Otago University Research Archive
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Mesenchymal stem cells homing to tumour sites: Cancer biology and therapy
No full textMesenchymal stem cells (MSCs) are bone marrow derived, non-hematopoietic stem cells, which home to sites of injury and inflammation. They have also been identified at tumour sites where they can have pro- or anti-tumourigenic effects. It is unclear which specific signalling molecules and proteases secreted by the tumour cells are responsible for mobilising MSCs to the tumour sites. However, as MSCs migrate to tumour sites, they could be utilised for cancer therapy, as engineered MSCs would be able to precisely deliver gene or protein treatment to tumour sites.
Here, the role of a protease, cathepsin D, in the homing of MSCs to tumour sites is described through in vitro studies. MDA-MB-231 and HT29 cells or their conditioned media were co-cultured in Boyden chambers with MSCs with or without protease inhibitors to analyse the migration and invasion of the MSCs. The migration and invasion of MSCs was induced by the cancer cells and their 3 day conditioned media. Since increased levels of proteases play a major role in regulation of the tumours, we focussed on identifying proteases which attract MSCs to the tumour cells. Migration was decreased with protease inhibitors pepstatin A and GM6001 in both the cell lines. Pepstatin A is a potent inhibitor of cathepsin D. Furthermore, cathepsin D expression was strongly detected in tumour cells when compared to the MSCs. To confirm the effects of cathepsin D, its expression was inhibited by siRNA which also resulted in a decrease in the migration and invasion of MSCs. The signalling pathway MAPK/ERK was found to be activated in MDA-MB-231 and HT29 cells and addition of pepstatin A reversed this activation to a significant level.
Wild-type FOXL2 and tissue inhibitor metalloproteinases (TIMPs) proteins were trialled to target cancer through engineered MSCs. To target adult type ovarian tumour granulosa (KGN cells), which is characterised by a single missense point mutation of the FOXL2 gene, the wild-type FOXL2 gene was overexpressed in KGN cells and MSCs using a tdTomato expressing lentiviral vector. Cisplatin and etoposide, the first line treatment drugs, were tested on KGN and MSC cells with and without wild-type FOXL2 expressing genes. The cancer cells co-culture incubated with MSCs expressing wild-type FOXL2 showed IC50 values of almost half when compared to cells with the mutated gene. Cell cycle distribution, DNA fragmentation and mitochondrial cell death evaluation showed significant G2/M arrest, DNA cleavage and mitochondrial permeabilisation in wild type expressing KGN cells when compared to mutant gene expressing KGN control cells. Angiogenesis inhibition by MSCs, demonstrated using HUVECs in tube formation assays, was also increased by co-culture with MSCs expressing the wild-type FOXL2 gene, compared to MSC-only controls.
TIMPs are the endoproteinases that inhibit matrix metalloproteinases (MMPs), which are partly responsible for tumour metastasis. TIMP genes were successfully cloned into a tdTomato-expressing vector and overexpressed in MSCs by lentiviral infection.
These findings provide the first evidence that cathepsin D is an important protease that plays a role in attracting MSCs to tumours. We demonstrated that cancer cells secreted cathepsin D which increases migration and invasion of MSCs and that the ERK signalling pathway may be involved in this MSC migration by cancer cells. In addition, we showed that wild-type FOXL2 gene expressing KGN cells were more susceptible to the drugs cisplatin and etoposide than the cells carrying only the mutated form of the gene, suggesting that it may be clinically useful to deliver the protein using MSCs as part of the treatment regime. However more in vitro and in vivo studies are required to confirm and optimise these results. We have therefore further explored the homing of MSCs to tumour sites and taken important early steps in developing targeted therapies for cancers that could be delivered by MSCs
The Therapeutic Potential of New Zealand's Coast: The role of personal experiences and education in shifting individual perceptions of therapeutic landscapes
No full textOver the past decade the therapeutic landscape framework has seen a number of critiques regarding its catch all approach to healing environments and the lack of discussion surrounding landscapes of harm. Geographers have highlighted that the framework generally assumes that when examining therapeutic landscapes research has typically focussed on the health-promotion elements, neglecting to discuss how the same environment acts in a health-limiting manner. Gaining a more in-depth understanding of how individuals interact with ‘therapeutic’ spaces, and the interactions they seek within them, will provide a better knowledge of how individuals perceive landscapes and individually place them on a continuum ranging from therapeutic to disruptive.
This research seeks to respond to these critiques of the framework and the gaps in the relevant literature through exploring the ways in which an individual’s personal experiences, education, and interactions with the physical environment at the coast shape the way in which they perceive, and therefore experience and interact with the coastal landscape. The research was carried out under a poststructuralist framework, utilising three research methods. These were focus group interviews, a solicited diary activity, and a final round of semi-structured interviews. Several key areas of interest emerged through this research. Primarily, the significant impact that personal experiences and place attachment play in the initial creation of therapeutic landscapes. It was found that for a landscape to be perceived as therapeutic by any individual a history of positive experiences and interactions were required to develop an initial sense of attachment. Second, education acted to supplement, or in limited cases work in place of, personal experiences in order to shape the coast as a therapeutic space. Finally, elements of the coast that were typically regarded as intrinsically health-enhancing, including the ocean and shoreline, were instead divisive elements in perceptions of the coast as a result of unique personal experiences.
Through exploring the impact of each of personal experiences, education, and the physical environment I will develop and argue the significance of two new concepts. The first, coastal yellow spaces, builds upon recent critiques focussing on geographers limited palettic discussions of space and seeks to recognise and acknowledge the unique health-enhancing potential of a broader range of landscapes. The second, the therapeutic continuum spanning the connection between therapeutic and disruptive landscapes, is developed in response to the assumption that therapeutic spaces are similarly therapeutic for all. Within this continuum I recognise landscapes as holding a degree of therapeutic potential, with an individual’s unique experiences and education shifting their perception of the landscape and determining its placement along the continuum. Both developments are suggested as tools with which geographers may better explore and understand the therapeutic and health-enhancing value of a range of landscapes
Structural insights into oncolytic Seneca Valley Virus capsid and receptor interactions
No full textOncolytic viruses (OVs) are replication competent agents that selectively target cancer cells. Seneca Valley Virus (SVV) is a newly-discovered oncolytic picornavirus which is classified as the sole member in the genus Senecavirus. SVV strain 001 has currently completed Phase I and Phase II clinical trials in pediatric solid tumors and small-cell lung cancer, respectively. Similar to other picornaviruses, SVV forms naturally occurring empty capsids (without genome), known as procapsids, which share the same antigenicity as full virions. Understanding the formation and structure of SVV procapsids could give insights into how to exploit them as virus-like particles (VLPs) for targeted in vivo drug delivery for cancer treatment. Recently, we identified the Anthrax Toxin Receptor 1 (ANTXR1), a membrane protein overexpressed in ~60% of types of cancer, as the high-affinity cellular recept or for SVV in cancer cells. However, the high-resolution information on SVV-ANTXR1 interaction sites remained poorly characterized, thereby hampering the potential to develop SVV mutant in future oncovirotherapy.
In this study, we purified SVV full capsids, procapsids and used cryo-electron microscopy (cryo-EM) to investigate the capsid architectures and receptor interactions. Our results show that both SVV full capsids and procapsids have a similar external structure, while on the interior the main differences were the missing genomic RNA in the procapsid and a disordered VP1 region. We also show that a cage of RNA serves to stabilize the inside surface of the full capsid, thereby making it more acid stable. In an attempt to improve SVV particle stability, we isolated a thermostable mutant of SVV by thermal selection assays. Cryo-EM analysis of SVV-56mt structure showed conformational shifts at the interpentameric surface that facilitated the formation of additional hydrogen bonds and aromatic interactions, which could serve as capsid stabilizing factors. In SVV-ANTXR1 complex, ANTXR1 decorates the outer surface of the SVV capsid and interacts with the surface exposed BC loop and loo p II of VP1, “the puff” of VP2 and “the knob” of VP3. Comparison of the receptor-bound capsid structure with the native capsid structure reveals that receptor binding induces minor conformational changes in SVV capsid structure, suggesting the role of ANTXR1 as an attachment receptor. Our results demonstrate that the capsid footprint on the receptor is not conserved in anthrax toxin receptor 2 (ANTXR2), thereby providing a molecular mechanism for explaining the exquisite selectivity of SVV for ANTXR1.
Findings from this study lay the foundation for the modification of the SVV procapsid to develop it for targeted in vivo delivery of therapeutics and to develop potent SVV mutants with specific cancer tropism
Young people (re)conceptualising digital citizenship: Constructing ways of being and doing citizen(ship) 'online'
No full textThis thesis explores how meaningful the concept of digital citizenship is to young people in Aotearoa New Zealand. In an increasingly digitally-mediated society, the way young people learn what it means to be a citizen online, and the behaviours consistent with belonging and connecting to digitally-mediated communities, are increasingly important. Digital citizenship, however, is an evolving concept. Digital citizenship arises when the inherent complexity of the notion of ‘citizenship’ intersects with the interrelational spaces offered by digital technologies and as a result makes possible new ways of being a citizen and doing citizen(ship) practices. In education, definitions of digital citizenship construct an ‘ideal’ digital citizen by outlining desired behaviours, dispositions, and skills, which normalise particular ways of being and doing. How meaningful idealised concepts are to young people, and whether definitions align with young people’s understanding of what it means to be a digitally-mediated citizen, has not been fully examined in New Zealand.
To explore how meaningful the concept of digital citizenship is to young people, this thesis operates at a theoretical junction, drawing upon multiple historical conceptualisations of citizenship (see for example, Heater, 2004; Mutch, 2005), understandings of discourses (Foucault, 1972), notions of space and place (Massey, 2005), and Bourdieu’s theory of practice (Bourdieu & Wacquant, 1992), specifically notions of capital and habitus. Taking a qualitative approach, I conducted focus groups and individual interviews with 28 young people, aged between 16 and 25, from diverse backgrounds. The resulting data were analysed using an iterative, inductive approach to explore young people’s meaning-making and ways of being and doing digital citizenship. These findings are presented in four parts that focus upon the way young people defined, shaped, located, and practised their citizenship and digital citizenship.
The findings show that digital citizenship is indeed, “many things to many people” (Vivienne, McCosker, & Johns, 2016, p. 15). While ‘digital citizenship’ was a new term for participants, they drew upon their understandings of citizenship to define digital citizenship as habitus (or ways of being) that, along with digital capital, is embodied through digitally-mediated practices. They located their digital citizen habitus through their sense of belonging and connectedness to places and spaces, and they embodied their digital citizen habitus through practices that reflected their lived realities. For these young people, digital citizenship was a fluid and nuanced process of digitally-mediated, participatory citizenship practices informed by everyday lived experiences. I argue that, if ‘digital citizenship’ is to be meaningful for young people, there is a need for educators to recognise young people as experts on their lived realities, to encourage reflection upon taken-for-granted digital practices and spaces, and to highlight the relational aspects of citizenship practices online and offline. While the young people in this study offered definitions of digital citizenship, creating a meaningful and shared concept requires a youth-centric approach that recognises everyday citizenship practices and empowers young people to co-construct ways of being and doing citizen(ship) in digitally-mediated spaces
Impacts of Research
No full textHigh quality research that changes the world is what we strive for at the University of Otago Division of Health Sciences. It’s also what governments around the world are asking for. They want greater accountability from researchers who must articulate how research investment benefits communities and
populations. Those benefits include health, societal, cultural, economic or environmental factors at the individual, whānau and community level. This booklet describes eight research impact case studies. They come from across the seven Schools in the Division of Health Sciences and highlight different stages of their impact journey. Each has been selected for making a meaningful contribution in a different field of research. These researchers are improving people’s health, changing local and central government policies, contributing to best practice guidelines, creating spin-off companies to commercialise research and building capability by training the next generation of scientists. By forming meaningful relationships with stakeholders, creating strong networks with other researchers and being advocates for policy change they are achieving success. We hope you enjoy discovering how their impact was achieved
Cellular bioassays as a clinical tool for monitoring sex steroids
No full textSex steroid production needs to be maintained within the physiological set point range, deviations outside of the normal range can be a symptom of- and risk factor for endocrine-related diseases (such as polycystic ovarian syndrome (PCOS), hypogonadism, breast- and prostate cancer). Testosterone is the dominant male sex steroid and estradiol (E2) is the most abundant and potent female sex steroid. Testosterone and E2 are mainly synthesised in gonads of both sexes. Testosterone and E2 induce a cellular response by diffusing into cells and binding to their respective nuclear receptor (androgen- (AR) and estrogen receptor (ER)), which upon activation, operate as transcription factors and regulators of gene transcription. It is clinically important to have an accurate measure of detection for testosterone and E2. The current clinical measures, which are immunoassay-based techniques, are under scrutiny due to their poor sensitivity and reproducibility, especially at low concentrations. Furthermore, although testosterone and E2 are the most prominent endogenously synthesised sex steroids, they are not the only molecules capable of activating their intracellular cognate receptor. Other endogenously synthesised androgenic and estrogenic precursor molecules to testosterone and E2 can also activate AR and ER, respectively. Furthermore, studies have shown that exogenous (anti-) estrogens and androgens sourced from synthetic and plant origin have potential to activate or inhibit AR and ERs.
A novel technique to identify all estrogenic and androgenic molecules within a sample is reporter gene bioassays (RGBs). RGBs are a cell-based technique that can measure the level of ER or AR activation by a test substance. Cells can be transfected with an expression plasmid to over express AR or ER/ and a reporter gene plasmid that harbours the response element specific to the receptor, which is upstream of a reporter gene. Alternatively, a cell line with endogenous AR or ER/ is transfected with a reporter gene plasmid only. Upon activation of AR or ER/, the receptor binds to its response element on the reporter plasmid to initiate gene transcription of the reporter gene, which is translated into an enzyme, such as luciferase. The reporter gene is measured following addition of a substrate to quantify the level of light emission, which is proportional to the level of receptor activation. Via this mechanism, all molecules that are capable of activating the receptor can be monitored.
The overarching aim of this study was to determine if RGBs can be used to measure clinically relevant samples. ER- and AR-RGBs hosted in T47D and HEK293 cells, respectively, were optimised to measure serum and plasma samples. The net serum estrogenicity and androgenicity measurement was compared with the data of the clinical standard immunoassay to determine if additional information could be provided. A major part of this PhD was the establishment of a clinical study, whereby serum from post-menopausal females was obtained for the purpose of measuring in the ER- and AR-RGBs. All patients recruited to the study started letrozole treatment and the aim of this clinical study was to determine if changes in ER- and AR bioactivity could be measured after the onset of treatment. Letrozole is an aromatase inhibitor that blocks the conversion of androgens into estrogens. Therefore, both ER- and AR-RGBs were utilised in a clinical setting to determine their clinical efficacy by comparing to the results of testosterone and E2 immunoassay data.
Overall, both ER- and AR-RGBs provided a readout for all serum and plasma samples tested, whereas only some samples had a detectable level of testosterone and/or E2 by immunoassay. This study shows that information to companion quantitative measures of E2 and testosterone can be derived from ER- and AR-RGBs
The immunomodulatory protein, GIF, encoded by orf virus
No full textInflammatory diseases cause considerable morbidity throughout the world. Examples of inflammatory diseases include rheumatoid arthritis, Crohn’s disease, atherosclerosis and asthma. Current treatments options are insufficient, and novel treatment options must be explored. Immunomodulatory proteins observed in nature could be repurposed as biotherapeutics, as a form of biomimicry.
Large double-strand DNA viruses encode a number of virulence proteins which are capable of dampening host immune response. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin-2 (IL-2) inhibition factor (GIF) is one such protein encoded by orf virus. Orf virus is a zoonotic virus which infects mostly sheep and goats. GIF is able to bind ovine and bovine GM-CSF and IL-2, but not human variants despite high sequence homology of both cytokines. The expression of virulence proteins including GIF allows orf virus to infect and re-infect individual animals and herds repeatedly.
This work reports on the establishment of an expression and purification protocol for the GIF protein, an examination of the structural and binding behaviour for GIF, as well as the exploration of a potential mechanism for GIF interacting with and inhibiting human GM-CSF.
Work consisted of establishing an expression and purification protocol of GIF in enough quantity for downstream experiments. Expression trials optimised the amount of DNA used in HEK 293-6E cell transfection, best day of harvest and the addition of supplements to the expression mixture. The purification protocol involved a three-step fast-performance liquid chromatography purification with an additional dialysis step to remove heat-shock protein 70 (HSP70) bound to GIF.
Binding and modelling studies of GIF were carried out to help explain its biological properties. Structural binding experiments confirmed that GIF is subject to N-linked glycosylation which is required for secretion from the cell. Enzyme-linked immunosorbent assays (ELISAs) confirmed that GIF did not bind human GM-CSF or IL-2, but was able to bind bovine GM-CSF. A cell proliferation assay with GM-CSF-dependent TF-1 cells revealed that GIF treatment reduced proliferation of cells treated with human GM-CSF compared to the no-GIF control, indicating that GIF may be able to have some form of interaction with human GM-CSF. We studied the interface of the GIF-ovine GM-CSF complex and observed the differences in residues between human and ovine GM-CSF. It was concluded that GIF might be able to form a transitory interaction with human GM-CSF, however this interaction is likely to be weak due to steric hinderance across the binding interface.
Further examination of the structural basis and binding behaviour of GIF is suggested for it to be repurposed as a biotherapeutic
Electric vehicle uptake in Dunedin 2018
No full textAnalysis of the Ministry of Transport’s vehicle fleet statistics shows that Dunedin has the high-est penetration of electric vehicles (EVs) of New Zealand’s main cities as at the end of 2018.
Dunedin has the largest proportion of pure electric vehicles per 1000 people (3.73), with its closest rival being Wellington (3.19).
When both pure electric (e.g. Nissan Leafs) and plug-in hybrid electric vehicles (e.g. Mitsubishi Outlanders) are counted, Dunedin still just comes out top (4.34), marginally ahead of Wellington (4.33) which has a greater proportion of PHEVs.
Dunedin also has the highest proportion of electric vehicles registered for personal use (as opposed to commercial or other), followed by Wellington and Christchurch. Dunedin’s personal registrations are mostly pure electric vehicles, at a far higher proportion per 1000 people (3.29) than all other cities (2.3 or less)
Isoreticulation of an adamantane-based lithium framework
No full textIsoreticulation is the “tweaking” of the structure of an already established metal-organic framework (MOF) to alter certain properties, while still maintaining the original parent topology. This work investigated the isoreticulation of the already established Li-MOF with the intention of enhancing gas uptake. The literature suggests that isoreticulation through the incorporation of Lewis basic sites (Chapter 2) and linear ligand extension (Chapter 3) are useful ways to enhance the uptake of gas.
A general overview of MOFs, i.e. what they are and specific design strategies is provided in Chapter 1. This is then followed by examples of isoreticulation, including the addition of Lewis basic sites and linear ligand extension. To this end, the versatility of the adamantane core is showcased, but the absence of literature methods describing the direct attachment of functionalised N-heterocycles is made known.
The utilisation of N-oxide chemistry and photochemistry towards the direct addition of N-heterocycles onto the adamantane core is discussed in Chapter 2. Both areas of investigation utilised a di-substituted adamantane precursor, 1,3-dibromoadamantane (1,3-DBA) or 1,3-carboxyadamantane (1,3-DCA), to facilitate the addition of two N-heterocycles. The N-oxide chemistry focused on the coupling of 1,3-DBA with various pyridine-N-oxide moieties. Many reaction conditions were tested, with each providing access to mono-substituted adamantane derivatives. The photochemistry explored light irradiation of 1,3-DCA with the N-heterocycles methyl nicotinate, methyl isonicotinate, 2-acetylpyridine and 5-methyl-2,2’-bipyridine carboxylate. The chemistry discussed provided access to both mono- and di-substituted adamantane derivatives. In both cases, access to the mono-substituted adamantane core proceeded with the retention of the unreacted precursor functional group, i.e. a bromine atom or carboxylic acid of 1,3-DBA or 1,3-DCA, respectively. Three robust compounds were synthesised using the photochemistry established: 1,3-bis(3-carboxypyridine)adamantane (L1), 1-carboxy-3-(3’-carboxypyridine)adamantane (L2) and 1-carboxy-3-(4-carboxypyridine)adamantane (L3).
Linear extension of 1,3-bis(carboxyphenyl)adamantane to the corresponding 1,3-bis(4-carboxyphenyl-4’-phenyl)adamantane (L4) is described in Chapter 3. Various synthetic routes towards the construction of L4 are discussed, all of which were successful.
Complexation of L1 and L4 with various metal salts is detailed in Chapter 4. When reacted with LiOH·H2O, both L1 and L4 provided isoreticulations of the previously established framework Li-MOF, referred to as IRLi-MOF and IRLi-MOF-L4, respectively. The complexation of L1 with Cu(BF4)2·H2O and Zn(OTf)2 provided a 2D3D interpenetrated framework (Cu-L1) and an infinite 1D zig-zag chain (Zn-L1), respectively
Regional differences driving organic matter and trace metal signatures reflected in temperate reef bivalve communities on the South Island, New Zealand.
No full textIncreases in anthropogenic activity along coastal zones has led to an influx of terrestrial particulate matter containing high levels of nutrients and contaminants to coastal ecosystems. In order to manage these detrimental impacts, it is imperative that we understand the factors that drive the spatial variation in pollutants at both regional and local scales. Bivalves are key marine players in the processing of organic matter providing a link between benthic, pelagic, and terrestrial habitats. To this effect, bivalves are recognized as sentinel organisms in evaluating marine ecosystem function as well as detecting pollutants associated with land-based inputs. Therefore, changes to the basal organic matter source pools supporting these species will result in inherent changes in organic matter sources supporting species further up the food chain. Comparisons between Marlborough Sounds, Tasman Bay, and Fiordland on New Zealand’s South Island, provide a unique study system containing gradients in anthropogenic impacts influencing the uptake of organic matter and accumulation of pollutants in bivalves.
The present study aimed to identify the influence of anthropogenic stressors in driving organic matter flux and trace element concentrations within bivalve communities in Marlborough Sounds, Tasman Bay, and Fiordland. We evaluated direct interactions between catchment modifications and salmon farming on feeding strategies, organic matter source pools, and trace element signatures of five individual bivalve species.
We established that feeding strategies reveal interspecific differences among sites, while intraspecific differences are reflective of small-scale differences in organic matter resource pools. Further, organic matter source pools shaped by catchment modification and salmon farms revealed the observed differences in proportions of macroalgae supporting primary consumers. Lower proportions of organic matter were correlated with a high degree of catchment modification. Salmon farms influenced nutrient delivery to the surrounding benthos to all subtidal species. However, the intertidal (Mytilus galloprovincialis) was uninfluenced.
Spatial difference in trace element concentrations tracked through basal organic matter source pools revealed the influence of catchment modification on the transmission of trace elements into bivalves. Further, aquaculture systems influenced the use of alternate basal organic matter sources (macroalgae versus phytoplankton) which were coincident with trace metal concentrations reflected in sediment-dwelling species.
Lastly, as mercury concentrations have not been well resolved for commercially important bivalves in South Island fisheries, using current mercury analysis methodology, we reported baseline mercury concentrations. Further, we illustrated the influence of catchment modification from forestry on spatial variability in mercury concentrations.
The contrast of the relatively developed catchments surrounding Marlborough Sounds and Tasman Bay with Fiordland’s relatively pristine forested catchment provides an important test of the links between anthropogenic activity and the contaminant loads reflected in coastal bivalves. Monitoring these coastal bivalves provided essential insight into the movement of particulate organic matter throughout food webs and their implications for ecosystem health