BIOMEDICAL SCIENCE RESEARCH SYMPOSIUM Wednesday 26 October 2016 - - PDF document

Wednesday 26 October 2016 10:00am to 2:45pm (Followed by prize giving and nibbles) Auckland Hospital, Level 12, Support Building, Rooms 599-12058 and 599-12080

BIOMEDICAL SCIENCE RESEARCH SYMPOSIUM

GENERAL INFORMATION The Biomedical Science Research Symposium will be held at the Auckland Hospital, Level 12, Support Building, Grafton, in rooms 599-12058 and 599-12080 on Wednesday 26th October from 10:00am to 3:00pm. All Honours students are expected to be present throughout the Symposium. There will be two parallel streams of presentations (see map for room locations). Each student will have 15 minutes to present a summary of his or her research and then 5 minutes for

• questions. Each session will have a chairperson and two official markers who will score each

presentation according to the three criteria shown below. The Board of Studies (Biomedical Science) is most grateful to Coherent Scientific and BD Biosciences for providing sponsorship for this event. Presentation Marking Criteria Content ....................................................................................................................................... /30 (Is the context of the work adequately defined) (Quality, quantity and level of information) Structure .................................................................................................................................... /30 (Does it develop as a clear, logical sequence of ideas and conclusions) (Is the audience left with a clear idea of the relevance of the work) Method and delivery ............................................................................................................. /40 (Oral and visual clarity and impact) (Pacing and audience engagement) (Did the presentation go significantly under or over time?) (Response to questions) Total Mark .................................................................................................................... /100 Note: Chairperson will warn presenter when 2 minutes and 1 minute remain. Running a little over time is OK but there will be a penalty if the presentation runs significantly over time. Room numbers for each of the venues and a schematic of their locations are given below.  Venue 1 - Room 599-12058  Venue 2 - Room 599-12080 Level 12 can be accessed via the main Auckland City Hospital entrance. Take the escalator to the Level 5 and turn right across the link from the new hospital to the old building. Take the lifts to the level 12. These lifts are located behind the Muffin Break.

VENUE LOCATION Auckland Hospital, Support Building, Level 12

Venue 2 12080 Venue 1 12058

Please use lifts nearer to 12080

MAP

LOCATION AT THE AUCKLAND CITY HOSPITAL

VENUE 1: 599-12058 Chairperson: Dr Scott Graham 10:00am Introduction Time Name Title Supervisor 10:10am Akshata Anchan Migration of melanoma cells across the Blood-Brain Barrier. Features that aid the extravasation

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melanoma cells into the brain Scott Graham Kate Angel Graeme Finlay Simon O’Carroll 10:30am Aquila Chua Determining the proliferative and lineage specific properties of CALM- AF10 minimal fusion induced leukemia by the colony forming cell assay Stefan Bohlander 10:50am Brady Cress Toward the inhibition of PI3Kγ membrane binding using small molecules Peter Shepherd Jack Flanagan 11:10am Hannah Darroch Exploring the functional heterogeneity between neutrophils generated during emergency granulopoiesis versus steady state Chris Hall 11:30am Kai-Cheng Deng Use of targeted Proteomics to predict activation of PR-104A in Human Leukaemias Yongchuan Gu Bill Wilson Lunch 12 noon (Room 599-12080) 1:00pm Vittoria Draghi Does rate

• f

rewarming after hypothermia affect white matter integrity after global cerebral ischaemia in the near-term fetal sheep? Laura Bennett Joanna Davidson 1:20pm Blaze Forbes Uptake-2 mediated dopamine transport in the nigrostriatal system: implications for L-DOPA therapy in Parkinson’s disease Janusz Lipski Peter Freestone 1:40pm Amy Gamage The roles of Placental Macrophages (Hofbauer cell) in placental vascular development Joanna James 2:00pm Andrew Jiang Modelling Parkinson’s disease in sheep Renee Hadley 2:20pm Il Hwan Lee Optimizing high throughput screening to discover small molecule inhibitors of a conserved sialic acid binding site in Staphylococcus aureus virulence factors Ries Langley Nibbles and Prize giving – 3:00pm (Room 599-12080)

VENUE 2: 599-12080 Chairperson: Dr Julie Lim 10:00am Introduction Time Name Title Supervisor 10:10am Jessica Liu Cardiac glycogen mishandling in high-fat diet-induced insulin resistance and obesity Kimberley Mellor 10:30am Courtney Lynch Investigating radio sensitivity determinants in head and neck cancer using CRISPR-Cas9 Francis Hunter Bill Wilson 10:50am Emma McCafferty Neuroanatomy of GABAA receptors in the normal and Huntington’s disease human cerebellum Henry Waldvogel Richard Faull 11:10am Julian Moxon The effectiveness of cannabinoid ligands in Melanoma and the reliability of commercial melanoma Cell Lines Michelle Glass Peter Shepherd 11:30am Olena Oryshchuk Investigation of the proliferative potential of the BCR/JAK2 fusion gene in primary murine bone marrow cells using an in vitro myeloid clonogenic assay Stefan Bohlander Lunch 12 noon (Room 599-12080) 1:00pm Thulani Palpagama Investigation of GABA signalling in an in vivo Alzheimer’s disease mouse model Andrea Kwakowsky Henry Waldvogel 1:20pm Louise Ramerz The functional characterisation of long non-coding RNA ZFAS1 in human breast cancer Marjan Askarian- Amiri 1:40pm Rebecca Woolley Development of ovine adenovirus as a cancer vaccine John Taylor 2:00pm Vithushilya Yoganandarajah Characterisation

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glutathione synthesis pathways in the rat cornea Julie Lim Nibbles and Prize giving – 3:00pm (Room 599-12080)

Venue 599-12058

Presented by: Akshata Anchan Supervisor: Dr Scott Graham and Co-supervisors: Dr Kate Angel, Dr Simon O’Carroll and Dr Graeme Finlay Title: Migration of melanoma cells across the Blood-Brain Barrier. Features that aid the extravasation of melanoma cells into the brain Abstract Background: Melanoma is an aggressive cancer with high propensity to metastasize to the brain. This however, requires disruption of the structural integrity of the blood-brain barrier (BBB), thought to occur through interactions

• f adhesion molecules between the brain endothelial cells and melanoma cells.

Literature surrounding this process using human-cell models is limited, therefore increasing the necessity to investigate the features that may aid cancer metastasis. Methods: This study researched for evidence showing barrier disruption of the BBB after addition of human melanoma cells. Electrical Cell-substrate Impedance Sensing (ECIS) was used to measure this as a decrease in resistance across the tightly adhered brain endothelial cells (hCMVECs). New Zealand melanoma (NZM) cells were also assessed for cell-surface expression of adhesion molecules using flow cytometry while their secretory profiles were assessed using cytometric-bead array. Results: Each NZ Melanoma line induced barrier disruption of hCMVECs within the first few hours of addition and all had high expression of CD44, CD147, CD146 and others but were negative for CD31. Interestingly, preliminary data suggests pronounced difference in a range of secreted factors by the melanoma cells which could influence barrier interactions. Discussion: Understanding the processes by which melanoma cells enter the brain across the BBB has big implications for therapy against aggressive metastatic brain cancers, of which there are no current FDA approved drugs targeting this aspect (migration). As accessibility of drugs to these cells is higher whilst in the blood, discovering targets prior to migration into brain is extremely valuable.

Presented by: Aquila Chua Supervisor: Professor Stefan Bohlander Title: Determining the proliferative and lineage specific properties of CALM-AF10 minimal fusion induced leukaemia by the colony forming cell assay Abstract Background: The CALM/AF10 fusion gene is correlated with a poor prognosis in leukemia patients. CALM is involved in clathrin-mediated endocytosis, while AF10 is a putative transcription factor. CALM/AF10 contributes to leukomogenesis by dysregulation of AF10 target genes. The CALM/AF10 minimal fusion gene (C/A-MF) contains only the domains required for leukemogenesis. The proliferative potential and lineage specificity of (C/A-MF) induced leukemia cells are yet to be characterized. Methods: Bone marrow (BM) cells from healthy C57BJ/6 mice (Healthy Group, HG) and C57BJ/6 mice with secondary transplanted leukaemia initiated by C/A- MF (Leukemic Group, LG) were seeded onto 35mm cell culture dishes containing growth factor supplemented methylcellulose medium (colony forming cell assay, CFC). After a 10 day incubation period, the colonies were scored, and the cells were harvested and replated in fresh medium. This replating process was performed 3 times. Cytospin and modified Wright-Giemsa staining was performed on cells obtained at the end of each plating. Results: The average colony count (standardized for number of cells seeded) was much higher in the LG than the HG at the end of the 2nd and 3rd plating (p value <0.05). Cytospin analyses at the end of the 4th plating revealed clear morphologic differences between the HG and LG, with the HG having mostly mast cells and the LG showing predominantly blast-like cells. Discussion: These results show that there are clear differences in the proliferation and differentiation potential between C/A-MF driven leukaemia and normal BM cells in the CFC assay.

Presented by: Brady Cress Supervisors: Dr Jack Flanagan and Professor Peter Shepherd Title: Toward the inhibition of PI3Kγ membrane binding using small molecules Abstract Background: In response to activation of some GPCRs, PI3Kγ triggers an internal cell signalling pathway that increases cell growth and metabolism. Inhibitors of this enzyme have a therapeutic effect in some cancers and immune disorders. PI3K binding to the membrane is critical for activity and blocking this interaction might provide an alternative mechanism of inhibition. This project implements a screening cascade to identify compounds with these properties. Methods: Published inhibitors of coagulation factors V and VIII membrane binding were screened for their ability to inhibit PI3Kγ catalytic activity with the substrate in a liposome format. Inhibitors were then tested in the presence of detergent and compounds with reduced activity were eliminated. Reducing agents were used to identify inhibitors that might function by covalent modification of the protein. A biophysical assay that reports directly on the protein membrane interaction was used to characterise interesting inhibitors. Results: Compounds were discovered that inhibited PI3Kg activity, but most were sensitive to detergent. The most potent compound caused 81% inhibition of PI3Kγ but this was blocked by thiol reducing agents. Of the inhibitors tested in biophysical assays, none showed clear evidence of blocking membrane binding through a protein-only mechanism. Discussion: The only compounds thought to block protein membrane interactions were discovered for coagulation factors. Their structures indicate they are likely to act by non-specific mechanisms. This was confirmed by the effects of detergent and reducing agents. PI3Kg catalytic activity can be blocked by a thiol reactive compound, indicating the presence of a reactive Cys in the protein.

Presented by: Hannah Darroch Supervisor: Dr Chris Hall Title: Exploring the functional heterogeneity between neutrophils generated during emergency granulopoiesis versus steady state Abstract Background: Neutrophils are a critical component of the innate immune system. Emergency granulopoiesis (EG) is an infection responsive process that generates large numbers of neutrophils as required. Exploiting a zebrafish model of EG it was discovered that EG-generated neutrophils within a previously infected host have enhanced bactericidal activity compared to steady state. The enhanced bactericidal activity of the EG-generated neutrophils may be a cell-autonomous feature or the result of signals from the infected host. Objectives: We will assess whether EG-generated neutrophils maintain their elevated bactericidal activity when transplanted into a non-infected recipient. Methods: A transgenic zebrafish line with fluorescently labelled neutrophils (Tg(lyz:DsRED2)) will be infected with bacteria to induce EG. EG-generated neutrophils will be isolated via FACS from infected larvae, and steady state neutrophils isolated from control larvae. FACS-isolated neutrophils will be transplanted into age-matched non-infected recipients and the bactericidal activity of these neutrophils will be measured using live confocal imaging. Results: We have shown that transplanted neutrophils are viable and maintain their bactericidal activity within the recipient larvae, validating this approach. To

• ur knowledge, this represents the first time this has been attempted in this

model system. We will discuss efforts currently underway to extend this approach to investigate EG-generated neutrophils to enable comparison with steady state. Discussion: This research will contribute to a greater understanding into this newly discovered feature of neutrophils to further the argument that neutrophils are functionally heterogeneous and can alter their activities to better suit the needs of the host.

Presented by: Kai-Cheng Kieren Deng Supervisors: Dr Yongchuan Gu and Professor Bill Wilson Title: Use of targeted Proteomics to predict activation of PR-104A in Human Leukaemias Abstract Background:PR-104A is a prodrug, activated by AKR1C3 (Aldo-Keto Reductase family 1C member 3) and under hypoxia, by POR (Cytochrome P450 Oxidoreductase) to DNA crosslinking metabolites. Recent preclinical studies demonstrated beneficial responses with leukaemias highly expressing AKR1C3. The aim of this study is to test whether quantitation of PR-104A reductases with targeted proteomics can predict PR-104A activation. Methods:AKR1C3 activity was quantitated in 18 human leukaemia cell lines using the fluorogenic probe coumberone. Post-mitochondrial supernatants of all lines were prepared for targeted proteomics mass spectrometry. HCT116 cells

• verexpressing POR or AKR1C3, and a subset of the leukaemia lines with differing

AKR1C3 levels were exposed to PR-104A for 4 hr under aerobic/anoxic

• conditions. PR-104A-derived DNA adducts were quantified, and 24 hr later DNA

damage response (γH2Ax) was assessed by flow cytometry. Results: Leukaemic cell line AKR1C3 activity ranged widely from 20 to 4000 pmol/106 cells/hr. DNA adducts from the reduced metabolites of PR-104A and subsequent γH2Ax formation increased in response to high expression of POR under hypoxia or AKR1C3 under oxia. The targeted proteomics assay currently is under characterisation and further optimisation for precision. Discussion: Relationships between enzyme expression, DNA adducts and γH2Ax in a subset of 5 cell lines suggest that AKR1C3 and POR levels predict activation

• f PR-104A under oxic and hypoxic conditions respectively. The next step is to

test whether the targeted proteomics assay for AKR1C3 predicts coumberone metabolism in the leukaemia panel, which will validate this assay for clinical use in a forthcoming clinical trial in T-cell acute lymphoblastic leukaemia.

Presented by: Vittoria Draghi Supervisors: Dr Joanne Davidson and Professor Laura Bennet Title: Does rate of rewarming after hypothermia affect white matter integrity after global cerebral ischaemia in the near-term fetal sheep? Abstract Background: Hypothermia is the only treatment for infants suffering hypoxic- ischaemic encephalopathy (HIE) but is only partially effective. Limited evidence suggests that slower rewarming after hypothermia may further improve

• neuroprotection. In this study, we aimed to determine the effect of rate of

rewarming after hypothermia on neural inflammation, myelination and axonal integrity. Methods: Chronically instrumented fetal sheep (0.85 gestation) were randomised into either sham-ischaemia-normothermia (n=8), ischaemia- normothermia (n=8), 72 hours hypothermia (n=8), 48 hours hypothermia (n=8),

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48 hours hypothermia with 24 hours slow rewarming (n=7). Immunohistochemistry was used to assess microglia and astrocyte number, myelination density and axonal integrity in the white matter. Results: Cerebral ischaemia was associated with a significant increase in microglial and astrocyte number, loss of myelin basic protein (MBP) and CNPase expression, and decreased axonal density and directionality in the intragyral and periventricular white matter compared to sham control (P.<0.05). Hypothermia for 72h significantly reduced microglial and astrocyte number, increased MBP expression and improved axonal integrity (P<0.05). Microglial activation was greater and MBP expression was lower with 48h vs. 72h hypothermia (P<0.05). Slow rewarming further reduced microglial activation versus 48h hypothermia (P<0.05), and increased CNPase expression vs 72h hypothermia (P<0.05), but had no greater effect on MBP or axonal integrity. Discussion: This study suggests that 48 hours of hypothermia is suboptimal, being associated with greater white matter inflammation, demyelination and axonal injury compared to 72 hours of hypothermia. Slow rewarming was associated with further improvement in white matter inflammation and myelination but not axonal integrity.

Presented by: Blaze Forbes Supervisor: Professor Janusz Lipski Title: Uptake-2 mediated dopamine transport in the nigrostriatal system: implications for L-DOPA therapy in Parkinson’s disease Abstract Background: Loss of substantia nigra pars compacta (SNc) dopaminergic neurons is a hallmark of Parkinson’s disease. The gold-standard treatment for Parkinson’s disease is L-DOPA (levodopa), a dopamine precursor, compensating for dopamine deficits. However, the efficacy of L-DOPA therapy wanes over time and side-effects develop. Extracellular levels of dopamine produced by L-DOPA are regulated by uptake via the dopamine transporter (DAT, or uptake-1), metabolism, and diffusion. Recent studies indicate that uptake-2 (monoamine transport mediated by OCT-3 and PMAT) may also contribute. We examined the effects of pharmacological uptake- 2 blockers on in vitro extracellular DA levels before and after L-DOPA application. Methods: Brain slices taken from healthy rats contained either the SNc or the

• striatum. Extracellular dopamine levels were measured by fast scan controlled

adsorption cyclic voltammetry, an electrochemistry technique validated in our lab previously. Changes in dopamine levels were evoked by applications of either dopamine or L-DOPA to the solution perfusing the slice. Uptake-2 was inhibited with decynium-22 (OCT-3/PMAT inhibitor) or with lopinavir (PMAT inhibitor). To isolate uptake-2 mediated effects, DAT was inhibited with cocaine and dopamine metabolism with pargyline. In vivo experiments are underway examining the effect of D22 upon L-DOPA- induced elevations of dopamine in the striatum in anesthetised rats with nigrostriatal lesions. Results: No additional dopamine increases were seen when uptake-2 blockers were applied during exogenous dopamine application. No significant changes in the amplitude or decay of L-DOPA-induced increases were observed in the presence of uptake-2 blockers compared to in their absence. Discussion: In vitro data have thus far provided no evidence of uptake-2 mediated effects on nigrostriatal dopamine handling in healthy animals. However, in the Parkinsonian brain, loss of dopaminergic neurons leads to changes in dopamine handling. It remains to be seen whether in vivo experiments in lesioned animals reveal uptake-2-mediated effects.

Presented by: Amy Gamage Supervisor: Dr Joanna James Title: The roles of Placental Macrophages (Hofbauer cell) in placental vascular development Abstract Background: Development of an extensively branched placental vascular network is critical for pregnancy success, as poor vascular development has been linked to IUGR (intrauterine growth restriction) and long-term health implications for affected babies. Little is understood about the cellular interactions regulating placental vascular development. A population of placental macrophages (Hofbauer cells (HCs)) exist within placental villi from early in gestation. Macrophages in other organ systems demonstrate angiogenic roles, thus we hypothesise that HCs may play a role in early placental vessels formation. Objectives: To isolate and characterise primary HCs from 1st trimester placentae and determine how HCs may influence placental angiogenesis. Methods: HCs were isolated from villous tissue by trialling a series of protocols employing different enzymatic digest protocols and temperature conditions, in conjunction with anti-CD14 magnetic bead cell selection. Isolated cells were characterised by flow cytometry. Results: Following the trial of 3 different protocols, the optimised protocol chosen gave an average yield of 70,000±50,000 cell per gram of villous tissue (n=7). Isolated cells comprised of both adherent and non-adherent cells. Adherent HCs displayed morphological characteristics expected of tissue- resident, activated macrophages including adherence to plastic, and a granular/vacuolated phenotype, and spanned an average length of 154μm±104μm. Isolated cells co-expressed CD163 and HLA-DR, demonstrating a molecular macrophage phenotype, and of being the desired HC population. Discussion: The mix of adherent and non-adherent cells isolated suggests that HCs may exist in different activation states within 1st trimester placentae. The

• ptimisation of HC isolation protocols in this work will enable their further

molecular and functional characterisation.

Presented by: Andrew Jiang Supervisors: Professor Russell Snell and Dr Renee Handley Title: Modelling Parkinson’s disease in sheep Abstract Background: Parkinson’s disease (PD) is a progressive neurodegenerative disorder that causes severe defects in motor function and cognitive ability. The most common genetic variant associated with PD involves a GGC(Glycine)- >AGC(Serine) single amino acid substitution in the LRRK2 gene (LRRK2 G2019S) that is found in ~5% of familial autosomal dominant PD cases. Sheep as a viable large animal model has gained significant attention for its precise modelling of human neurodegenerative disorders. The aim of my research is to develop the protocols for CRISPR-Cas9 ovine genome editing and ultimately introduce the LRRK2 G2019S mutation into sheep fibroblasts in preparation for the development of an ovine PD model. Methods: To facilitate CRISPR-Cas9 site specific mutagenesis, co-transfection of guide RNA (gRNA), Cas9 protein and single stranded oligodeoxynucleotide (ssODN) donor construct containing the G2019S mutation was performed. Different titrations of each CRISPR reagent was tested in order to compare and

• ptimize editing efficiency. Editing efficiencies were determined through Illumina

MiSeq sequencing. Results: I have established preliminary evidence via a specific PCR assay that editing has occurred at some level. I am waiting for my MiSeq sequencing results, for which I’ll be able to determine the best combination of gRNA, Cas9 protein, ssODN donor template for specific genome editing. Discussion: I have identified the preferred gene and mutation to target for the development of a large animal model of PD. Preliminary evidence indicates that precise genome editing of the ovine LRRK2 gene has occurred. Taken together this is a good first step towards the construction of an ovine Parkinson’s model.

Presented by: Ilhwan Lee Supervisor: Dr Ries Langley Title: Optimizing high throughput screening to discover small molecule inhibitors of a conserved sialic acid binding site in Staphylococcus aureus virulence factors Abstract Background: Staphylococcus aureus is an important human pathogen responsible for a wide range of diseases with high resistance to antibiotics. This has promoted researchers to investigate novel treatment options such as targeting virulence factors. This approach aims to reduce the pathogenicity of the organism allowing the immune system to clear the infection. The virulence factors of interest are SSL11, SSL5, CHIPS, FLIPr with a highly conserved sialyl- lewis x binding site. SSL11 in particular binds to P-selectins and interfere with neutrophil migration to the site of infection. It is our hope that inhibitors of the sLeX binding site will promote improved immune response. Methods: Mass Spectrometry (MS) SSL11 was enzymatically biotinylated in order to immobilise on streptavidin coated wells. A pool of 80 compounds from ChemLibrary were incubated and assayed via triple-tof MS in positive ion mode. AlphaScreen SSL11 was conjugated to the acceptor and BSA-sialyl-lactosamine to the donor bead. The beads were incubated with compounds from ChemLibrary and measured using Enspire plate reader. Results: MS results showed 40 of 80 compounds were detectable with low

• errors. Most data correlated well to the competition assay performed previously

by Dr. Ries Langley. Alphascreen assay is still continuing to be performed at the moment of this abstract. Discussion: A significant challenge during the project was ATP concentration during biotinylation which could lead to precipitation of SSL11. Some compounds not detectable in positive ion mode of MS are most likely due to strong negative charges and will be helpful to run a negative ion mode in the future to explore this possibility.

Venue 599-12080

Presented by: Jessica Liu Supervisor: Dr Kimberley Mellor Title: Cardiac glycogen mishandling in high-fat diet-induced insulin resistance and obesity Abstract Background: Cardiovascular disease is the leading cause of mortality in diabetes. Diabetic hearts have elevated glycogen levels in contrast to other tissues, despite having impaired insulin signalling. Altered cardiac-specific glycogen handling mechanisms may be associated with diabetic diastolic dysfunction development, providing insight for future advancements in treatment. Aims: To investigate changes in cardiac glycogen content regulation in a high-fat diet-induced model of obesity and insulin resistance. Methods: Male Sprague Dawley rats fed either a control or a high-fat diet (HFD) for 15 weeks were monitored for body weight, food intake and blood glucose levels (BGLs). At 13 weeks of feeding, rats were subjected to a glucose tolerance test (GTT). Heart weights and tibia lengths were measured after 15 weeks. Glycogen assays were performed while RT-qPCR and western blots were used to assess key proteins implicated in glycogen metabolism. Results: After 15 weeks of feeding, HFD-fed rats had significantly higher body weights while BGLs and food intake were unchanged. GTT results showed glucose intolerance in HFD-fed rats and heart weights were significantly greater. Cardiac glycogen content was elevated despite decreased glycogen synthase protein levels and unchanged GABARAPL1 mRNA, a marker for autophagy-lysosomal glycogen breakdown. Discussion: These results show that HFD-feeding elicits obesity, glucose intolerance and cardiac hypertrophy. HFD-induced elevated cardiac glycogen levels were not linked to changes in enzyme-regulated synthesis or lysosomal

• breakdown. Further investigation of the cause and consequence of excess

glycogen in the diabetic heart is now warranted.

Presented by: Courtney Lynch Supervisors: Dr Francis Hunter and Professor Bill Wilson Title: Investigating radiosensitivity determinants in head and neck cancer using CRISPR-Cas9 Abstract Background: Radiotherapy is a major treatment modality of head and neck squamous cell carcinoma (HNSCC). Cell-intrinsic radiosensitivity, particularly due to gene expression, is thought to contribute to radiotherapy outcome. Discovering genetic determinants of radiosensitivity provides potential for molecular diagnostics and pharmacological radiosensitisation. Methods: Genome-scale CRISPR-Cas9 knockout (GeCKO) libraries were

• ptimised by lentiviral titrations, cryopreservation viability assays and ploidy
• analysis. Characterisation of 28 HNSCC cell lines was undertaken to select

informative models for GeCKO screens. GeCKO libraries were generated in the UT- SCC-74B and SiHa cell lines via lentiviral transduction. Radiosensitivity was assessed by clonogenic assays, cell counts and phase-contrast microscopy. Proof-

• f-principle screens with 6-thioguanine were performed with each library before

proceeding to daily fractionated ionising radiation screens. sgRNA cassettes from the surviving cultures were PCR-amplified for NGS. Analysis of relative sgRNA abundance used the MAGeCK, RIGER and HiTSelect packages. A parallel investigation will associate global RNAseq data with radiosensitivity using Pearson correlations and differential expression analysis. Results: Transduction of mixed-ploidy UT-SCC-74B selected for a diploid sub-

• population. Treatment of the GeCKO libraries with 6-TG selected for outgrowth of

resistant clones. Sequencing of the 6-TG resistant SiHa library showed enrichment of the genes HPRT1, NUDT5 and SLC43A3. Analysis of the UT-SCC-74B 6-TG-resistant library and differential expression studies is ongoing. Discussion: 6-TG treatment showed enrichment of genes previously implicated in resistance. Radiosensitivity screens with the UT-SCC-74B library are in

• progress. Future steps include radiosensitivity screens with another suitable cell

line from the characterised panel and increasing the statistical power of the RNAseq study.

Presented by: Emma McCafferty Supervisors: Associate Professor Henry Waldvogel and Distinguished Professor Richard Faull Title: Neuroanatomy of GABAA receptors in the normal and Huntington’s disease human cerebellum Abstract Background: The cerebellum is a region of the brain that is involved in fine motor control and higher brain functions. Recent studies have revealed that the cerebellum degenerates in Huntington’s disease (HD), which may contribute to the disease’s symptoms. Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the cerebellum, and mediates its effects through the GABAA receptor (GABAAR). The GABAAR is a pentameric complex assembled from a variety of 19 subunits, and different combinations of these subunits produce the physiological and pharmacological properties of the receptor. Methods: Free-floating immunoperoxidase and immunofluorescent techniques were used to investigate the localisation of different GABAAR subunits in three normal and three HD post-mortem human cases. Results: The findings from this study revealed a widespread and heterogenous distribution of GABAAR subunits in both the normal and HD human cerebellar

• cortex. The α1, β2/β3, and γ2 subunits were localised to Purkinje cell processes, cell

bodies and processes of interneurons in the molecular layer, and the glomeruli of the granular layer. The α2, α3, α5, and α6 subunits showed medium expression in the molecular layer and the glomeruli of the granular layer, with varying degrees of expression in Purkinje cells. No obvious qualitative differences were observed between normal and HD cases. Discussion: The localisation of GABAAR subunits in the human cerebellum agrees with some, but not all, findings seen in animal studies. This study forms the morphological and neuroanatomical basis of GABAAR receptor composition in the human cerebellum, and assists in our understanding of the neurochemical changes that may occur in this region in HD.

Presented by: Julian Moxon Supervisors: Associate Professor Michelle Glass and Professor Peter Shepherd Title: The effectiveness of cannabinoid ligands in Melanoma and the reliability of commercial melanoma Cell Lines Abstract Background: As rates of melanoma continue to rise in countries around the world, new avenues must be explored in order to develop more effective treatments that can target a greater range of melanoma subtypes. For some time, cannabinoids have been used in a palliative role in the management of cancer; however in recent years, they have also had their antiumour potential

• investigated. Cannabinoid receptors, particularly CB1, have shown to be

expressed in multiple cancer subtypes, including melanoma cells, where application of agonists has facilitated cell death. Methods: Agonist stimulation and PCR techniques were employed in order to investigate cellular viability, receptor signalling, and receptor expression in a number of primary and commercial melanoma cell lines. Results: 18 Primary human melanoma cell lines were treated with the cannabinoids THC (10uM), CBD (10uM) and CP 55,940 (1uM). Surprisingly, and in contrast with published literature, no meaningful level of cell death was

• detected. This prompted us to check for receptor expression by PCR and found

that none of the lines had detectable mRNA for either CB1 or CB2 receptors. As most previous work had been carried out on commercial lines, we then examined

• these. While some of these lines expressed receptor, activation with cannabinoid

ligands did not alter cell viability. Current studies are underway to determine if the receptor is functional through cell signalling assays. Discussion: This study began with the idea that we would first replicate existing results, before looking to expand on and characterise the antitumour properties

• f cannabinoids in melanoma. However, the results instead suggested that

cannabinoids do not mediate cell death in melanoma and that the commercially available lines are a poor representation of those cell lines obtained from tumour biopsies.

Presented by: Olena Oryshchuk Supervisor: Professor Stefan Bohlander Title: Investigation of the proliferative potential of the BCR/JAK2 fusion gene in primary murine bone marrow cells using an in vitro myeloid clonogenic assay Abstract Background: Bone marrow (BM) hematopoietic stem have the capacity to self- renew and differentiate into all lymphoid and myeloid lineages. This property is employed to study leukemogenesis. The colony forming cell (CFC) assay, providing a semisolid medium, allows investigation the proliferation and differentiation pattern of BM stem and progenitor cells. The purpose of the present project was to study whether the BCR/JAK2 fusion gene, found in various types of leukaemia, is able to alter a proliferative and differentiation potential of primary of primary murine BM cells. Methods: Murine BM cells were transduced with retroviruses carrying MIG- BCR/JAK2 or MIG vectors for the experimental and control arms of the CFC assay,

• respectively. In addition, untransduced BM cells were utilised as a mock control
• arm. All cells were sorted based on EGFP expression prior to the assays. Every 10

days the CFCs were evaluated, then replated and analysed in cytospin

• preparations. Western blot was used to determine the production of intact

BCR/JAK2 protein. Results: The integrity of the retroviral constructs was confirmed with restriction enzyme digestion and sequencing. The expression of the of the BCR/JAK2 was verified with western blot. Preliminary CFC assay results showed a transient expansion of BCR/JAK2- transduced BM cells (day 10 – day 20), however, these cells did not proliferate long term. Cytospin preparation showed enhanced differentiation into mast cells of the BCR/JAK2 transduced cells. Discussion: In contrast to the previously reported cytokine-independent proliferation of BCR/JAK2-transduced Ba/F3 cells, our results indicate that signals capable of inducing the growth in the latter system are not sufficient to induce long-term proliferation of the of BCR/JAK2-transduced mBM cells. Potential reasons of discrepancy as well as limitations of the study will be discussed.

Presented by: Thulani Palpagama Supervisor: Dr Andrea Kwakowsky and Associate Professor Henry Waldvogel Title: Investigation of GABA signalling in an in vivo Alzheimer’s disease mouse model Abstract: Background: Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter of the central nervous system. Changes of the GABAergic system have been identified in the progression of Alzheimer’s disease (AD). GABA is synthesized by glutamic acid decarboxylase (GAD65/67), packaged in vesicles through vesicular GABA transporter (VGAT), and acts on GABAA and GABAB

• receptors. GABA clearance relies on GABA transporters (GATs) and is metabolised

by GABA transaminase (ABAT). AD is characterized by accumulation of neurotoxic beta-amyloid (Aβ) and impaired cognitive function. Objectives: In this study, we have characterised molecular changes of GABA signalling components in the hippocampal CA1, CA3 and dentate gyrus (DG) regions of aged, male wild-type mice injected bilaterally with Aβ1-42 in the CA1 of the hippocampus. Methods: 3 days after stereotaxic Aβ1-42 administration, prior to cell loss the mice were either transcardially perfused with 4% paraformaldhyde and brains sectioned on a sliding freezing microtome for immunohistochemistry, or the CA1, CA3 and DG regions were dissected and snap-frozen for Western blotting. Results: We found no significant changes in the expression at protein level of the GABA signalling components, GAD65/67, VGAT, alpha1,2,3,5, beta1,2,3, gamma2 GABAA receptor subunits, GAT1, GAT3, BGT-1, and ABAT in the CA1, CA3 and DG region of the mouse hippocampus in mice administered with Aβ1-42 compared to naïve control and ACSF injected control mice. Discussion: These results suggest that the GABAergic system is relatively well preserved prior to Aβ1-42 -induced cell loss in this in vivo AD mouse model.

Presented by: Louise Nicole C. Ramirez Supervisor: Dr. Marjan Askarian-Amiri Title: The functional characterisation of long non-coding RNA ZFAS1 in human breast cancer Abstract Background: ZFAS1 is a long noncoding RNA (lncRNA) differentially expressed during mouse mammary gland development and in human breast cancer. ZFAS1 is a ribosome-bound lncRNA associated with monosomes and light polysomes. Further analysis indicated its association with the small subunit of the ribosome. 50% ZFAS1 knockdown in MDA-MB-468 human breast cancer cells by short hairpin RNA revealed its co-expression with pre-ribosomal RNA transcript (45S), and decreased phosphorylation of ribosomal protein S6, suggesting a role in ribosome biogenesis. Since ZFAS1 is highly expressed and extremely stable, a more efficient knockdown method to further elucidate its function is required. CRISPR/CAS9 technology manipulates genes at the genomic level, hence offering a solution to this challenge. Methods: We conducted CRISPR/CAS9 targeting of exons 2, 3, 4 and 5 of ZFAS1. Exon-specific CRISPR constructs were designed, cloned, and transfected to MDA- MB-468 cells. CRISPR/CAS9-mediated cleavage of ZFAS1 exons was verified, and ZFAS1 expression and function in the CRISPR/CAS9-edited cells was examined. Results: We have confirmed the cleavage of three out of four targeted exons and shown that single cleavage by CRISPR/CAS9 in ZFAS1 did not lead to any significant changes in its expression in MDA-MB-468. We have demonstrated that ribosome biogenesis in these cells is similar to control cells, suggesting point editing of ZFAS1 has not affected its function. Discussion: Our experiments have demonstrated the feasibility of CRISPR/CAS9 targeting of ZFAS1. However, true functional ZFAS1 knockdown may only be achieved with a dual CRISPR gRNA strategy simultaneously targeting the 5' and 3' ends of ZFAS1.

Presented by: Rebecca Woolley Supervisor: Dr John Taylor Title: Development of ovine adenovirus as a cancer vaccine Abstract Background: Ovine adenovirus (OAdV) is a promising vector for cancer immunotherapy due to its ability to stimulate antigen-specific immune responses in humans, who lack neutralising immunity to the virus. Survivin is a ubiquitous tumour antigen essential for the growth and survival of many human cancers, making it an attractive target for vaccines designed to promote T-cell immunity. This project aimed to develop a recombinant OAdV vector encoding survivin and, in parallel, an immortalised ovine cell line for scalable propagation of the virus. Methods: A survivin-encoding expression cassette incorporating IRES-driven eGFP expression was constructed for insertion into the OAdV genome at an intergenic site. Different primary ovine cell lines were transfected with plasmids that encode hTERT- and SV40LT, in order to generate an immortalised cell line. Results: Construction of the Survivin-IRES-eGFP OAdV vector is ongoing. Fibroblasts derived from foetal and adult sheep and foetal sheep kidney cells transfected with hTert and SV40-LT were isolated after dual antibiotic selection and screened for telomerase activity and expression of LT by RT-PCR. Discussion: Recombinant viral vectors enhance the presentation of tumour antigens to the immune system. They promote strong activation of cytotoxic T lymphocytes and enhance their avidity for cancer cells. OAdV is an effective vector in animal and cellular models due to its ability to infect a wide range of cell types, high immunogenicity and robust transgene expression, despite widespread pre- existing immunity to human AdV. A novel, OAdV-survivin vaccine is a promising candidate for cancer immunotherapy, deserving of further investigation in preclinical trials.

Presented by: Vithushiya Yoganandarajah Supervisor: Dr Julie Lim Title: Characterisation of glutathione synthesis pathways in the rat cornea Abstract Background: Given its anterior positioning in the eye, the cornea is increasingly susceptible to oxidative damage. As a result, it possesses a robust antioxidant defence system. Glutathione (GSH) is one of the principal antioxidants used by the cornea to protect itself from oxidative damage. However, little is known about the molecular pathways involved in the accumulation of GSH via the import of precursor amino acids required to synthesise GSH. The objective of my project was to identify and localise transporters involved in the uptake of amino acids required for GSH synthesis in the rat cornea. Methods: Membrane protein fractions prepared from rat cornea were separated by SDS-gel-electrophoresis and probed with isoform-specific antibodies for glutamate transporters (EAAT1-5), glutamine transporters (ASCT2 and LAT1) and glycine transporters (GLYT1-2). Immunohistochemistry was also performed

• n rat corneal sections and confocal microscopy used to localise transporters to

different corneal regions. Results: Western blotting revealed glutamate, glutamine and glycine transporters to be expressed in the rat cornea. Immunohistochemistry showed transporters to be localised to the outer corneal epithelium but not the stroma or inner endothelial layer. Discussion: The identification of the isoform-specific amino acid transporters validates the existence of an intracellular GSH synthesis pathway in the rat corneal epithelium. This suggests that other regions of the cornea utilise different mechanisms for accumulating GSH. This information is important in the design of targeted strategies to enhance antioxidants in the cornea to prevent against

• xidative damage, corneal swelling and loss of sight.