenced by distance to or from log stack for all four ports. Tauranga displayed the fastest historic gross load rate (JASm³/hour) yet the slowest productive cycle time. Gross load rate is influenced by delays, volume per cycle, and productive cycle time. The difference in productive time and gross load rate could therefore be assumed to be from increased volume per cycle and/or reduced delays in Tauranga. Exporters are fined for loading slower than scheduled. This cost is greater when shipping rates are high as fines are based on shipping rates. A 5% increase in loading efficiency can save the exporter US$11,000 per vessel at historic maximum shipping rates.

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  • Native forest monitoring : tracking changes in native forest remnants.

    Arnold, T. A. H. (2016)

    Undergraduate thesis
    University of Canterbury Library

    Native forest monitoring is undertaken by forest companies as a requirement for certification of their forests by groups such as the FSC. It is important for companies to be able to track changes that are occurring to native forest remnants that are often spread throughout their operational plantation forest estate. Pan Pac tasked me with completing their 2016 native forest monitoring programme and review the results that have been collected since the programme was implemented in 2002. The objective of this was to both gain a better understanding of how the composition of the remnants in their5 estate is changing and to make recommendations on how the programme could be improved in the future. The majority of the 11 Permanent Sample Plots (PSPs) measured were in good or stable condition, several of which showed strong regeneration of the understory over the past 14 years. Three of the sites have been affected by heavy ungulate browsing (deer and/or goats), which has resulted in the continued suppression of the understory vegetation. While all current canopy layers of the PSP have not changed significantly, current and future disturbance such as ungulate browse could result in a change in composition from the current forest makeup. Ungulate browsing has been identified as the biggest driver of change in the native forest remnants within Pan Pac’s estate. To further examine to magnitude of this, exclosure plots could be established in impacted remnants to assess the effect of removal of browsing pests as a basis for Pan Pac to make decisions about future ungulate control. Continued monitoring of native forests is key to be able to identify as well as understand what is happening with native forest remnants. Tracking composition change is important as it allows the forest manager to target management practices such as ungulate control to combat non-natural changes that are occurring.

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  • Comparing performance of seedlot types in the Kaingaroa Forest using ground pilots and aerial LIDAR : comparing the performance of open-pollinated, control pollinated and clonal seedlots in a plantation trial in the Kaingaroa forest utilising airborne LIDAR.

    Henderson, Theo J. A. (2016)

    Undergraduate thesis
    University of Canterbury Library

    Problem: As more improved planting stock such as clones and genetically improved seedlings are introduced to the market it is important to properly understand the benefits of each production type. Various breeding programmes make claims around performance of their seedlots but there is a shortage of literature around the performance of these production types in a plantation setting for most production species. Approach: One seedling, two cuttings, and 7 clonal varieties were compared in a plantation setting on a single site. The stand was measured via five permanent sample plots (PSPs) per seedlot. The seedlots were categorised by material production type and compared using pair-wise analysis to find statistically significant differences. The seedlots were then compared individually to find any intramaterial differences. Available aerial LIDAR was then used to estimate tree height for the total seedlot area and establish whether this was an accurate estimate. Average LIDAR height was then used to estimate tree height for each of the five PSPs to establish whether this would improve the prediction of heights and permit its use for large-scale evaluation of genetic material. Results: Categorising seedlots by material type there was no statistical difference for height performance but there was for DBH and basal area. Clones and open-pollinated seedlots showed superior performance over controlled-pollinated material, but not different from each other. Clones showed reduced height variability over non-clones. DBH and basal area variability was also reduced but the difference was only statistically significant versus open-pollinated seedlots. Comparing seedlots individually there was large variation in performance and variability within material types, with clones showing some superiority and non-clones inconsistent improvements. The LIDAR tree height model for whole seedlot area showed to be a significant predictor average PSP height but poorly predicted CV. Predicting PSP area provided with LIDAR improved correlations over whole stand predictions for both values. Implications: The performance superiority for clones over other production types in this trial is not as pronounced as previously suspected. Clones do, however, provide a more uniform crop. The LIDAR tree height model could be used for further analysis but not for height variability without further improvement. Result validity was, however, reduced by the lack of trial replication and randomisation. This is the key limitation and makes guaranteeing improvements are due to improved genetics (not environment) problematic.

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  • Factors which influence corewood stiffness in radiata pine.

    Jones, Grace (2016)

    Undergraduate thesis
    University of Canterbury Library

    Increasing stocking and competition with weeds significantly increased Hitman estimates of stiffness at the significance level α=0.05. Accuracy of models predicting Hitman from TreeTap measurements can be improved by customizing them for particular silvicultural regimes and diameter at 1.4m (DBH). Controlled factors: genetics, wind sway and fertilizer use, did not significantly influence Hitman estimates of stiffness. Tree height did not significantly influence stiffness estimates, but including DBH in prediction models improved models of stiffness estimates. Stiffness in 10 year old Pinus radiata stems was studied in an experiment with the following factors: genetics, herbicide/fertilizer use, stocking and wind sway. Acoustic velocity was used as an estimate of modulus of elasticity (MOE) and was estimated using 2 different tools: Hitman, a resonance based tool used on 2m log sections, and TreeTap, a time-of-flight based tool used on 1.2m outer-wood sections of standing trees. DBH and tree height were also recorded for each tree. Green density was measured using submersion in order to use the formula: MOE = green density∗ acoustic velocity² Stiffness estimates from TreeTap were strongly correlated with Hitman estimates, but were about 30% higher on average. The relationship between stiffness estimates from these tools changed with weed competition and with stocking. No significant difference in stiffness was found between the northwest and the southeast sides of the stems when using the TreeTap tool, and an average value for each tree was used for subsequent analyses. These findings are similar to those from other studies carried out on different sites, and to a previous destructive sample at the same site. There were a few major outliers, but despite these the final model relating TreeTap and Hitman estimates was significant (P<0.0001). Weed competition and stocking significantly affected the intercept (P=5.71e-05 and P=1.08e 05 respectively) of a model predicting Hitman values from TreeTap estimates of stiffness.

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  • Nanopore Sequencing of RNA from Breast Cancer Genes

    de Jong, Lucy Clair (2016)

    Undergraduate thesis
    University of Otago

    Abnormal mRNA splicing can disrupt gene function and influence the course of disease. Analysis of abnormal splicing is an important part of determining whether a particular genetic variant found in the population is pathogenic or not. However, to correctly identify abnormal splicing, we must first understand what is normal. This project assessed the isoforms of the genes BRCA1 and BARD1, which are particularly relevant to the onset of breast cancer. BRCA1 is a tumour suppressor gene implicated in breast cancer onset. BARD1 codes for a protein that interacts with BRCA1 and produces a smaller mRNA transcript. Normal exon skipping events have been identified for both BRCA1 and BARD1, however, current methods are unable to reliably identify full transcripts. This has resulted in knowledge of individual exon skipping events but often does not tell us whether multiple events occur in the same transcript. The MinION nanopore sequencer (Oxford Nanopore Technologies), uses a nanopore to produce long-read, single molecule sequences. This has great potential for identifying multiple long isoforms, which is not practical using current technologies. The aim of this project was to examine the ability of the MinION to identify mRNA splicing patterns of transcripts derived from BRCA1 and BARD1. All mRNA from a normal lymphoblastoid cell line was converted to cDNA and targeted genes of interest were amplified by polymerase chain reaction (PCR). All potential isoforms generated from BRCA1 and BARD1 were then pooled and analysed using the MinION sequencer. After trialling many different analysis methods, the read data was analysed using the BLAST-like Alignment Tool (BLAT) with two outputs, a tabular and a graphical format. The tabular format grouped reads into potential isoforms, while the graphical format allowed visualisation of these isoforms and identified the exon/intron boundaries. Using both these formats 34 BRCA1 isoforms and 39 BARD1 isoforms were identified, 24 and 17 of which were potential novel isoforms respectively. Two of these novel isoforms from the BRCA1 dataset (Δ10-17 and Δ11q21) were further verified using Sanger sequencing. This was a proof of principle research project that demonstrated the potential use of the MinION nanopore sequencer for successful characterisation of multiple mRNA isoforms. This research has successfully identified a number of novel isoforms from the BRCA1 and BARD1 genes using the MinION sequencing device.

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  • Development of Novel Triadin-Based Biosensor for the Quantification of H2O2 in the Cardiac Dyad Microdomain

    McLachlan, Julia Jocelyn (2016)

    Undergraduate thesis
    University of Otago

    Release of Ca2+ from the sarcoplasmic reticulum (SR), mediated by the activation of the ryanodine receptor (RyR2), initiates myocardial contraction. This process of Ca2+-induced Ca2+ release is confined within a 20 nm region termed the dyad, located between the transverse tubule and the SR. Architectural properties of the dyad restrict diffusion of Ca2+ and other intracellular messengers to create a unique, subcellular microdomain. Previous research has shown that endogenous producers of reactive oxygen species (ROS) are abundant within the dyad, and have attributed alterations in Ca2+ release with the oxidation of RyR2. However, the spatiotemporal dynamics of ROS within this microdomain remain unquantified due to an absence of tools that enable subcompartmental ROS resolution. Therefore, this research aimed to utilise the recent development of genetically encoded redox biosensors, capable of ROS detection, to create a biosensor tethered to the N-terminus of the dyadic protein, triadin. A Human Embryonic Kidney (HEK) 293 cell model was used to characterise two ROS (H2O2) sensors, HyPer3 and roGFP2-Orp1, in vitro to determine the biosensor more suitable for the triadin-based construct. Using single-cell imaging, HEK 293 cells transiently transfected with HyPer3 or roGFP2-Orp1 were superfused with a range of oxidising and reducing agents. Based on these data, roGFP2-Orp1 exhibits a greater dynamic range, increased sensitivity, and faster oxidation-reduction kinetics than HyPer3, and thus was selected for the triadin-based construct. In parallel, single-cell Ca2+ imaging was used to confirm that co-expression of triadin has a neutral effect on RyR2 function. HEK 293 cells expressing RyR2 WT or RyR2 WT + triadin were loaded with the cytosolic Ca2+ indicator, Fluo-4 AM and superfused with 0.1–1 mM Ca2+ to activate RyR2 WT. These data found a non-significant (p = 0.9) difference in Ca2+ release in RyR2 WT (61.9 ± 3.1%) and RyR2 WT + triadin (60.3 ± 3.5%) cells exposed to 1 mM Ca2+. To create roGFP2-Orp1-triadin, overlap extension PCR was used to generate an SfaAI restriction site that enabled roGFP2-Orp1 to be subcloned into the triadin plasmid. However, the development of roGFP2-Orp1-triadin was unsuccessful. This research highlights a novel method for H2O2 quantification within the dyadic microdomain. Characterisation of HyPer3 and roGFP2-Orp1 revealed that roGFP2-Orp1 exhibits the sensitivity and reversibility required for an in vivo biosensor. In addition, co-expression of triadin has a neutral effect on the properties of RyR2-mediated Ca2+ release, which indicates that triadin is an appropriate tether for the dyadic localisation of roGFP2-Orp1. Future directions include the generation of an animal model to further understand the spatiotemporal dynamics of ROS in relation to alterations in Ca2+ release in vivo.

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  • Anatomical Characterisation of Prolactin Receptor Expression in the Developing Mouse Brain

    Boyes, Kendra Michelle (2016)

    Undergraduate thesis
    University of Otago

    Prolactin signalling through the prolactin receptor plays a significant role in many physiological processes. The diverse biological functions attributed to prolactin are largely dependent on the distribution of its receptor. In particular, the distribution of the prolactin receptor has been well characterised in the adult brain of rodents, but has yet to be fully documented in the developing brain of neonates. To determine the role of the prolactin receptor in the developing brain of neonates, we have developed a transgenic mouse in which cre-recombinase is expressed under the control of an internal ribosomal entry site (IRES) in the long form of the prolactin receptor gene (PrlrL-IRES-Cre). The PrlrL-IRES-Cre mouse has been crossed with a cre-dependent tau-GFP reporter line. In this study tau-GFP is expressed wherever the prolactin receptor gene has been transcribed. The tau-GFP associates with microtubules in neuronal processes, which is important, because in addition to identifying neuronal cell bodies expressing the prolactin receptor, we were able to study the projections of prolactin-responsive neurons for the first time. Post natal day (PND) 1 (n = 8), PND 14 (n = 8) and PND 28 (n = 6) male and female mouse brains were perfused using 4% paraformaldahyde, cut in 20 micron serial sections and thaw mounted onto slides. To visualise PrlrL expressing cells, immunohistochemistry was performed. Sections were incubated with rabbit polyclonal anti-GFP (green fluorescent protein) (A-6455, Life Technologies), for 48 hours at 4℃, followed by an incubation with goat anti-rabbit biotinylated secondary antibody (BA-1000, Vector) for 3 hours. Labelled cells were visualised using nickle enhanced 3,3'-diaminobenzidine (DAB), and identified using an Olympus AX70 light microscope. Staining showed minimal expression in any part of the brain of PND 1 mice, whereas staining could be identified in the choroid plexus, anterior ventral periventricular nucleus and arcuate nucleus of PND 14 and 28 mice. Interestingly, minimal staining was observed in the amygdala and no staining was observed in the paraventricular nucleus in any of the ages examined. These are areas that exhibit high levels of PrlrL expression in adult mice. The increase in PrlrL expression may indicate an age dependent upregulation of the prolactin receptor through the availability of the PrlrL gene, likely driven by hormonal changes associated with puberty. The age-associated increase in PrlrL expression in the anteroventral periventricular nucleus and arcuate nucleus in prepubertal animals suggests a developmental role for prolactin in these regions.

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  • Effects of maternal immune activation on brain arginine metabolism and microglia during neonatal neurodevelopment

    Zhang, Jiaxian (2016)

    Undergraduate thesis
    University of Otago

    Schizophrenia is a debilitating chronic mental disorder with prominent frontal and hippocampal dysfunction. While the exact cause of schizophrenia is poorly understood, a number of factors, including neurodevelopmental disruption, have been linked to the aetiology of the disease. Maternal immune activation (MIA) is a neurodevelopmental model of schizophrenia based on epidemiological evidence that prenatal exposure to infections increases the risk of schizophrenia in adulthood. This model often uses a single systemic administration of the synthetic cytokine inducer polyinosinic:polycytidilic acid (polyI:C) during mid-gestation (e.g., gestation day 15) to induce MIA in pregnant rats. A number of anatomical, neurochemical and behavioral features of schizophrenia are evident in the adult MIA rat offspring. Accumulating evidence implicates altered metabolism of L-arginine, an amino acid with a number of bioactive metabolites, and microglial dysfunction in the pathogenesis of schizophrenia. Recent research has shown altered brain arginine metabolic profile and microglial dysfunction in the adult, as well as adolescent, MIA rat offspring. However, does a single MIA insult during the early stage of neurodevelopment affect brain arginine metabolism and microglia has not been addressed previously. We hypothesized that MIA during neonatal neurodevelopment might alter brain arginine metabolism and affect microglial maturation. To test these hypotheses, we designed the following three experiments to investigate how polyI:C-induced MIA affected brain arginine metabolism and microglia in rats at the age of postnatal day (PD) 2. Twenty-four pregnant Sprague-Dawley rats were given a single tail vein injection of polyI:C (4.0 mg/kg; MIA) or saline (control) on gestation day 15 (n = 12/group). Experiment 1 quantified the tissue concentrations of L-arginine and its nine downstream metabolites (L-citrulline, L-ornithine, glutamate, glutamine, GABA, agmatine, putrescine, spermidine and spermine) in the forebrain of male and female control and MIA offspring (n=12/sex/group) at PD2 using high performance liquid chromatographic (HPLC) and liquid chromatography/mass spectrometric (LC/MS) assays. There were no significant differences between the control and MIA groups in both sexes for the ten neurochemical variables measured, suggesting that MIA did not have a global effect on brain arginine metabolism at this age. Since schizophrenia is associated with prominent frontal and hippocampal dysfunction, Experiment 2 measured tissue concentrations of L-arginine and its nine downstream metabolites in the frontal cortex and hippocampus of male and female control and MIA offspring (n=8/sex/group) at PD2. There were increased levels of L-arginine, glutamate, putrescine, spermidine and spermine and glutamate/GABA ratio, but decreased glutamine/glutamate ratio, in both male and female MIA offspring relative to the controls in a region-specific manner. These results provide the first evidence that a single MIA insult during early neurodevelopment altered arginine metabolism in the frontal cortex and hippocampus at the neonatal stage. Human genetic studies have identified schizophrenia risk genes encoding neuronal nitric oxide synthase (nNOS), one of the key arginine metabolising enzymes. Experiment 3 compared nNOS immunoreactive profiles and microglial migration and maturation in the brains of control and MIA offspring in both sexes (n = 4/sex/group) at PD 2. Immunohistochemistry revealed markedly increased number of nNOS-positive cells in the somatosensory cortex, striatum and the CA3 and dentate gyrus sub-regions of the hippocampus, but not frontal cortex, in the MIA offspring relative to the controls in both sexes. Moreover, there were delayed microglial migration and maturation in both male and female MIA offspring when compared with the controls. This study, for the first time, demonstrates that a single MIA insult alters the brain arginine metabolism and leads to the up-regulation of nNOS and abnormal development of microglia at the age of PD 2. These findings further support the involvement of L-arginine metabolism and microglia in the pathogenesis of schizophrenia. The changes during early neonatal neurodevelopment may contribute to neuronal and behavioral dysfunctions observed in the juvenile and adult MIA rat offspring.

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  • Does prolactin act directly on AgRP neurons in the arcuate nucleus?

    MacLeod, Morgan Anna (2016)

    Undergraduate thesis
    University of Otago

    The development of a positive energy balance occurs during pregnancy to support the growth of fetal and maternal tissues, and to increase energy stores in preparation for the metabolic demands of lactation. Leptin levels increase as pregnancy advances, along with appetite and fat deposition. This is unusual, as in the non-pregnant state, leptin decreases food intake and increases energy mobilization. Therefore, leptin is not being recognized during pregnancy. Leptin-responsive neurons, such as Agouti-related peptide (AgRP) and Pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus, do not respond normally to leptin during pregnancy. It is likely that changes in gestational hormonal profile have a role in mediating leptin resistance. Increased plasma prolactin is observed during pregnancy and, in addition, there are prolactin receptors in the same regions of the brain that control food intake. Prolactin is also known to have an orexigenic effect. We hypothesise that prolactin is a mediator of this increased appetite and resistance to leptin during pregnancy. The aim of this study was to examine whether prolactin directly targets leptin-responsive neurons in the arcuate nucleus. We examined the AgRP neurons, known to be involved in stimulating food intake. Transgenic mouse models and double-label immunohistochemistry were used to look at whether prolactin action is observed in neurons that express AgRP. In separate experiments, AgRP-Cre-tdTomato mice and AgRP-Cre-tauGFP mice were injected with prolactin and perfused. The brains were used to detect prolactin-induced phosphorylation of STAT5, an indicator of prolactin action. The sections were then double-labelled to identify AgRP neurons with labeling of either the tdTomato transgene or GFP respectively. AgRP neurons were readily detected in the arcuate nucleus. Many pSTAT5-stained nuclei were also detected in the arcuate nucleus of prolactin-treated animals, however, none of the AgRP neurons showed pSTAT5-positive nuclei. To confirm these results, we looked at conditional deletion of the prolactin receptor in AgRP neurons using AgRP-PRLR flox/flox mice. We looked at differences in food intake and body weight between these knockout mice and wildtype littermates, no significant differences were observed. We also used immunohistochemistry to look at GFP expression. These mice would be expected to show Cre-induced expression of GFP if the prolactin receptor was normally expressed in the AgRP neurons. vGAT-Cre-Prlr flox/flox transgenic mice were used as a positive control as previous work suggests that GABA neurons express the prolactin receptor. While GFP was readily detected in the positive controls, no staining of any neurons was visible in the non-pregnant, pregnant or lactating brains from AgRP-PRLR flox/flox mice, indicating that the prolactin receptor gene is not normally expressed in AgRP neurons. From these experiments we were able to conclude that prolactin-induced pSTAT5 does not colocalise with AgRP neurons, and that the prolactin receptor gene is not expressed in AgRP neurons. Therefore, the results obtained did not support our hypothesis that prolactin-induced increases in food intake are mediated by direct actions of prolactin by AgRP cells, and showed that prolactin does not directly act upon AgRP neurons to mediate leptin resistance during pregnancy.

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  • Creating a New Code: Artificial Creation & Erasure of Epigenetic Memory in Mammalian Cells

    Wang, Andrew Hue-Yen (2016)

    Undergraduate thesis
    University of Otago

    ABSTRACT Cytosine methylation is perhaps the most dynamic and best-studied form of epigenetic modification. Occurring predominantly in the CG dinucleotide context within mammalian genomes, it is essential for normal embryonic development, X chromosome inactivation, genomic imprinting and transposon silencing. The uniqueness of CG methylation lies in its ability to be maintained following cell division in the absence of the signal which created it. Together, the global distribution of CG methylation forms an “epigenetic memory” that contributes to the differentiation and maintenance of distinctive somatic cell fates. Artificial manipulation of cytosine methylation (known as “synthetic epigenetics”) could potentially improve the creation and differentiation of developmentally potent cells, which will be instrumental in the advancement of regenerative medicine. I have undertaken two experimental projects investigating the potential of synthetic epigenetics, whereby epigenetic modifiers of non-mammalian provenance were overexpressed in mouse embryonic stem cells (ESCs). In plant genomes, methylation occurs at CHG nucleotides (where H is any base other than G) in an analogous manner to mammalian CG methylation, due to the action of the CMT3 methyltransferase. Thus, in the first project, I created transgenic cell-lines with inducible overexpression of Arabidopsis CMT3, with the aim of creating methylation at CHG nucleotides. Overexpression of CMT3 was performed in three mouse embryonic stem cells (mESC) lines; two wild-type lines (E14 and V6.5) and a DNA methylation depleted line known as (DNMT-TKO). My experimental results to date suggest that CMT3 alone is inadequate for CHG methylation establishment in mammalian genomes, however, increased levels of methylation in the CG context were detected, suggesting it may have previously unappreciated maintenance capacity in this context. The second experimental project explored the nature of DNA methylation removal. Cytosine methylation can be actively removed from DNA by the Ten eleven translocation (TET) enzymes, which oxidise 5-methylcytosine (5mC) to 5 hydroxymethylcytosine (5hmC), and further oxidised derivatives 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Exactly how these analogues are returned to unmodified cytosine is unknown, partly because there are currently few molecular tools to control the transition between 5hmC, 5fC and 5caC. Unpublished in vitro observations suggest that Ten eleven translocation (TET) enzymes from the amœba Naegleria gruberi are far more effective at iteratively producing 5fC and 5caC, compared to mammalian homologues which produces 5hmC predominantly (T.P. Jurkowski, personal communication). I created mouse embryonic stem cells with inducible expression of Naegleria TET and mutated Naegleria TET variants, and used bisulphite sequencing to assess their relative ability to demethylate DNA. I found that the amœba TET variants were ineffective at demethylating DNA compared to mammalian TET controls. Unexpectedly, some constructs even showed significant increased methylation. I hypothesise that the Naegleria TET variants I created act as non-functional competitors of endogenous TET when overexpressed in mammalian cells. While this likely precludes their widespread use in the field of synthetic epigenetics, they still may provide a useful experimental tool for further study of TET enzyme biology.

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  • Functional Role of Uric Acid in Cardiac Stem Cell Homeostasis

    Cheakhun, Cindy (2016)

    Undergraduate thesis
    University of Otago

    In New Zealand, cardiovascular disease (CVD) affects 1 in 20 adults and is the leading cause of mortality. Cardiac pathophysiology along with diabetes mellitus is historically associated with glucose metabolism, but recently it has been suggested that elevated serum uric acid (SUA) could be a major driver of these complications. Cardiac stem cells (CSCs) are a new therapy approach for CVD, however, the therapeutic effects are usually mixed. A major contributing factor could be the necrotic environment that the stem cells are being isolated from or transplanted back into. Elevated SUA could contribute significantly to the necrotic environment. Hence, our goal was to characterise the effects of elevated SUA, reflected by changes in extracellular uric acid (EUA) on cardiac progenitor cell (CPC) function in order to improve stem cell therapy, as CPCs are also investigated for use in stem cell therapy and are similar to CSC in terms of physiology and function. We characterised the uric acid (UA) transporter expression profile in human CPCs including known renal transporters: ABCG2, MRP4, GLUT9, URAT1, OAT1, OAT2, OAT4 and NPT4, using qPCR. Human CPCs as well as mRNA from human heart tissue express UA efflux transporters ABCG2 and MRP4 to a greater extent than UA influx transporter GLUT9, irrespective of diabetic status. The expression of GLUT9, ABCG2 and MRP4 were also confirmed at the protein level through western blot analyses of proteins extracted from CPCs. This transporter profile in human CPCs support our new concept of a ‘cellular uric acid homeostasis’ (CUAH), and the fact that a disturbance of CUAH, as in diabetes mellitus, may be detrimental to the function of CPCs and ultimately affect stem cell therapy. From functional assays, it appears that CPCs are sensitive to changes in EUA concentrations. By the end of UA treatment, 300μM UA treatment, which is considered normal SUA, had the most viable cells. The amount of viable cells decreased with 200μM UA and 500μM UA significantly (p<0.01). In sum, the results support the idea that CUAH is crucial for the functionality of CPCs, and therefore changes in CUAH, or the environment that stem cells and progenitor cells are transplanted into can impair their ability to integrate into the host tissue, and therefore decrease the efficacy of stem cell therapy.

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  • Sex-differential expression of microRNAs during mammalian neurodevelopment

    Szakats, Susanna Katharine (2016)

    Undergraduate thesis
    University of Otago

    Pervasive sex differences between male and female brain development have been implicated in neurodevelopmental disorders featuring a distinct sex bias, such as autism. To further our understanding of sex differences we hypothesised that sex-differential microRNA expression occurs in the developing brain. MicroRNAs are powerful regulators of gene expression whose function is essential for normal brain development, and are thought to mediate development of sex differences. Small RNA sequencing was performed with RNA isolated from E15.5 mouse brains (n=3 per sex), representing early brain differentiation. Comparison of microRNA expression identified 12 microRNAs that were differentially expressed between sexes (p<0.05). Differences in expression were validated using real time quantitative RT-qPCR for miR-10, and miR-205 and miR-5099. miR-10 is encoded within the Hox gene complex, a cluster of genes conferring positional identity to structures along the anterior-posterior axis of the developing embryo. As miR-10 is thought to be controlled in the same spatially co-linear manner as the Hox genes, we anticipated that it has a distinctive expression domain. Using in situ hybridisation with a locked nucleic acid (LNA)-probe targeting miR-10, strong staining for miR-10 expression was found in the hindbrain and anterior spinal cord of embryos at E15.5. Not only did we investigate known microRNAs, we also analysed novel microRNAs aligning to the mouse Anti-Müllerian Hormone AMH gene. Previous experiments identified a non-coding RNA (ncRNA) transcribed at this locus, bearing hallmarks of a primary microRNA. Subsequent preliminary small RNA sequencing at E12.5 (n=1 per sex) showed microRNA profiles at the AMH locus unique to each sex. Using RT-qPCR validation, female-specific expression of a novel microRNA aligning to exon 3 of AMH was determined. Interestingly, an increase in microRNA expression occurred in mouse lines where the exons 1 and 2 of the AMH gene were deleted. This observation lead to identification of a putative repressor element within exon 2, thought to negatively regulate expression of ncAMH. Our findings indicate that sex-differential expression of microRNAs occurs in the developing mouse brain during a key stage of brain patterning. The identified microRNAs are known to influence development and neurological functions, and therefore their expression in a sex-differential manner may contribute to sex differences established in the developing brain.

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  • Efficacy of Prototype Vaccines for Prevention of Tuberculosis

    Sandford, Sarah (2016)

    Undergraduate thesis
    University of Otago

    Tuberculosis (TB) is a pulmonary disease caused by Mycobacterium tuberculosis, which killed 1.5 million people in 2014. The current TB vaccine, live attenuated Mycobacterium bovis bacille Calmette Guérin (BCG), effectively protects against neonatal TB yet has variable efficacy against pulmonary TB in adults. Consequently, a more effective preventive vaccine is needed. This project aimed to characterise the ability of two innovative new vaccines to improve antigen delivery and induce protective mucosal immune responses against mycobacterial infection in mice. The first vaccine approach employed sustained release of protein antigen together with adjuvants to enhance immunogenicity. To that end, a vaccine comprising the mycobacterial antigen 85B (Ag85B) with poly lactic-co-glycolic acid nanoparticles in a thermoresponsive chitosan gel was developed. This vaccine was hypothesised to prolong antigen exposure, enhancing the development of an effective immune response, and reducing the need for booster vaccinations. The second approach was designed to activate mucosal immune responses, using non-pathogenic Lactococcus lactis as a vector. Genetically engineered to overexpress the model antigen Ovalbumin (Ova) on its surface via a Group A Streptococcus serotype M1 Pilus (L. lactis PilM1-Ova), this prototype vaccine was hypothesised to improve antigen delivery to the mucosal immune system. The chitosan gel vaccine containing Ag85B, and the L. lactis PilM1-Ova vaccine were administered separately to groups of mice. For both vaccines, the activation state and cytokine responses of antigen-specific T cell populations were detected using flow cytometry. Compared to BCG, the chitosan gel vaccine did not induce higher frequencies of activated, proliferating, cytokine-producing, or memory Ag85B-specific T cells, and failed to protect against an intranasal BCG challenge. By contrast, L. lactis PilM1-Ova induced more activated, proliferating, cytokine-producing and memory Ova-specific T cells than recombinant BCG expressing Ova (BCG-Ova). Although L. lactis PilM1-Ova was not protective against intranasal BCG-Ova challenge, this may have been due to slow in vivo growth of the recombinant BCG-Ova. This work suggests that the chitosan gel vaccine requires further development to improve immunogenicity, for example by selecting a different antigen or antigen dose. L. lactis PilM1 shows promise as a vaccine delivery system for TB and is worthy of further studies, ideally with expression of an endogenous mycobacterial antigen to measure vaccine-induced antigen-specific T cell responses and protection against TB.

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  • The Influence of Exercise and Hyperlipidaemia on Breast Cancer

    Buss, Linda (2016)

    Undergraduate thesis
    University of Otago

    Exercise reduces the risk of breast cancer development, and improves survival in breast cancer patients. However, the underlying mechanisms of this protective effect remain to be fully elucidated. It is unclear whether exercise can attenuate or modify the pro-tumour effects of obesity and related conditions, such as hyperlipidaemia, on breast cancer growth. The main aims of this study were (1) to develop a relevant, tumour-bearing mouse exercise model and (2) to determine the effect of exercise and hyperlipidaemia on the breast tumour microenvironment. We hypothesise that exercise attenuates the negative effect of hyperlipidaemia through ‘normalisation’ of the tumour microenvironment. Hyperlipidaemic ApoE-/- and wild-type (WT) C57BL/6 mice with orthotopic EO771 breast tumours were randomly assigned to intermittent or continuous voluntary wheel running or sedentary control. When tumours reached maximum size, mice were sacrificed and the serum, organs and tumours removed for analysis of tumour cell proliferation, immune infiltrate, circulating inflammatory factors, perfusion, microvessel density, hypoxia, HIF-1α protein level and GLUT-1 protein expression. This was done by immunohistochemistry, immunofluorescence, ELISA and Western blotting. Although exercise and hyperlipidaemia did not significantly impact tumour growth rate, exercising mice had significantly reduced body weights. Tumour-bearing mice showed a significant increase in serum monocyte chemoattractant protein 1 (MCP-1) compared to non-tumour-bearing mice (p<0.05), but perfusion was not significantly altered. Further studies are necessary to clarify and confirm these results, as this study was limited both by a short exercise and tumour-bearing time period. This study identifies a number of key considerations in the design of future preclinical exercise studies in tumour-bearing mice. In addition, our results provide evidence for the potential value of MCP-1 as an exercise-regulated, prognostic biomarker in mouse models, and indicate that hyperlipidaemia normalises the microenvironment of the tumour.

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  • A Validation Study to Detect Markers of Proliferation Adequacy in the Endometrium

    Burn, Rosamond Sarah (2016)

    Undergraduate thesis
    University of Otago

    Throughout the human female menstrual cycle, the endometrial lining of the uterus transitions from a non-receptive proliferative state to a receptive secretory state. This transition is required to prepare the endometrium for an embryo to successfully implant. Transitioning of the endometrium is governed by various genes, but the cellular processes are poorly understood. Unfortunately, for some women this process is compromised, resulting in the endometrium being inadequately primed for implantation and conception repeatedly fails. This is known as recurrent implantation failure (RIF). The aim of this study was to identify different markers within the functionalis layer of the endometrium that are important for implantation success. Qualitative and quantitative protein analysis was used to compare endometrial tissue biopsies between women with different fertility phenotypes. Biopsies were collected from women attending a single fertility clinic, with collection completed during the early-secretory (pre-receptive LH+2) phase of the menstrual cycle. Cryo-sectioning was used to cut tissue sections, revealing glandular epithelium, luminal epithelium, and stromal cells. Immunofluorescence and SDS-PAGE combined with Western blots were used to assess protein expression. Expression levels of 25 different proteins previously reported to be involved in endometrial proliferation that feature in endometrial focal adhesion or cell cycle pathways were compared between four cohorts; Fertile (n=5), RIF(n=6), Research (n=6), and Other (n=4). Quantitative PCR was also used to detect relative mRNA expression levels in the endometrium between the internal references (ACTB and CYC1) and the target genes (EGFR, PCNA, PGR, and MCM2). It was found that endometrial protein profiles differ during the early-secretory, LH+2, phase of the menstrual cycle, between women who are fertile and women with clinically defined RIF. Immunofluorescence results showed a statistically significant increase of CCNA, PCNA, MKI67, and SMAD3, and decrease of IL1R1, in the fertile cohort in comparison to the RIF cohort. Western blot also showed statistically significant up-regulation of PCNA in the fertile group, as well as PGR and YWHAZ. Gene expression profiles did not differ between fertile women and women with RIF, however there was a significant reduction of PGR and PCNA in the Research and Other groups in comparison to the Fertile and RIF groups. In this pilot study, protein and gene analysis was able to detect alterations of expression in groups of women with different fertility phenotypes. Further exploration of these markers could help determine their possible role in endometrial dysfunction in RIF women and establish a clinical panel to indicate endometrial preparation adequacy.

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  • Investigating the anti-invasive properties of dexamethasone in Fn14 positive triple negative breast cancer.

    Harris, Joshua (2016)

    Undergraduate thesis
    University of Otago

    Triple-negative breast cancer (TNBC) is an aggressive disease subtype with no current targeted systemic therapies due to a lack of actionable molecular targets. Efforts to identify therapeutic targets in TNBC have revealed that Fn14, a cytokine receptor, is over-expressed in most invasive breast cancers (1, 2) but not in healthy breast tissue. Fn14 has been shown to activate canonical NF-κB signalling (3), which in turn, transactivates the Fn14 promoter (3) leading to an autoregulatory loop that may drive invasive capacity (2) in breast cancer. We hypothesised that repression of the Fn14/NF-κB feedback loop may reduce Fn14 expression and invasive capacity in breast cancer. As corticosteroids are known to attenuate NF-κB activity through glucocorticoid receptor alpha (GR⍺), we postulated that corticosteroids might impact Fn14 expression. We investigated this likelihood in three TNBC cell lines positive for GR⍺ and Fn14 (BT-549, MDA-MB-436 and SUM149PT). Following 24h treatment with the corticosteroid, dexamethasone (DEX), we observed a decrease in Fn14 protein expression. In parallel, DEX reduced invasive capacity in vitro by an average of 40% with the most significant and consistent response in BT-549 cells. The DEX-mediated reduction in Fn14 expression and invasive capacity are likely due to a repressive relationship between GR⍺ and NF-κB. However, the potential mechanisms of repression are complex and poorly understood in breast cancer. To investigate this mechanism, we first used immunocytochemistry to visualise the spatial location of GR⍺ and NF-κB (p65) in BT-549 cells after 24h of DEX. Our results showed rapid nuclear translocation of GR⍺ and subtle nuclear exclusion of p65. We quantified this observation using western blotting over a 24h time course with DEX using subcellular fractionation of BT-549 cells. These data suggest that a repressive physical interaction between GR⍺ and p65 is unlikely, due to the opposite movement of transcription factors between cellular compartments. Interestingly, IκB⍺, an inhibitor of NF-κB, exhibited a subtle DEX-mediated increase in protein expression over 24h, corresponding with the cytoplasmic retention of p65. Our data, therefore, supports a model that repression of NF-κB signalling may be achieved through de novo NF-κB inhibition. Additionally, BT-549 cells transfected with an NF-κB-response element/luciferase reporter plasmid showed an intriguing increase in luciferase activity following 24h of DEX. It was later shown that this may have been caused by DEX-induced glucocorticoid resistance due to the downregulation of GR⍺ after DEX treatment. These results demonstrate a novel mechanism for Fn14-mediated invasion. However, investigation in additional cell lines is required. The induction of DEX-mediated glucocorticoid resistance also questions the clinical significance of glucocorticoid based therapy in the treatment of TNBC.

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  • Hepatitis B in Oman, risk factors and sequelae

    AlHarthi, Rahma (2016)

    Undergraduate thesis
    University of Otago

    Background Hepatitis B is a major public health problem worldwide. The prevalence of hepatitis B is dependent on the modes in which it is transmitted. There are two common modes of hepatitis B virus (HBV) spread: vertical (mother to neonate) and the horizontal (via infected blood or body fluids). Chronic infection with HBV can progress to liver cirrhosis and liver cancer (hepatocellular carcinoma; HCC). Oman is regarded as an intermediate endemicity region and has had neonatal vaccine against HBV since 1990; however, little research has been conducted in Oman regarding risk factors for hepatitis B and its contribution to end stage liver disease and HCC. Aims  To identify the prevalence of major risk factors for acquiring hepatitis B in Omani patients currently infected with HBV (positive hepatitis B surface antigen (HBsAg)).  To estimate the contribution of hepatitis B to liver cirrhosis in Oman. Methods The prevalence of major risk factors was identified by interviewing HBsAg positive patients using a standard questionnaire. Patients were recruited from outpatient clinics at two tertiary referral hospitals in Oman’s capital city Muscat. Data on patients with liver cirrhosis admitted to two tertiary hospitals in Muscat over a period of seven years was abstracted from medical records. The diagnosis of cirrhosis was confirmed using defined criteria and the aetiology confirmed from the results of diagnostic tests including HBV serology. This data was analysed to estimate the contribution of HBV to cirrhosis in the cohort. Results For the first objective, 279 patients were interviewed. The number of male and female patients was similar, and 75.5% of the participants were aged 20 – 39 years. Antenatal screening was the most common means of detecting HBV infection in women and prior to blood donation was the most common means of identifying HBV infection in men. With respect to HBV transmission risk factors, intra-familial contact with HBV infected persons and behavioural risks such as body piercing (females) and barber shaving (males) were more common than nosocomial risk factors. Knowledge about HBV infection was scarce among our participants. For the second objective, we identified records from 419 patients with cirrhosis. The median age was 59 years and males accounted for two thirds of the total studied population. 97.1% of patients were of Omani ethnicity. There was evidence of previous or current HBV infection (positive anti-bodies to hepatitis B core antigen) in 51.3% of the cirrhotic patients. 21.5% had active HBV (positive HBsAg). Of the patients with current HBV 91.2% were infected with HBV alone while 8.8% were co-infected with hepatitis C virus (HCV). Hepatitis C was present in 30.5% of cirrhotic patients and nearly half of those patients had evidence of past exposure to HBV. When stratified by gender, HBV infection was more common among male cirrhotic patients compared to females. Conclusions This study found that risk factors for HBV infection in Omani patients include direct contact of infected individuals within a family and exposure to high-risk behaviours such as piercing and barber shaving. Reducing vertical and horizontal transmission of hepatitis B in Oman could be improved by the implementation of routine antenatal screening of pregnant women and a greater focus on contact screening respectively. Future work is required to determine whether the association with behavioural risk factors is causal, particularly piercing and shaving at barber shops. If confirmed, relatively simple and effective interventions could be developed to reduce the risk of horizontal transmission related to these activities. We found that third of the patients identified with liver cirrhosis had past exposure to HBV and 20% had evidence of chronic infection. Most patients were of older age and male sex. This group of patients may benefit from antiviral therapy to prevent decompensation and regular surveillance for early diagnosis and treatment of HCC. Further research is required to assess the role of other exposures (alcohol, co-infection with other viruses) in the prognosis of hepatitis B to cirrhosis in Oman.

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