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Expanding the Footprint of Orogenic Gold –Trace Elements in Sulphides
No full textThe Macraes orogenic gold deposit in Central Otago, New Zealand was formed within the Otago Schist in the Early Cretaceous during long-lived terrane accretion and crustal thickening because of subduction along the Gondwana margin. This gold deposit is primarily composed of refractory, sulphide-hosted gold with a minor component of quartz vein-hosted free gold. The mineralising fluids are thought to have originated through metamorphic dehydration of rocks at depth at the greenschist to amphibolite facies transition and this fluid scavenged gold from the metamorphic pile. The gold-bearing fluid was subsequently focused, and gold and gold-bearing sulphides were deposited along various shears of the Hyde-Macraes Shear Zone (HMSZ).
The conventional method of gold exploration across Otago and elsewhere in the world is via the use of fire assay and/or portable x-ray fluorescence (pXRF) to search for anomalous pathfinder elements like arsenic which are detectable in soil and rock at much higher concentrations than gold. This method of exploration is highly effective at identifying and evaluating large regional-scale, mineralised structures such as the HMSZ.
This study examines the sulphides of the Macraes deposit directly to determine the concentration of trace elements (such as gold and arsenic) and establish their spatial dependency, zonation and inter-relationships. This project makes use of optical petrography, itrax, energy dispersive spectroscopy (SEM-EDS) and laser ablation-inductively coupled plasma-mass spectroscopy (LA-ICP-MS) to determine how the concentrations of trace elements behave with respect to each other and distance from a known ore zone in drill core.
With these data, we seek to allow exploration within a discontinuously mineralised structure to vector towards hydrothermal sulphides rich in gold rather than metamorphic sulphides and by extension, characterise distal and proximal hydrothermal sulphides in mesoscopic structures such as graphitic shears.
The itrax proved itself to have potential as an assaying tool since even at rapid count times and a relatively coarse sampling interval, arsenic counts were found to have a distribution that mimicked the results of traditional assay. With a longer count time and finer sampling interval, gold count distributions were useful for identifying potential grains of free gold in the core material that would easily have been missed by traditional core logging and assay. When compared to elements that may interfere with the gold count signal, such as tungsten and lead, it was found that gold could be somewhat reliably distinguished, this, when combined with the presence of free gold grains in the vicinity of high gold peaks means the itrax has the potential to reliably identify gold distributions in core.
Sulphide-gold (within the sulphide structure) has a strong positive correlation with arsenic in pyrite and weaker correlations with copper, silver, bismuth and tellurium, sulphide-gold was found to not have an obvious correlation with lead, nickel or zinc. Micronugget gold (discrete particulate gold within sulphides) appears to be more abundant in arsenic-poor regions of sulphides, suggesting that substitution of arsenic forsulphur in pyrite may promote incorporation of sulphide-gold and prevent micro-nuggets from forming due to changes in the surface charge conditions of the sulphides. When the concentrations of sulphide-gold, arsenic, copper, and zinc are plotted against depth, there appears to be a systematic increase in sulphide gold concentration in regions of whole rock enrichment and the ore zone can be defined with greater detail using the methodologies set out in this this work than with traditional whole-rock methodologies. In addition to enhanced ore zone definition, this methodology could aid in exploration since regions with rare but auriferous sulphides can be detected and pursued. There also appeared to be a reduction in base metal concentrations in the ore zone suggesting potential in locating regions of base metal dearthity in sulphide-focused exploration
Stimuli-driven heterocycle synthesis by quenching the (aza)quinone methide of self-immolative prodrugs
No full textA prodrug is an inactive compound that is converted into therapeutically active drug by enzymatic or chemical transformation. The research in this field is constantly aiming to develop new prodrugs to improve the physicochemical and pharmacological properties of the drug cargo being delivered. A self-immolative linker can play an important role in prodrug design as it acts as a spacer between trigger and payload, and enables rapid release of the active payload (drug) upon activation of the trigger. However, the by-products of the linker generated after activation are often neglected, and is particularly problematic for a commonly used self-immolative linker, p-aminobenzyloxycarbonyl (PABC) and p-(hydroxyl)benzyloxycarbonyl (PHBC). With these two linkers there is potential toxicity associated with the in situ formation of a quinone or azaquinone methide. It is the electrophilic nature of the methide that poses many potential toxicity problems in vivo, such as alkylation of DNA and proteins, and liver toxicity associated with depletion of antioxidants (e.g., glutathione).
The primary aim of this thesis was to develop a self-immolative linker that could capture the electrophilic azaquinone methide (by-product) before it can react with biological nucleophiles. The designed linkers incorporate a strategically placed nucleophile that can trap the electrophile as it is formed in situ, providing a detoxification mechanism (Chapters 2-5) or a method for in situ heterocycle synthesis (Chapter 6). To address the toxicity associated with azaquinone methide, model tripartite prodrugs consisting of a trigger, self-immolative linker (containing a built-in nucleophile) and a model drug or probe (leaving group) were developed.
In Chapter 2 a general synthetic approach to the new methide-trapping self-immolative linkers was developed. A multistep procedure was designed, facilitating the nucleophilic handle (an amine) to be introduced onto the self-immolative linker part of the prodrug in close proximity to the electrophilic methide, such that the methide would be quenched by the proximal nucleophile via an intramolecular cyclisation. The cyclisation step involved a 1,6-addition to the azaquinone methide to generate an N-benzyl-protected tetrahydroisoquinoline (THIQ) 209. The synthesis was designed with a cyclic anhydride 175 as the key intermediate, that could be used to introduce a diverse range of nucleophiles by a simple ring-opening reaction. The versatile design in the synthesis also allowed for alternate stimuli-responsive prodrug trigger groups to be installed, including a nitro group (Chapter 2), amide group (Chapter 3), azide group (Chapter 4) and boronic ester group (Chapter 6). Following the successful synthesis of the cyclic anhydride 175 in Chapter 2, a series of nitro-functionalised self-immolative linkers conjugated to the model drugs via a carbamate, carbonate or ether bond were synthesised (177-183). Using a HPLC assay developed during this project, each of the prodrugs were activated using Zn/AcOH as a reductive stimulus of the nitro group, producing THIQ 209 after the in situ trapping of the azaquinone methide. THIQ 209 was also synthesised and isolated by silica gel column chromatography (69% yield) and its structure confirmed using 2D NMR experiments. HPLC and LC-MS-monitored activation studies of two prodrugs (177 and 182) were conducted in the presence of a competing nucleophile, glutathione (GSH), and the amount of THIQ 209 generated was quantified and compared to the experiment in the absence of the GSH nucleophile. The results demonstrate that the secondary amine nucleophile appended to the linker is able to trap the methide in preference to a thiol nucleophile (GSH), providing proof-of-concept for a reactive methide prodrug detoxification strategy. A limitation of this experiment was that, for Zn/AcOH-mediated reduction, the reaction mixture was more acidic than that expected in a biological system, leading to reduced nucleophilicity of the methide trap (an amine) and the competing thiol (GSH). In an attempt to mimic in vivo reduction, activation of prodrugs 177 and 182 were attempted with nitroreductase enzyme, but preliminary experiments suggested that the enzyme used was inactive and did not reduce the nitro group.
In order to investigate the prodrug activation and nucleophilicity of the methide trap and the competing thiol under more neutral conditions (pH 7.4), amide-functionalised prodrugs 219-220 were synthesised and examined for enzymatic activation using penicillin G amidase (PGA) (Chapter 3). The prodrugs 219 and 220 were subjected to activation by enzymatic hydrolysis, and using a HPLC-monitored assay the amount of cargo released and THIQ 209 generated was measured in the absence and the presence of a highly nucleophilic thiol, N-acetyl cysteine (NAC). Without the thiol, the prodrugs displayed excellent 1,6-self-immolation, releasing the model drug 166 (⁓ 90% in 24 h) and generating THIQ 209 (⁓ 78% in 24 h). In the presence of the thiol, the amount of model drug released (⁓ 88% in 24 h) and THIQ 209 generated (⁓ 78% in 24 h) was relatively unaffected by the competing thiol, supporting the results from Chapter 2. To provide further confirmation for the in situ formation of 65 and to rule out the generation of any thiol or water (another potential competing nucleophile) adducts, LC-MS studies were executed. Only low levels of thiol and water adducts were identified by LC-MS (no detection by standard HPLC), demonstrating that the intramolecular cyclisation outcompetes the intermolecular reaction, even in the presence of 1 mM (2.5-fold excess) and 4 mM (10-fold excess) NAC.
In Chapter 4 the synthesis of azide-functionalised prodrugs 247, 248, and 249 carrying benzyl amine (184), 7-hydroxycoumarin (166) and ciprofloxacin ester 238 as model drug cargo was carried out. The azide-prodrugs were triggered (reduced) with NaSH, a hydrogen sulfide donor, to mimic overexpression of H2S, commonly associated with inflammatory conditions. A 5-fold and 20-fold excess of NAC was used as a competing nucleophile for the azaquinone methide, and the formation of THIQ 209 and release of model drug 166 were quantified by HPLC assay and further analysed by LC-MS. The prodrugs showed promising percentage release of the model drug coumarin 166 and the generation of THIQ 209 in the absence of competing nucleophile, forming THIQ 209 (⁓ 75% in 24 h), and releasing model drug 166 (⁓ 87% in 24 h). In the presence of nucleophile, the amount of THIQ 209 (⁓ 73% in 24 h) generated and release of model drug 166 (⁓ 84% in 24 h) showed that the intramolecular reaction is more favoured, even in the presence of strong competing nucleophile.
The prodrugs synthesised in Chapters 3 and 4 (221, 248, 249), drug cargo (166 and 238), and the THIQ 209 were investigated for cytotoxicity in vitro against an epithelial breast cancer cell line (4T1) and a normal cell line (Madin-Darby Canine kidney; MDCK). The concentrations tested ranged from 1.95-1000 µM (Chapter 5). The prodrug 248, released drug 166 and THIQ 209 did not display cytotoxicity towards the 4T1 cancer and MDCK normal cell lines. The prodrugs 221, 249 and ciprofloxacin ester 238 displayed moderate cytotoxicity (IC50 = 279 µM (221), IC50 = 274 µM (249), IC50 = 152 µM (238)), towards the cancer cell line, whereas prodrug 249 and ciprofloxacin ester 238 exhibited relatively high cytotoxicity against normal cell lines (IC50 = 158 µM at 48 h (249), IC50 = 62 µM at 48 h (238)).
In Chapter 6 two acyclic prodrugs 288 and 317 that could lead to the in situ synthesis of THIQ bioactives with anti-tubulin activity and P-gp pump inhibition were identified. For the proof-of-concept, the boronic-ester-functionalised prodrug 288 was designed that could potentially release one drug following oxidation-mediated activation and produce a THIQ 277 that acts as a P-gp efflux pump inhibitor (co-drug type delivery). However, after successfully completing nine of the sixteen proposed synthetic steps, selective bromination at the 4-position of aryl acid 231 was unsuccessful. An alternate synthetic approach in which synthesis of anti-tubulin inhibitor (THIQ) 318 could be achieved was designed, however, due to time constraints, the synthesis was not executed as part of the current thesis
Community Insurance Versus Compulsory Insurance: Competing Paradigms of No-Fault Accident Compensation in New Zealand
Get PDFThis article presents a history of New Zealand’s accident compensation scheme as a struggle between two competing normative paradigms that justify the core reform of the replacement of civil actions for victims of personal injury with a comprehensive no-fault scheme. Under ‘community insurance’, the scheme represents the community taking moral and practical responsibility for members who are injured in accidents, while for ‘compulsory insurance’ the scheme is a specific form of compulsory accident insurance. Understanding the history of the scheme in this way helps explain both the persistence of the scheme and important changes made to it by different governments
Synthetic studies towards spiroaspertrione A
No full textIsolated from the Aspergillus species TJ17 of fungi, spiroaspertrione A features a novel spiro[bicyclo[3.2.2]nonane-2,1′-cyclohexane] carbocyclic core and was shown to possess fascinating antibacterial activity, including the potentiation of oxacillin against methicillin-resistant Staphylococcus aureus. This thesis describes several strategies aimed towards the first total synthesis of the meroterpenoid
Muttonbirds and modernity in Murihiku : continuity and change in Kāi Tahu knowledge
No full textStories of culture contact and change in New Zealand are, in James Clifford's words, "structured by a pervasive dichotomy: absorption by the other or resistance to the other." Expressed another way, Paige Raibmon argues that colonial binaries of authenticity continue to structure our understandings of indigenous people. In this thesis, I use te hopu tītī ki Rakiura, the customary harvesting of tītī (Puffinus griseus/"muttonbirds") by southern Kai Tahu from islands adjacent to Stewart Island, and he mahi pōhā (the kelp-bags used to traditionally store preserved tītī) , as a way to illustrate both the introduction and limitations of these colonial categories.
Influenced by recent historiography from India and British Columbia, as well as the work of sociologist Shmuel Eisenstadt, I argue that muttonbirding is best understood not as a Kāi Tahu rejection of modernity, but as a key component of an alternative Kāi Tahu modernity. To help locate both continuity and change in Kāi Tahu understandings and uses of the natural world in Murihiku (the area of southern New Zealand south of the Waitaki River and east of the Waiau River) and Foveaux Strait, this dissertation draws from recent work in economic history and the history of science. In so doing, I am attentive to both episteme (propositional knowledge or "knowledge what") and techne (prescriptive knowledge or "knowledge how") associated with the tītī harvest. Being a muttonbirder myself, I am able to read archival references to muttonbirds and muttonbirding particularly closely. These include letters and published works by Rev. J. F. H. Wohlers, the first foreign resident missionary in Foveaux Strait, who established a mission on Ruapuke Island in 1844 which he oversaw until his death in 1885. While the dissertation draws from a wide range of official records and print culture sources, my personal connections have allowed me to draw on private diaries and photographs of the harvest and pre-season preparations relating to pōhā. Bringing together this range of material allows me to offer a carefully localised reading of cultural development which makes frequent reference to people from whom I am directly descended. The dissertation as a whole emphasizes the centrality of whakapapa in structuring the Māori past.
I am of the opinion that my approach to Māori history, particularly the distinction I draw between episteme and techne, can be usefully deployed in analysing the response of other Kāi Tahu communities, and indeed those of other Māori, to the challenges and opportunities presented by colonisation from the mid nineteenth-century. Moreover, I believe that my dynamic interpretation of what constitutes Māori culture in the present-day ought to be considered by tribal groups directing efforts at cultural revitalisation
Identification of vesicles, their contents, and how they may contribute to oocyte quality and female fertility
No full textFor decades oocyte organelles have undergone considerable investigation as a way to understand their contribution to reproductive competence. However, despite their frequent observation in oocyte studies, little attention has been paid to an understanding of oocyte derived vesicles. It is understood that vesicle volume appears to decline throughout oocyte maturation which suggests the possible use, and importance of its contents during this process. While few have fortuitously identified the contents of oocyte vesicles, the lack of reproducible evidence and assertive conclusions prevents any formal certainty of both their role and what they contain. Based on these limited findings, the aim of the current study was to not only identify oocyte derived vesicles, but to uncover and recognise the composition of said vesicles in a centrifuged oocyte model in order to explore their attributes that may be further utilised to improve oocyte quality and thus, fertility.
Sheep oocytes sourced from the abattoir were either in vitro matured or maintained at germinal vesicle stage before centrifugation to segregate organelles by density into lipid, vesicle, mitochondria, and smooth endoplasmic reticulum layers. Transmission electron microscopy of fixed and sectioned oocytes was performed for an analysis of organelle segregation, and to confirm the location of the organelle layers. Potential vesicle contents were investigated via confocal microscopy where the application of LysoTracker Red, Fluo-4-AM and Nile Red stains were used to detect for acidic organelles, calcium stores and lipids respectively. Stains observed through confocal microscopy were correlated to their respective organelle layer established through transmission electron microscopy.
Mature oocytes were significantly more able to layer their organelles, following centrifugation, in comparison to their immature counterparts (p<0.0001). Furthermore, the ability of organelles to layer appeared to be unaffected by either ewe age, or whether oocytes were surrounded by a layer of supporting cumulus cells. Fluorescent imaging of oocytes upon the application of LysoTracker Red or Fluo-4-AM appeared to show no evident staining in relation to vesicles, moreover, visibly highlighting lysosomes and mitochondria respectively. Comparably, Nile Red was expectantly observed to emit fluorescence within the lipid droplets themselves, however, a weaker yet notable emission was additionally apparent within that of the vesicle layer. Bright spot analysis identified two dissimilar lipid profiles between these organelles.
The findings of the current study bear no evidence that vesicles are acidic storage sites, nor do the results support the potential of oocyte derived vesicles to possess calcium. The ability of Nile Red to identify chromatically distinct lipid profiles between the vesicle and lipid droplet regions suggests that oocyte derived vesicles are likely to harbour an additional lipid source that is utilised during the process of oocyte maturation, and hence may contribute to the overall developmental competence of the oocyte
A gaping hole: Oral health care for dependent older people
Get PDFFollowing decades of improved oral health care, many people in New Zealand now reach older age with their own teeth. However, dependent New Zealanders (those who require regular care support through home-based care or aged residential care facilities) have poor oral health, suggesting that they are not receiving adequate oral health care. This affects their overall health and quality of life in ways that may be painful, debilitating or fatal. These problems are exacerbated by shortfalls in oral health services, which are frequently unaffordable and/or inaccessible.
This thesis explores the feasibility and acceptability of publicly-funded oral health care provision for dependent older people in New Zealand among key informants from the oral health and aged care sectors. The New Zealand oral health and aged care systems are compared and contrasted with those from Canada, Australia, the United Kingdom and Ireland. These comparable countries face similar issues in cost and access barriers for dependent older people, and have various approaches that could be applied in the New Zealand setting. The research was framed by Guay’s (2005) three-point model of addressing demand, expanded by Smith (2010) to include awareness. Semi-structured interviews were conducted with eight key informants, comprising experts in dentistry, aged care, health policy and older people’s advocacy.
This research confirmed that dependent older New Zealanders’ oral health is in a state of crisis. Oral health policy has not been sufficiently developed or implemented to guide oral health care for older people. Preventive oral health care is insufficient for purpose, while cost and access barriers frequently prohibit clinical treatment. The oral health and aged care workforces both require improved conditions, regulation, training and interdisciplinary alliances to meet dependent older people’s oral health needs. While the workforce is interested in making these changes, centralised public funding is necessary for their implementation, and to reduce cost barriers for patients. Accessibility can be helped through a combination of in-house, mobile and domiciliary care provision, and more widespread training in special care dentistry. Any oral health care programme must foreground the human rights of older people, disabled people, and other socioeconomic minorities such as Māori and Pasifika people.
This thesis concludes that publicly-funded oral health care for dependent older people is not only acceptable but urgently necessary, and highly feasible if effective political strategies are developed for its implementation. It therefore recommends a call to action by oral health and aged care professionals, older people’s advocates, disability rights organisations, and others concerned about dependent older people’s health and wellbeing
The role of cytoskeletal F-actin in homomeric and heteromeric ∆N-TRPV1 and TRPV4 mechanotransduction.
No full textHypertension is one of the leading health concerns facing our population today, however the mechanisms underlying it are not fully understood. This highlights the need to investigate other potential regulators of blood pressure. One regulator of blood pressure is the magnocellular neurosecretory cells (MNC) in the hypothalamus which control the release of vasopressin (AVP) to increase blood pressure. MNCs are intrinsically osmosensitive, so can be activated by changes in osmolality which cause cells to shrink (hyperosmolality) or to swell (hypoosmolality). The intrinsic osmosensitivity of MNCs are controlled by transient receptor potential vanilloid (TRPV) channels. The present study focused on TRPV4, which is active during hypoosmolality, and ∆N-TRPV1, which is active during hyperosmolality. Although TRPV4 and ∆N-TRPV1 have both shown the potential to form heteromers, there is limited research regarding this and whether this could contribute to the mechanical activation of TRPV channels. Therefore, the aim of the present study was to determine the activation of homomeric and heteromeric ∆N-TRPV1, TRPV4 during mechanical stress and to identify whether changes in the F-actin cytoskeleton affect their mechanical activation.
Experiments were conducted on Human Embryonic Kidney cells (HEK293) transfected with ∆N-TRPV1, TRPV4 or both ∆N-TRPV1 and TRPV4 and incubated for 10-20 hours. The activity of ∆N-TRPV1, TRPV4 and putative ∆N-TRPV1/TRPV4 was evaluated using single-channel patch-clamp in a cell attached configuration. The channels were investigated with and without pressure using the parameters of current-amplitude, conductance, open probability (NPo) and area under the curve (AUC). Pressure was applied through the recording pipette in the form of either positive (30 cmH2O) or negative (-30 cmH2O) pressure via a water column. In order to investigate the effect of the F-actin cytoskeleton on the channels Cytochalasin D (CytD) was applied to the cells for 40 minutes prior to recordings. Channels were then subjected to single-channel patch clamp and assessed using the same parameters as the untreated channels.
Evidence regarding the potential of ∆N-TRPV1 and TRPV4 subunits to combine to form a heteromer was indicated by the localisation of ∆N-TRPV1 and TRPV4 DNA in HEK293 cells. This evidence was further supported by the different current-amplitudes and conductance at 0 cmH2O. This indicated likely evidence of ∆N-TRPV1 and TRPV4 subunits being able to combine to form a functional channel in a HEK293 cell. ∆N-TRPV1 and TRPV4 were both shown to be mechanically activated by cell shrinkage, whereas putative ∆N-TRPV1/TRPV4 showed variable changes to mechanical activity during cell shrinkage. Neither ∆N-TRPV1, TRPV4 or ∆N-TRPV1/TRPV4 had any significant evidence to indicate mechanoactivation to cell swelling. F-actin depolymerisation was shown to reduce the current-amplitude and conductance of both homomeric and heteromeric ∆N-TRPV1 and TRPV4. Putative ∆N-TRPV1/TRPV4 was shown to have an increase in mechanosensitivity to positive pressure but the mechanosensitivity of ∆N-TRPV1 and TRPV4 was decreased when F-actin was depolymerised.
While these findings suggest that ∆N-TRPV1 and TRPV4 can form a mechanically sensitive channel in HEK293 cells, whether this channel is present in MNCs remains unknown. Furthermore, as the polymerisation of F-actin was shown to impact the mechanosensitivity of ∆N-TRPV1/TRPV4, putative ∆N-TRPV1/TRPV4 has the potential to play a role in AVP release during hypertension
Eleventh Annual Report of the Perinatal and Maternal Mortality Review Committee
Get PDFThis report considers perinatal and maternal mortality and morbidity from 1 January to 31 December 2015; perinatal mortality from 2007 to 2015; maternal mortality from 2006 to 2015; and babies with neonatal encephalopathy from 2010 to 2015.
In this report the PMMRC have also introduced data and discussion related to Maori. We need to focus our lens on outcomes for Māori mothers and infants, as the inequity between Maori and non-Māori continues.
The outlying causes of stillbirth and neonatal death among babies of Māori mothers are spontaneous preterm birth, antepartum haemorrhage, maternal conditions (mostly diabetes-related), and hypertension.
There is a significantly higher, almost double, maternal mortality ratio among Māori mothers than New Zealand European mothers. Tragically, Māori women are over-represented among maternal suicides. The main contributory factors amongst these deaths continue to be barriers to access and/or engagement with care, which the PMMRC will be working with the sector to improve.
Also new to the PMMRC report is the work of the Maternal Morbidity Working Group (MMWG). In May 2016 we welcomed this working group dedicated to reducing maternal morbidity to the PMMRC. The MMWG transitioned to the Commission from the existing Severe Acute Maternal Morbidity (SAMM) research group based at the University of Otago. Supported and funded by the Ministry of Health, the group will be active through to June 2019. The MMWG is responsible for nationally reviewing incidences of women who are pregnant or have recently delivered who are also very ill, and developing quality improvement initiatives alongside the maternal health services. This group is supporting the work of the PMMRC to improve the quality and experience of maternity care for women, babies and wha¯nau, informed by robust, consistent, reportable and women-centred maternal morbidity review.
The perinatal related mortality rate in 2015 is the lowest reported since the PMMRC began collecting data in 2007 and is significantly lower than the rate for the years 2007–2014 combined. We are pleased to report a statistically significant reduction in fetal deaths (stillbirths and late terminations of pregnancy combined) from 2007 to 2015, and an ongoing statistically significant reduction in stillbirths. The neonatal death rate has not changed significantly in New Zealand from 2007 to 2015, and note that the PMMRC have indicated that this will be a key are of investigation for 2017–2018. There have been significant reductions in neonatal mortality in the United Kingdom (UK), Australia and Scandinavia, and we will look to learn from those experiences
The role of Mirna in response to irradiation in a rectal cancer cell line model
Get PDFNeoadjuvant radiotherapy/chemoradiotherapy (N-RT/CRT) is frequently used to treat rectal cancer due to its ability to reduce tumour bulk prior to surgery. It has been shown to significantly reduce the recurrence of cancer and improve the anal structure integrity post-surgery. However, radiotherapy is often associated with severe adverse effects, drastically reducing the quality of life. Additionally, some patients are subjected to these adverse effects with no positive outcomes as 30% have no response to N-RT/CRT. Therefore, it is essential to understand why tumours respond differently to a treatment. One way to explore this is to look at the molecular characteristics of cancers such as miRNAs involved in regulating gene expression and response to irradiation exposure. This study investigates the expression of miRNAs in response to irradiation exposure in a rectal cancer cell model.
This study used the rectal cancer cell line SW837 to research miRNA as biomarkers of radiosensitivity. Usually, colorectal cancer research is conducted with the faster, more stable colon cancer cell lines. However, we believed researching a rectal cancer cell line would be more representative of rectal cancer because rectal cancers are genetically distinct from colon cancers. The characterisation of irradiation effects on cell viability was important to investigate as our lab had never worked on radiating cells before. SW837 is also relatively underrepresented in research. Therefore, optimisation of all assays was conducted to lay the foundation for future research. After irradiation characterisation, we studied the expression differences in a pre-selected miRNA panel of interest when exposed to different irradiation doses in our SW837 cells.
When characterising the cell viability, death, and apoptosis of SW837 cells exposed to different irradiation doses, a longer incubation post-irradiation incubation time produced the greatest distinction between doses. This was concluded that irradiation-induced cell responses occur the greatest after a full cell cycle has occurred. Therefore, an incubation time of at least 72-hours was ideal as it was found that SW837 cells had a doubling time of 90-hours. Western blotting was used to detect γ-H2AX, a sign of irradiation damage. However, it failed to be detected using western blotting, highlighting the importance of correct protein loading.
miRNA expression changes did not result in any statistically relevant data due to a lack of repeats. However, interesting changes were observed in two of the miRNA. miRNA-185-5p showed a decreased expression with increased irradiation dosing, while miRNA-23a-3p showed the opposite. Overall, though these results were not statistically backed, interesting changes to expression have started to appear, indicating areas to research. Furthermore, this study has optimised many assays to further research in our lab regarding irradiation exposure in cell lines