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Arachnid ecology in New Zealand, exploring unknown and poorly understood factors.
No full textSpiders (Araneae) are an important ecological component of most terrestrial ecosystems. Comparatively, New Zealand spider ecology is poorly understood, particularly with regards to how spiders have been affected by the influx of new organisms entering an otherwise isolated habitat. This thesis aimed to observe the impact of vegetation structural complexity and mammalian predation on coastal Otago spider abundance and diversity, focusing in particular on the seasonal loss of vegetation complexity on introduced deciduous trees and the impact of introduced rodents. In addition, in order to non-destructively sample spiders in a predator-free ecosanctuary, a novel no-kill trap was designed and tested.
The native New Zealand green mistletoe (Ileostylus micranthus) provides a continuous area of evergreen foliage on introduced deciduous trees throughout the winter. Recording invertebrate numbers from mistletoe samples and comparing them to host plant samples provided insight into how invertebrate populations react to loss of structural complexity on introduced deciduous trees during leaf fall. There was no significant difference between mistletoe and host plant invertebrate populations during the summer, but during the winter, invertebrate numbers on deciduous hosts were significantly lower in comparison to evergreen hosts and to mistletoes on either plant host type.
A novel, non-kill spider trap was developed to assist with sampling in environmentally sensitive areas, such as ecosanctuaries. Dubbed the Sanctum Aranearum, the Sanctum utilizes structural complexity to attract spiders and other invertebrates to settle within the trap, which can then be readily collected without killing by-catch. The Sanctum was constructed from a white cuboid bucket with windows cut near the surface to allow invertebrates to enter. The internal structure included large and small plastic tubes and wire imbedded in sand. The Sanctum was tested against pitfall traps as a close analogue to compare capture results and found that the Sanctum was able to capture a higher abundance of invertebrates and a greater diversity of arachnids.
The impact of predation by introduced rodents was assessed by sampling spiders at the Orokonui Ecosanctuary, near Dunedin, Otago, while it underwent a rodent elimination program. This elimination programme provided an optimal chance to observe how spider populations would be affected by the removal of rodents from a habitat. Sancta were utilized due to the sensitive nature of the ecosanctuary. A nearby Queen Elizabeth II National Trust conservation area with comparable vegetation was used as a control site to compare spider populations, as rodents were known to be present. Analyses were unable to separate the impact of rodents from potential seasonal effects on spider populations. It was noted that larger native spiders were more abundant in Orokonui than at the control site, but lack of historical data made any conclusions purely speculative.
This thesis has made a novel contribution in terms of understanding the role of mistletoes as over-winter refugia for invertebrates. The findings in Chapter 2 have a multitude of implications regarding how urban and agricultural invertebrate populations survive winter with the introduction and proliferation of many deciduous plants to New Zealand. Both beneficial and pest species could potentially utilize mistletoes and further research is required.
The novel Sanctum Aranearum presented in Chapter 3 and successfully deployed in an ecosanctuary in Chapter 4 is a valuable addition to arachnological methods and further work to explore the potential field uses for it are already being planned
Characterisation of developmental cues in early postnatal AgRP neurons
No full textBody weight regulation and the control of food intake are topics that continue to receive much attention as countries around the world grapple with alarming rates of obesity and the associated poorer health outcomes. Central to this has been two antagonistic populations of neurons, the agouti-related protein (AgRP)-expressing neurons and the proopiomelanocortin (POMC)-expressing neurons, which originate from the hypothalamic arcuate nucleus (ARC). Over the past two decades, there has been increasing interest in how these neurons develop, and how developmental insults may impact on their formation and later life function. As a component of this, it has been recognised that the adipocyte hormone leptin plays a critical role in modulating the development of the projections of AgRP and POMC neurons to other brain regions associated with body weight regulation. In rodent models, much of this circuitry elaboration occurs during the first few postnatal weeks.
The research presented in this thesis followed two lines of enquiry, with the aim of better understanding the normal postnatal development of AgRP neurons in a mouse model. Initially, I focused on the role of leptin in these neurons’ development, by using qPCR to investigate whether several putative transcriptional targets of leptin signalling, Ephb3, Fez1, Ngfr, Plxnb1, and Unc119, were indeed leptin regulated in postnatal day 8 ARC. However, no evidence was found to support this hypothesis. In the second half of this thesis, the novel in situ hybridisation technique, RNAscope, was used to survey the hypothalamic expression of three ligand-receptor pairs, Ntn1 and Dcc, Wnt5a and Fzd5, and Slti1 and Robo1, that I reasoned may be involved in AgRP neuron elaboration. All three pairs were found to be expressed in the early postnatal hypothalamus, and various patterns of expression were described in the ARC and paraventricular hypothalamus on postnatal days 2, 8, 12 and 16.
As the incidence of obesity in the developed world reaches epidemic proportions, understanding the normal development of the neural circuitry responsible for regulating food intake and maintaining energy balance is of increasing public health importance
Climatology of Vapour Transport associated with New Zealand Droughts
No full textDrought is a natural hazard which has a wide range of impacts across multiple sectors. While a universal or exact definition of drought is problematic, it is relatively straightforward to classify drought according to the impacted section of the hydrological cycle. Here, hydrological drought is chosen as the focus, as the ecological and socio-economic impacts become more severe as a drought propagates into a hydrological drought. Restraints on hydrological data availability often necessitate the use of standardised indices based on meteorological data, such as the Standardised Precipitation and Evapotranspiration Index (SPEI), as a proxy for hydrological drought. Despite the varied definitions of drought and the complexity in the spatio-temporal development of drought, decreases in precipitation remain a shared characteristic across all drought types, including hydrological drought. Decreases in precipitation are frequently linked to wider atmospheric processes which can act to block the movement of atmospheric moisture, preventing the supply of moisture needed to precipitate out. Thus decreases in precipitation are an outcome of a disruption to the typical pattern of moisture transport over a particular region.
Across New Zealand drought research has remained focused on the spatial and temporal expression of drought. With most precipitation across the country coming from the ocean, a focused study of how this transport of moisture is interrupted during drought events could reveal important information on atmospheric causes of drought occurrence. In the present study, the relationship between drought and atmospheric moisture is investigated across New Zealand and regionally. The 3 month accumulation period of the SPEI (SPEI-3) shows utility in tracking hydrological drought onset, while its ability to capture the impact of snowfall and groundwater processes is questionable across the East Cape, East Coast of South Island and Central Otago regions. An environment to climate approach identifies the relationship between drought and atmospheric moisture featuring declining westerly moisture transport, with a weaker connection to moisture flux indicating the significance of PET to the East Cape, East Coast of South Island and Central Otago regions. A climate to environment approach, using the Self Organising Map (SOM) method, supports the initial findings.
Collectively, the results provide a first look at atmospheric moisture transport over New Zealand during drought events, revealing important information on possible atmospheric mechanisms associated with drought development such as blocking activity or changes in jet stream movement. Further, this thesis highlights the utility of the SOM method for atmospheric research over New Zealand and provides a basis for further detailed investigation, while the analysis of the SPEI-3 has important implications for the New Zealand Drought Index and its possible usage for monitoring hydrological drought
Filamin B hinge-1 in joint development
No full textIn this study I aim to evaluate how the actin binding protein filamin B influences the development of cartilaginous structures of the skeleton, that when mutated, causes a range of disorders impacting large joints, limb development and craniofacial aspects of the body. Joints are an essential part of any organism as they allow for movement and are therefore extremely complex mechanical structures. The soft tissue elements of the joints are important because they provide these joints the ability to withstand immense forces from movement. Little is known about how filamin B is involved in the formation of the soft tissues in the developing joint and therefore the lack of understanding of how filamin B variants lead to the dislocation of the large joints seen in Larsen syndrome and other filamin B related syndromes. Joint development is critical in analysing how filamin B is involved in the formation and rare disease are an extremely valuable tool in approaching this. Rare genetic diseases such as Atelosteogenesis types I (AOI), Atelosteogenesis type III (AOIII), Boomerang dysplasia (BD) Spondylocarpotarsal synostosis syndrome (SCTS) and Larsen syndrome (LS) can all show different aspects of development that has been disrupted from various mutations within the filamin B gene. These are able to provide indications on what section of the filamin B protein, then when perturbed, leads to developmental abnormalities. In this study I used two mouse models tagged with the fluorophore mCherry and eGFP to track the development of joints in mice, in relation to filamin B both with and without the protein’s hinge-1.
My research found that both forms of hinge-1 included filamin B and hinge-1 excluded filamin B are found during development. It was also shown that red puncta indicating hinge-1 excluded filamin B is present within the joint capsule between the forming femur and tibia at E15.5. These red puncta were observed in the cells adjacent to the cellular membrane. During development the red puncta were exclusively defined to the joint spaces within all developing legs examined at E15.5. It was also shown that the critical point for knee joint ligament formation is between mouse embryonic time points E15.5 and E16.5
Investigating the activation of nucleus accumbens projective paraventricular nucleus of the thalamus during cue-food reward conditioning
No full textWhen a previously neutral cue is learnt to predict a food reward, the presence of the cue itself can elicit the motivation to obtain the food. The nucleus accumbens shell (nAccSh) in the mesolimbic system is involved in generating motivation. It is known that paraventricular thalamic (PVT) neurons project to nAccSh. However, how it may interact with the nAccSh in conditioning is not well understood. Therefore, I investigated the activation of nAccSh-projective PVT neurons after the conditioning. It is hypothesised that nAccSh-projective PVT neurons are activated by conditioning.
Male Wistar rats were injected in nAccSh with a retrograde adeno-associated viral vector (AAV) containing a channelrhodopsin (ChR2)-green fluorescent protein (GFP) construct. This labelled the membrane of all neurons projecting to the nAccSh, including PVT neurons. The rats were then randomly divided into conditioning group (light stimulus predicting the release of a sugar pellet, n=4) and control group (light not predicting sugar pellet, n=3). After the conditioning experiment rats were euthanised, and the brains were sliced. The PVT was stained for GFP and cFos (an indirect marker for neuronal activation), using fluorescent immunohistochemistry.
The nuclear cFos staining counts were similar across the anterior, medial and posterior PVT [two-way ANOVA, F(2,15)=1.1, p=0.3458] without a group effect [F(1,15)=0.98, p=0.3376]. Co-localisation of GFP-labeled nAccSh-projective PVT neurons and cFos nuclei was seen in both groups across PVT regions. The analysis showed a trend towards a regional difference in the count of co-localised cells [F(2,15) =3.4, P=0.0584]. However, it was not significantly different between the groups [F(1,15)= 0.5, p=0.4574].
These findings suggest that different PVT regions can be activated by repetitive exposure to stimuli and food rewards, regardless of their predictive relation. Pavlovian conditioning may not necessarily activate nAccSh-projective PVT neurons. However, this preliminary study requires more replicates to fully test the hypothesis
Development of detection methods for Legionella longbeachae in clinical and environmental samples
No full textLegionella longbeachae is the predominant causative agent of Legionnaires’ disease (LD) in New Zealand with a peak infection period during the spring and summer months. This is a plant-associated microbe that can be found throughout the environment and is associated with potting mix and compost. LD is difficult to diagnose because of difficulty with obtaining respiratory samples.
Acid pre-treatment followed by culture is commonly used for the isolation of Legionella species in both clinical and environmental samples but has limited sensitivity because of high contamination loads. Detecting volatile organic compounds (VOCs) in breath is a minimally invasive technique that could be used to investigate patients with possible LD.
The aims of the current study were to:
a) develop isolation methods to improve identification of L. longbeachae in clinical and environmental settings,
b) develop an immunomagnetic separation (IMS) method to improve culture and PCR,
c) identify possible VOCs using gas chromatography-mass spectrometry (GC-MS) that can be used as diagnostic biomarkers for LD.
d) investigate potting mix productions for the presence of L. longbeachae,
e) study the potential influence of physico-chemical characteristics of potting mix products that permit L. longbeachae to survive,
To develop the IMS method, a polyclonal antibody was raised in rabbits by immunising them with heat-killed L. longbeachae antigens with several booster injections. The antibody produced was separated from serum and purified by ion-exchange chromatography. This polyclonal antibody had high specificity and sensitivity in capturing L. longbeachae, with minimum cross-reactivity with other species. This antibody was coupled to immunomagnetic beads and used to separate organisms in stored respiratory samples for both culture and PCR. Feedstock samples used in the manufacture of the potting mix were taken from three sites around New Zealand for PCR and culture.
Culture results of sputum samples were significantly improved by using IMS. qPCR was used for this preliminary screening of environmental samples. The results showed that bark and bark-containing products had the highest number of PCR positive samples for L. longbeachae.
Culture results for these PCR positive samples were negative. Several isolation methods were developed to improve the culturability of L. longbeachae from environmental samples. The results showed that the combination of the Legionella selective GVPC antibiotic suspension with IMS reduces the contamination of cultures with other organisms and also improves the recovery of L. longbeachae from the samples.
Physico-chemical methods were used to determine the elemental make up of a subgroup of feedstock samples. The results showed that there were higher concentrations of boron and sulfur in PCR negative bark samples than PCR positive samples.
Finally, the headspace of the culture of L. longbeachae was pre-concentrated and analysed by GC-MS to generate a volatile profile of these bacteria. A peak of interest was found in this profile. Further work needs to be undertaken to identify the precise chemical structure of this compound.
In conclusion, this work has demonstrated that the rate of positive cultures of stored sputum samples is higher with IMS separation. This result needs validating in prospective studies in a diagnostic laboratory to determine if it can become a useful diagnostic tool. The sensitivity of culture of heavily contaminated environmental samples can be improved using IMS and GVPC decontamination and these techniques can be validated in future studies. Addition of elements such as sulphur and boron to potting mix should be evaluated as a method of manufacturing a safer potting mix product. Further studies are needed to precisely identify the chemical structure of a VOC identified and clinical trials conducted to determine whether this is of diagnostic value
Targeted Epigenetic Editing Using a dCas9- SunTag Methylation System
No full textDNA methylation is a stable and somatically heritable epigenetic process. It is classically understood as a gene silencing mechanism, whereby hypermethylation of gene promoters is largely associated with transcriptional silencing. However, emerging research in the past decade has found a different association, particularly in the context of cancer and metastasis. These studies have demonstrated a seemingly paradoxical correlation between high levels of promoter methylation and subsequent transcriptional activation. As such, we are yet to fully unravel the complex mechanism underlying this relationship. This warrants further investigations to better understand this dynamic molecular mechanism and thus its implications in cancer pathobiology and metastasis.
Previously established methods of manipulating DNA methylation have largely involved the use of chemical drugs, such as decitabine. These drugs act globally on the epigenome and are thus non-specific. Importantly, as they are inhibitors of DNA methylation, they cannot definitively demonstrate that an increase in promoter methylation is directly responsible for an increase in gene expression. Therefore, by using a CRISPR-based system for targeted epigenetic editing, the precise mechanism of paradoxical gene activation by DNA methylation can now be investigated.
Our group has recently demonstrated this paradoxical correlation in a study characterising epigenetic changes in metastatic melanoma. The Early B-cell Factor 3 (EBF3) gene promoter region was found to be substantially hypermethylated in metastatic cell lines compared to paired, primary cell lines. This increase in methylation was also found to be associated with a subsequent increase in gene expression. These findings suggest that EBF3 in human melanoma cell lines is a good candidate gene to investigate this complex relationship.
Here, I aim to establish a methylation editing system and apply it to the EBF3 promoter region. This is a three-component CRISPR system incorporating the SUperNova Tag (SunTag), which allows for the recruitment of multiple effector proteins. In this project, I use the DNA methyltransferase 3A (DNMT3A) effector protein to induce active methylation at a target locus within the EBF3 promoter region in melanoma cell lines.
Methylation changes have been evaluated using methylation-specific Illumina iSeq sequencing. Variable levels of targeted methylation have been observed, of up to 38.0% absolute methylation change between edited and unedited samples. This project represents important progress in investigating the promoter methylation of EBF3 in more detail, as well as laying out an essential platform to further investigate methylation changes and causal mechanisms of gene expression alteration in future studies
Gene expression variability in breast tumour development
No full textBreast cancer is the most common female malignancy and presents a major health issue both worldwide and in New Zealand. Individuals with a family history of either breast or ovarian cancer have an increased lifetime risk of breast cancer. Furthermore, approximately 10% of breast cancers are thought to be caused by rare inherited genetic variants, of which variants in BRCA1 and BRCA2 are the most prominent. Individuals carrying a pathogenic BRCA1 or BRCA2 variant have up to a 87% and 84% lifetime risk of developing breast cancer compared to 12% for the general population.
BRCA1 and BRCA2 functions are critical to genomic stability, which is essential to maintaining a healthy cell state in all tissues. However, there is a lack of disease risk in the majority of non-breast and ovarian tissues. Additionally, there are thousands of different sequence variants across each gene that present distinct risk profiles ranging from benign to pathogenic. A large proportion of these are considered variants of uncertain significance (VUS) resulting in no additional information to aid clinical management. The effect of pathogenic variants on gene expression is a potential source of information that can help to identify genes involved in tissue-specific risk and a phenotype that can be used to help classify VUS.
Previous studies have explored BRCA1- and BRCA2-associated gene expression profiles, however, there has been a lack of consistency between genes identified as associated with BRCA1- and BRCA2 variant status. These studies have typically focused on identifying differences by comparing the mean level of gene expression, however, the variability of gene expression is also under genetic control and has been under-explored in BRCA1- and BRCA2-associated tissues.
BRCA1 and BRCA2-associated gene expression variability was calculated across three familial breast tumours datasets. Additionally, as BRCA1-associated tumours are typically subtyped as basal-like, gene expression variability was calculated between basal and non-basal breast tumours in four datasets. BRCA1- associated and basal-like tumours exhibited greater global gene expression variability compared to familial breast tumour with no BRCA1 or BRCA2 pathogenic variant (BRCAx) and non-basal tumours. By comparison, the mean level of gene expression was similar between all tumour-types. Three genes (DSC3, EN1 and IGF2BP3) were found to be differentially variable in BRCA1-associated and basal-like breast tumours compared to BRCAx and non-basal tumours, respectively. RNA in situ hybridisation (RNA ISH) was used to explore EN1 expression in 503 breast tumour cores, including 151 BRCA1-associated tumours. Consistent with mircoarray analyses, RNA ISH found the expression of EN1 was variable in BRCA1-associated tumours.
Two breast cell lines were used to measure gene expression variability in BRCA1 heterozygous models. In contrast to tumour analysis, MCF-10A BRCA1mut/+ cells displayed less global gene expression variability compared to wild type MCF-10A cells. Additionally, BRCA1 knock-down in MCF-7 cells showed no change in gene expression variability. The knock-down of BRCA1 was, however, associated with a decrease in luminal and an increase in basal marker expression. To further explore these observations, CRISPR-Cas genome engineering was used to generate a MCF-7 cell that harboured a BRCA1 pathogenic variant. Similar to BRCA1 knock-down models, MCF-7 BRCA1 mutant cells exhibited a decrease in expression of the luminal marker ESR1. However, immunocytochemistry was unable to detect the basal maker CK5/6 in any of the MCF-7 mutant clones.
Rare high risk breast cancer variants displayed an association between genotype and gene expression variability. Therefore, I also investigated the relationship between common risk variants and gene expression variability by conducting variable expression quantitative trait loci analysis (veQTL). This analysis showed that of the 181 published common breast cancer risk variants, 27 had breast-specific veQTL associations with 60 genes. One variant, rs11075995, was associated with four genes (CYP11B1, CYP17A1, HSD3B2 and STAR), all of which were involved in C21-steroid biosyntheisis.
These studies successfully demonstrated the potential utility of gene expression variability analysis to identify candidate breast cancer risk associated genes. The application of this approach may lead to a better understanding of the mechanisms involved in the development of disease. Furthermore, these methods may transform how researchers interpret the results form published and future genome-wide association studies
Type 2 diabetes and the dental pulp
No full textType 2 diabetes (T2D) is an international health burden. Globally, it is the most common chronic disease and its incidence is increasing. Type 2 diabetes mellitus is a metabolic proinflammatory disorder characterised by chronic hyperglycaemia resulting in an altered immune response and delayed healing. Patients with T2D are common in general dental practice. Oral complications of T2D are well recognised, particularly associated with periodontal disease but the influence of hyperglycaemia on the dental pulp is unclear.
Inflammation and immune responses of the dental pulp are similar to those in other connective tissue in the body and are mediated by several cellular and molecular factors to minimise harmful effects induced by the irritating factors. Hyperglycaemia affects body tissues through a non-enzymatic process known as glycation and the accumulation of irreversible advanced glycation end products (AGEs). AGEs accumulation is responsible for diabetic complications such as thickened connective tissue and increased tissue inflammation. AGEs exert noxious effects on tissues through an advanced glycation end-product receptor (RAGE).
Normal tissue and a regulated inflammatory response contribute to healing. The histological, immunological and inflammatory changes in human clinically normal dental pulp in T2D patients are limited. Improved knowledge and understanding of these changes may assist clinicians in planning care to manage patients with T2D diagnosed with pulp disease.
Hypothesis & Objectives
The hypothesis of this thesis is that T2D affects the clinically normal dental pulp by altering histological, immunological, and inflammatory responses. To test this hypothesis, the current study has three main objectives. The first is to evaluate the histological, immunological and inflammatory changes in the clinically normal dental pulp of patients with T2D. The second objective is to evaluate the glycation process in the dental pulp by evaluating AGEs and their receptors RAGE, Galectin-3 (Gal-3), and the related inflammatory response. The third is to establish an in vitro diabetic model and evaluate the effect of high glucose concentration on human dental pulp cell (hDPCs) behaviour and gene expression.
Methods
Ethical approval for this study was gained from the University of Otago Human Ethics Committee (Health) (Project Reference H16/069 and Project Reference H18/077) and Māori consultation was entered with the Ngāi Tahu Research Committee. Clinically normal (healthy) and extracted permanent teeth were collected from T2D (n=20) and non-diabetic (n=20) participants for the first and second objectives.
To achieve the first and second objectives, histological staining, immunohistochemistry (IHC), immunofluorescence (IF), double immunofluorescence (DIF) and quantitative polymerase chain reaction (qPCR) were used. Following extraction, teeth from T2D (n=10) and non-diabetic (n=10) participants were cut transversely below the cementoenamel junction (CEJ), formalin fixed, decalcified in 10% ethylenediaminetetraacetic acid (EDTA), and paraffin embedded. Sections were stained with haematoxylin and eosin (H&E), Massons trichrome, Van Gieson (VG) and silver reticulin stains for histological evaluation. Other sections were used for IHC using anti-TLR2, anti-TLR4, anti- CD4, anti-CD68, anti-CD83, anti-FOXP3, anti-interleukin (IL)1!, anti-IL6, anti-tumour necrosis factor (TNF)-", anti- AGEs, and anti-RAGE. Three sections were used for IF using anti-AGEs and other five sections were used for DIF using anti-RAGE with anti-CD4 and anti-vimentin.
Remaining teeth from T2D (n=10) and non-diabetic (n=14) participants were used for gene expression analyses. Immediately after extraction teeth were sectioned transversely below the CEJ, and the coronal pulp was removed for ribonucleic acid (RNA) extraction. Messenger RNA (mRNA) levels for AGE, RAGE, S100A12 and NF-#B were determined using TaqMan assays.
For the cell culture experiments, coronal pulp tissue was excavated from mature unerupted third molar teeth extracted from healthy adults (n=4) and cell lines generated using the explant method. An in vitro diabetic model by using different glucose concentrations mimicking blood glucose concentrations was used to evaluate the effect of normal and high glucose on viability and gene expression of hDPCs at 1, 3 and 5-day time-points.
Data Analysis
Histological slides and IHC samples were scanned by an Aperio Scanscope CS2 image capture device, analysed under light microscopy and digitised with ImageScope. Data analysis was performed qualitatively and semi-quantitatively (number of positive cells/image area) to assess the histological changes and the protein expression in the specimens.
The DIF sections for RAGE, vimentin and CD4 were viewed and images were taken using an EVOS M5000 inverted fluorescent microscope. The slides were examined, and the individual and merged proteins in non-T2D and T2D dental pulp were qualitatively identified. The staining was examined, and proteins were qualitatively identified, and merged in non-T2D and T2D dental pulp.
The mRNA expression analysis was performed using comparative quantification cycle (Cq) values. The fold difference (FD) for each gene was calculated between the two groups. Data analyses were performed with GraphPad Prism®, using a Student’s t- tests at P-value <0.05.
The viability data analysis in cell culture was performed using GraphPad Prism and Excel software for Mac OS Catalina (Version 10.15.4). The data are presented as mean and standard deviation (SD). A Student’s t-test was used and the differences between the groups were considered significant when P-value <0.05.
The gene expression in cell culture was performed by using the comparative Cq method to calculate the difference in the mRNA expression between the 5.5mM and 25mM D-glucose concentrations at the different time-points.
Results
Histological changes were observed in normal dental pulp of participants with T2D compared to healthy controls. T2D resulted in a dental pulp that was less cellular, less vascular, evidence of thickened blood vessel walls, increased pulp calcification, increased collagen and decreased elastin deposition. The positive cell count/area showed increased expression of CD68 (P<0.001), CD83 (P=0.04), and decreased expression of FOXP3 (P=0.01) in T2D dental pulp compared with non-T2D samples. The positive cell count/area analysis also showed that the cytokines were significantly increased in T2D (IL1! (P=0.01), IL6 (P<0.0001), IL17 (P<0.0001) and TNF-" (P=0.01)). The glycation process was significantly increased in the dental pulp of T2D as evidenced by increased IHC expression of AGE (P<0.0001) and RAGE (P=0.02). The qPCR results showed that the expression of RAGE, S100A12, NF-#B and COL1A1 mRNA genes was significantly increased in the dental pulp of T2D compared with the non-T2D samples (P<0.0001), while no significant changes were detected for Gal-3 mRNA expression. The glycation process was also assessed by IF and DIF which showed increased accumulation of AGE in the extracellular matrix (ECM) and around blood vessel walls. DIF showed co-localisation of RAGE and vimentin and co-localisation of RAGE and CD4, indicating RAGE was expressed on fibroblasts and CD4+ve cells.
The results of the study using an in vitro diabetic cell culture model showed that high glucose concentration was associated with a reduced viability rate of hDPCs with increasing time. Furthermore, the used highest glucose concentration resulted in increased expression of collagen type III (COL3A1) and increased mineralisation due to the increased expression of ALP over time. The cell culture mRNA showed that high glucose resulted in increased gene expression of RAGE and decreased expression of Gal-3.
Conclusions
T2D leads to changes in dental pulp morphology, altered immunosurveillance and increased cytokine expression in the dental pulp. The glycation process was clearly evident in the pulp from T2D patients and may be responsible for the changes observed. These changes in clinically normal dental pulp in T2D patients may influence the healing response following pulpal injury and may affect the treatment plan and treatment outcome for these patients
An Exploration of Mental Health Promotion in New Zealand: A Qualitative Study
No full textMental health promotion employs a strengths-based approach and focuses on promoting positive mental health. The current level of knowledge and reporting about mental health promotion efforts occurring in New Zealand is limited. Much of the attention is focused on national initiatives, with little attention or understanding of other health promotion interventions that may be more community focused or smaller in scale. Given this dearth, and the growing rates of mental ill-health in New Zealand, it is important to develop a comprehensive understanding of mental health promotion action in New Zealand.
This thesis sought to explore what health promotion practitioners are currently doing to address mental health in New Zealand, in terms of both content and practice. Semistructured interviews were conducted with fifteen health promotion practitioners employed at various organisations in New Zealand. Participants were selected using a combination of maximum variation and snowball sampling methods. Interview transcripts were thematically analysed using both inductive and deductive data analysis methods.
Interviews with health promotion practitioners revealed a diverse range of mental health promotion action occurring within New Zealand. The majority of programmes/projects were focused downstream, at the individual level, on developing personal skills. Mental health promotion programmes/projects involving community action, the reorientation of health services, and/or building healthy public policy were reported far less frequently. The findings of this study also revealed that health promotion practitioners in New Zealand have a sound knowledge and understanding of mental health and best practice health promotion. However, interviews highlighted various factors, operating at the systems level, that restricted health promotion practitioners from being able to translate this knowledge into their practice. Health promotion practitioners reported operating within short-term and prescriptive contractual agreements, a tertiary-focused health care system, a fragmented field, and a limited workforce, which created significant challenges for their mental health promotion practice. Particular practices impacted included health promotion practitioners’ equity- and Treaty-based practice and their evaluation capacity.
Overall, the findings from this research offer important insights into mental health promotion action occurring in New Zealand. They highlight a number of opportunities for future mental health promotion action, practice and research. Most pertinently, they highlight the need for system level action to address the system level factors restricting health promotion practitioners’ practice