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The Divergent Trafficking Pathways of the Ca2+ activated K+ channels KCa2.3 and KCa3.1
No full textThe small- and intermediate-conductance Ca2+-activated K+ channels KCa2.3 and KCa3.1 regulate a plethora of physiological processes: including water and electrolyte transport in polarised epithelia, neuronal firing, and vascular tone. Critical for KCa2.3 and KCa3.1 function is the regulation of the number of channels at the plasma membrane; however, the mechanisms that regulate the trafficking of KCa2.3 and KCa3.1 to and from the plasma membrane are still poorly understood. There are several marked differences in the trafficking pathways of these two channels, for example, KCa2.3 has previously been established to recycle back to the plasma membrane after endocytosis; however, there is conflicting evidence in the literature if KCa3.1 recycles back to the plasma membrane.
This research project has focused on the regulation of KCa2.3 and KCa3.1 by two multiprotein complexes: exocyst and retromer. Exocyst is an eight-protein complex that tethers post-secretory vesicles to the plasma membrane. Additionally, exocyst has previously been established to regulate the trafficking of several proteins to the basolateral membrane of polarised epithelia. whereas the retromer complex is associated with the endosomal compartment and been demonstrated to regulate the retrieval and recycling of many membrane-bound proteins back to the plasma membrane, including ion channels. By using a combination of cell surface biotinylation, immunoblotting, and Ussing chamber electrophysiology, this project aimed to determine if exocyst and retromer regulated the trafficking of KCa2.3 and KCa3.1.
Knockdown of the exocyst subunit Sec3 significantly reduced KCa3.1-specific current (n = 5, P < 0.01) and significantly reduced the basolateral membrane population of KCa3.1 in FRT epithelia (n = 4, P < 0.05). These data suggest that the exocyst complex is required for the delivery of KCa3.1 to the basolateral membrane of polarised epithelia.
Stabilisation of the retromer complex with the pharmacological chaperone R55 increased the KCa2.3 population at the cell surface (n = 3, P < 0.05). Additionally, siRNA-induced knockdown of the retromer subunit VPS35 or the retromer-associated protein SNX3 decreased KCa2.3 levels at the cell surface (n = 3 each, P < 0.05). These data suggest the retromer regulates recycling of KCa2.3 back to the plasma membrane. Furthermore, cell surface levels of a mutated KCa2.3 channel, with a short deletion in the N-terminal domain, was not affected by R55. Surprisingly, stabilising retromer decreased the basolateral population of KCa3.1 (n = 4, P < 0.01) and KCa3.1 specific current; suggesting, that retromer does not regulate the trafficking or recycling of KCa3.1. Cumulatively, these data suggest, for the first time, that retromer is involved in the recycling of KCa2.3, but not the genetically related KCa3.1 channel
COGNITIVE FUNCTIONING AND SOCIAL UNDERSTANDING IN YOUNG AND OLDER ADULTHOOD
No full textPast research sees consistent age-related declines across many measures of cognitive
functioning and social understanding. As such, the present study aims to investigate the
age-related links between these measures. I examined this by giving young and older
adults a series of tasks relating to cognitive functioning (statistical learning, working
memory, processing speed, and fluid IQ) and social understanding (emotion recognition
and theory of mind [ToM]). Additionally, I gave them nine social psychology
questionnaires, and a Cyberball task measuring participants’ understanding of when
they were being ostracised. These measures were included to find potential links
between cognitive, social understanding, and prejudice measures. Five hypotheses were
established: 1. Young would outperform old on measures of cognition and social
understanding, 2. Statistical learning might underlie emotion recognition in older adults,
3. Working memory underlies younger adults statistical learning, 4. Older adults will
have higher RWA due to age-related declines in social understanding performance. 5.
Better cognitive ability in older adults will result in better performance on the Cyberball
task. Younger adults were better on all cognitive and social understanding measures,
except for statistical learning. There were correlations between cognitive functions and
a decline in social understanding. After controlling for cognitive decline there was still
an age-related decline in social understanding. For both young and older adults, there
were associations between RWA and ToM, whereby higher RWA scores correlated
with poorer ToM ability. Finally, older adults’ ability to judge the number of passes
they had received in the Cyberball task was correlated with a better working memory
and less sensitivity to rejection. Findings from this study reveal interesting links
between cognition and social understanding that future research should examine further
Evaluation of volatile flavour compounds in blue cheeses by linear and non-linear chemometric approaches
No full textUnderstanding which volatile compounds discriminate between blue cheeses is important to maximise product quality, facilitate innovation, and determine authenticity. However, identification of discriminant volatiles between blue cheeses has been hindered by methodological limitations and the presence of non-linear volatile behaviours during blue cheese ripening. To address these challenges, an end-to-end analytical workflow was optimized and a new non-linear data analysis approach using self-organizing maps and entropy-based feature selection (SOM-EFS) was adapted from the field of machine learning. To validate the SOM-EFS methodology, its predictive performance was compared throughout the thesis with an established linear chemometric approach. Three untargeted volatile fingerprinting experiments, each using solid phase microextraction, gas chromatography, and mass spectrometry, were conducted to identify discriminant volatiles i) between blue cheese varieties; ii) as a function of ripening time in two varieties of blue cheese; and iii) between cheeses produced using different Penicillium roqueforti strains, curd compositions, and ripening times/temperatures. The non-linear SOM-EFS technique effectively discriminated between samples and yielded better predictive performance than the linear models. However, there were interpretive synergies by utilizing the two approaches in tandem. Across all experiments, it was determined that alcohols especially, but also esters, ketones, and sometimes even hydrocarbons, served as effective discriminant compounds in blue cheeses. As a result of the first experiment, 1-nonene and 2,6-dimethylpyridine were reported for the first time in blue cheeses. The second time-series experiment confirmed, and effectively modelled for the first time, the non-linear volatile behaviours in blue cheeses during ripening. The second study also identified two distinct patterns of methyl ketone and secondary alcohol generation and interconversion during blue cheese ripening. The third experiment demonstrated that differentiation of blue cheese volatile fingerprints is driven mostly by selection of the Penicillium roqueforti strain, followed by ripening time, then curd composition, while temperature was found to have little effect on the development of volatiles. This thesis has furthered the understanding of which volatiles differentiate blue cheeses both by variety and as a function of Penicillium roqueforti strain, curd compositions, ripening temperatures, and ripening times. It has also contributed a new non-linear data analysis tool, SOM-EFS, that can be applied more generally in the fields of food and flavour chemistry
Biomaterial-Based Delivery of a Growth Factor and Cytokine Combination to Address Wound Healing Complications
Get PDFWound healing is a highly complex process that requires coordination of growth factors, cytokines, and cell types, in order to progress from an acute wound to a resolved scar. Healing complications, such as chronic wounds and scarring disorders, comprise a substantial health burden, and are linked to dysregulation of key growth factors and cytokines across multiple phases of healing. Standard care practices for these disorders are non-specific and often ineffective, and biologic treatments attempting to restore growth factor or cytokine balance to the wound have largely failed in the clinic, despite promising preclinical evidence.
This thesis aimed to identify why delivery of growth factors and cytokines for wound healing complications has thus failed, and to evaluate strategies for improving their delivery using biomaterials. The first of two comprehensive literature review summarised the fact that the large-scale clinical failure of growth factor and cytokine delivery to wounds is likely due to the additive effects of poor and variable trial design and reporting, the incompatibility of topical administration, and issues relating to regulation and manufacturing. Biomaterials are an emerging novel strategy for tailored phasic and multiphasic release of proteins to wounds and have been proposed to address some issues identified with topical delivery. A second comprehensive literature review concluded that a multifaceted approach using multiple proteins within a composite biomaterial system may potentially be more therapeutically efficacious than previous topical delivery of single proteins in the clinic. However, there is still a lack of evidence that biomaterials actually provide proteolytic protection and structural support within the wound, and accurate comparisons to prove that they have improved efficacy compared to topical delivery of growth factors and cytokines. Limitations in this area of research are centered around translatability, with inconsistent model choices and non- human relevant outcome measures. Careful consideration needs to be applied when selecting which growth factors, cytokines and biomaterials to use, and these choices need to relate to targeting of multiple phases of wound healing and appropriate release kinetics.
The second part of this thesis focused on development and testing of a biomaterial- based composite VEGF-E and vIL-10 delivery system to enable phasic regulation of wound inflammation, repair, and scarring. A biphasic release pattern was observed in vitro, with burst release of vIL-10 from the alginate hydrogel by day 1, and cumulative release of VEGF- E from lipid cubosome particles over 6 days. With only 52% release of VEGF-E and 2.3% release of vIL-10, more optimisation of protein loading is required. The lipid component of the system exhibited toxicity in human keratinocytes in vitro, but the hydrogel was well- tolerated when applied to murine skin wounds, with no significant adverse effects observed.
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Retention of hydrogels to the wound surface and cellular integration within the hydrogel were poor, so modifications are required to improve adherence and cell adhesion.
Overall, this study highlighted many challenges in the development of growth factors and cytokines for the treatment of wound healing and scarring indications. A clear need was identified for streamlining of the preclinical and clinical pathways leading to approval. A more comprehensive and methodological approach to the development of growth factor and cytokine delivery systems that target the phasic dysregulation of chronic wounds or excessive scars was recommended as a strategy to facilitate the approval of much needed therapies for human wound indications
The potential for environmental sustainability-driven dietary change to improve health: a systematic review with modelling analyses
No full textWhat we eat is important to our health and longevity. Unfortunately current diets are a major contributor to the burden of non-communicable disease, with interventions for individuals and the food environment required to improve dietary intakes. Interventions motivated by individual-health status however, may have unintended consequences such as feelings of shame and stigma, resulting in poor adherence. Because healthy dietary patterns are often of low environmental impact, there is potential to frame healthy diet messages around environmental sustainability. I have conducted a systematic review to identify what environmental sustainability-driven scalable initiatives aimed at improving dietary intakes have been undertaken previously. The results from this review identified the scarcity of such initiatives, providing justification for the use of simulation modelling to estimate the potential impact of population-wide environmental sustainability-driven dietary change on health outcomes, environmental measures, and costs to the individual.
One of the primary contributors to increased greenhouse gas emissions and land use is food production, however the environmental burden varies considerably between food groups. The largest contributor to greenhouse gas emissions within the diet is red meat production. Conversely, some of the lowest contributors to greenhouse gas emissions within the diet are those food groups often promoted in red meat replacement, such as legumes, soy, nuts and seeds. From a health perspective high red and processed meat intakes are associated with increased risk of premature mortality, heart disease, type 2 diabetes, and certain cancers, while legume, soy, nut, and seed intakes are protective.
To better understand the effects of red and processed meat replacement in Aotearoa New Zealand, I have modelled five scenarios and their expected effect on health and environmental outcomes, as well as grocery cost to the individual in a multistate lifetable model using Adult Nutrition Survey data. The scenarios were in line with Heart Foundation (1) or EATLancet (2) recommendations, or a replacement with minimally-processed plant-based alternatives (3), ultra-processed plant-based alternatives (4), or cellular meat (5). The change in food group intake for each scenario was calculated, with the nutrient adequacy of each scenario considered. Replacing red and processed meats, regardless of the scenario, would have beneficial consequences in terms of health and health system costs, reduced health inequities, and greenhouse gas emissions, with some scenarios reducing the expected grocery cost to the individual. Replacing red and processed meat intakes did not result in sub optimal intakes of key nutrients associated with red meat, such as protein, iron, zinc, or vitamin B12.
Findings extend and confirm current dietary advice to reduce red and processed meat intake. For the greatest health and environment benefits, as well as greatest cost saving to the individual of the scenarios modelled, red and processed meat replacement should first and foremost be with minimally-processed plant-based foods such as legumes. Replacement with ultra-processed plant-based alternatives was costlier to the individual and increased the sodium content of the diet. Future research should consider if environmental sustainability-driven, rather than health-driven, interventions prompt dietary change, and how food environments may promote behaviour change to encourage minimally-processed plant-based eating patterns
Indigenous Peoples and Employment Law: the Australasian model
Get PDFThis paper examines the relationship between indigenous values and employment law in New Zealand and Australia, with some comparative reference to the position in North America and in relation to international standards.
Since the 1980s, indigenous values have emerged as a dynamic in the first world workplace, particularly in indigenous enterprises, and enterprises and organisations with a strong indigenous element or connection. One manifestation of this is the recent proliferation of indigenous and aboriginal chambers of commerce and business associations in Australasia and North America. The reasons behind the emergence of this dynamic are political, social and, particularly in North America, economic.
On the whole, trade unions have not been the driver of this development, but have responded to it by incorporating it, usually into their own social justice agendas, but also by taking the opportunity to acquire new members. In North America, the advent of the lucrative casino gaming industry onto tribal lands since the 1980s spawned a sudden interest in union organising on reservations, as well as Indigenous resistance to those efforts. In New Zealand and Australia, there has been increased recognition of Indigenous culture in employment agreements and the general law, which largely stems from the social, political and increasing economic influence of Indigenous consciousness
Image processing for MARS spectral CT
Get PDFThis thesis reports on the developments made to give improvements in the MARS image reconstruction chain. The work described in this thesis helps all MARS users to produce high-quality material images that can offer an improved diagnosis in the medical imaging field. The thesis describes two main areas of research. Firstly, an existing algorithm was implemented in C++ to calculate the number of photons for each pixel position across the detector used in the MARS system. Secondly, speed and usability improvements were made to the MARS material decomposition (MD) software.
The thesis describes the coding aspect of the x-ray source model behaviour of the MARS scanner according to a parameterised semi-analytic source model developed using a series of Monte Carlo simulations. Photon counts are calculated at each pixel using these parameters and implemented the source model in a form that is compatible with the existing image reconstruction software. However, some additional changes will need to be applied to the current reconstruction algorithm to support the high energy resolution provided by the model. This development will help to accurately provide the energy and position of incident photon counts, which is required for future polychromatic material reconstruction algorithms. The developed source model is now being used in simulation software that can produce a MARS-like data-set from an ideal set of material volumes.
The thesis also details the improvements made in the current MARS MD software package that is used by the current MARS scanner users as a primary material analysis tool. Other members of the team are investigating hybrid material reconstruction, which combines the MD and reconstruction into a two-step process. In this case, the speed of the MD software is crucial as it would be run per-iteration and our current reconstruction has 900 iterations. The former MD algorithm uses non-negative least square equations to find the approximate solutions. The algorithm used in this thesis eliminates the need for iterations in the non-negative least square equations via direct analytic methods. The work completed in the MARS MD software led to some improvement in the performance of the algorithm and also a reduction in the computation times. The results of the developed version were also compared with the former MD to confirm the efficiency of the software
The impact of unpredictability on the biomechanical variables of gait and mobility-based participation of children with cerebral palsy
No full textWalking is a primary human motor function which normally develops in the first 18 months of life. Children with cerebral palsy have impairments that interfere with their walking ability. The International Classification of Functioning, Disability and Health (ICF) defines participation as involvement in a life situation. Limitations in walking ability contribute to decreased levels of participation. Participatory environments are complex, unique to the participant, and often include unpredictable circumstances. For example, an unexpected change of direction eliminates the ability to make a pre-planned motor adjustment, requiring compensatory movement adaptation. Little is known about motor response to unpredictability, particularly in children. Measurement of response to unpredictability and the relationship to participation levels requires availability of relevant measurement tools. The overall aim of this thesis is to understand children’s responses to unpredictability while walking and how those responses may be related to mobility-based participation.
Methods:
This thesis involved four processes to better understand motor response to unpredictability and participation: 1) a literature review led to the development of a framework to conceptualize the impact of unpredictability while walking; 2) an empirical study to determine response to unpredictability in children who are typically developing; 3) a scoping review to survey the literature for Clinical Gait Assessment Tools (CGAT) that measure unpredictability and participation; and, 4) a survey of practicing physiotherapists to investigate how CGATs are being used.
A novel model, the Context of Walking (CoW), was developed to conceptualize the impact of unpredictability. Two methodological studies informed the main empirical study that investigated the impact of mid-gait unpredictability on children who are typically developing (n=13). The scoping review of nearly 500 articles identified CGATs used in research studies. An electronic survey of paediatric physiotherapists was conducted to understand clinicians’ priorities for usage of CGATs.
Results:
When exposed to unpredictable circumstances, children demonstrate alterations in gait biomechanics: decreased gait speed, decreased step length, increased double limb support time, increased stance time and increased turn velocity. Over 80 CGATs were identified by the scoping review; 72% measured the ICF domain activity and 12% measured participation. Utilization of CGATs in the research literature was centered on those that measure activity. The survey study of 101 clinicians found that only three of the thirteen most commonly used CGATs have a measure of participation, and all but one measured the activity domain. The study also found that many clinicians have awareness of the large number of available CGATs but have never used them.
Conclusions:
Unpredictability requires gait adaptations that are like those of people at risk for fall. It is possible that children with cerebral palsy may limit exposure to participatory environments to avoid gait requirements that they cannot efficiently or safely manage. Available CGATs are limited in their measure of participation and do not measure response to unpredictability. To address the problem of unpredictability, it is necessary to develop targeted intervention programs and appropriate measurement tools to determine if response to unpredictability improves with intervention, and whether such improvement results in a higher level of participation
Long-term cultural barriers to sustaining collective effort in vaccination against COVID-19
Get PDFThis study attempts to shed light on long-term cultural barriers to strengthening collective action in COVID-19 vaccination. I propose that rugged individualism, characterized by emphasis on self-reliance and strong antipathy to government intervention, is linked to a greater prevalence of resistance to inoculation against the novel coronavirus across American counties. The main hypothesis of this paper rests upon the premise that a culture of rugged individualism is conducive to the emergence and persistence of mistrust, political polarization and distrust in science, which undermine collective effort in vaccination. Using subnational data for the United States, I consistently find evidence that rugged individualism, captured by long-run exposure to the westward-moving frontier in American history, has a positive influence on the predicted share of the population that is hesitant towards voluntary vaccination against COVID-19. In addition, individualistic counties tend to suffer from under-vaccination, captured by lower rates of the population that has been fully inoculated against COVID-19. More broadly, the findings suggest that rugged individualism is an impediment to resolving collective action problems, notwithstanding the widely acknowledged positive impact of individualistic cultures on innovation and long-run economic performance