Molecular Medicine and Pathology

Cancer pharmacogenomics- has it gone silent?

Supervisor

Assoc Prof Nuala Helsby
Dr Kathryn Burns
Dr Soo Hee Jeong

Discipline

Molecular Medicine and Pathology

Project code: MHS076

Understanding the genomic differences between individuals may help us improve the safe and effective use of medications and other therapies. 5-fluorouracil (5-FU) is a chemotherapy drug used in the treatment of breast and colorectal cancer. Unfortunately a substantial proportion of patients are at risk of life-threatening normal tissue toxicity after standard doses of this drug. An enzyme called dihydropyrimidine dehydrogenase (DPD) is involved in the elimination of this drug from the body. Some patients have inherited versions of this enzyme that are inactive and they are at high risk of toxicity. However, there is often no evidence of inherited loss of DPD function in most cases of 5-FU toxicity. One alternative is that some patients have low levels of this enzyme due to epigenetic silencing of the gene.

The project aims to determine the most appropriate conditions to reproducibly and accurately detect inter-individual differences in the amount of DPD enzyme (protein) in freshly collected human cells. This will help us to accumulate evidence for 'silencing' of DPD expression in individuals who have not inherited low activity genetic variants.

Functional characterization of non-protein coding genes in cancer

Identifying adult stem cells in bone

Supervisor

Brya Matthews

Discipline

Molecular Medicine and Pathology

Project code: MHS183

Bone has a tremendous healing capacity, and fractures generally heal to restore the original functionality of the bone. However, in severe cases, or in patients who are elderly or have other complications healing may be delayed or blocked. The periosteum is a rich source of stem cells that go on to form bone, however, there are no well-established ways to identify these cells. In addition, the processes by which they activate and differentiate are also poorly defined. In this project we will evaluate growth of different potential stem cell populations. We will also examine events following injury by histology and immunohistochemistry.

You will perform or be exposed to techniques including: cell culture, tissue dissection, flow cytometry and cell sorting, immunohistochemistry, image analysis.

Hands on experience in some lab techniques preferred. Students who are considering continuing with a research-based degree (Honours or Masters) are particularly encouraged to apply.

Expression and up-regulation of receptor for advanced glycation end-products as a mediator of beta cell damage in diabetes

Supervisor

Dr Shiva Reddy

Discipline

Molecular Medicine and Pathology

Project code: MHS116

We are witnessing a dramatic rise in the incidence of type 1 and type 2 diabetes. Such a rapid increase cannot be driven solely by genetic changes in affected individuals but strongly implicates certain environmental factors as being causal. The ready availability of processed foods we ingest may be an important contributing factor through generation of advanced glycation end products(AGEs).
We propose that during both forms of diabetes, beta cell damage may be mediated following binding of dietary-derived AGEs or intrinsically-generated ligands to specific receptors on beta cells, known as receptor for advanced glycation end products (RAGE). RAGE expression on beta cells can be up-regulated in the presence of AGEs, leading to activation of a cascade of downstream beta cell damaging signalling pathways.
In this research we will develop and apply multi-label immunohistochemistry to pancreatic sections from deceased diabetic subjects to test the hypothesis that the expression of RAGE, and some of its key ligands are increased in beta cells during the prediabetic and diabetic phases, and seek molecular evidence of beta cell damage and death.
Demonstration of increased levels of RAGE in the beta cell prior to and after onset of diabetes could pave way towards developing small molecule inhibitors of RAGE, leading to a reduction in beta cell inflammatory damage and amelioration of diabetes and its serious secondary complications.

Techniques: multi-label immunohistochemistry, molecular pathology, advanced microscopy, digital imaging, immunology and endocrinology

The role of nitric oxide and related oxidants in beta cell damage during type 1 diabetes

Supervisor

Dr Shiva Reddy

Discipline

Molecular Medicine and Pathology

Project code: MHS173

The mechanisms of beta cell inflammation and damage during type 1 diabetes remains elusive. Although immune-mediated processes are intimately involved during the destructive process, little is known about the initiating events and the early destructive processes that ensue within the beta cell that lead to the differing trajectories of disease onset.

Several studies have shown that nitric oxide (NO), under the influence of certain cytokines and other adverse factors may be produced within the islet cells, following up-regulation of inducible nitric oxide synthase (iNOS), in and around the diabetic islet. NO, at high levels, is toxic to the beta cell. It may also combine with superoxide anions to produce the more potent peroxynitrite which can nitrate tyrosine residues of key proteins within the beta cell, impair beta cell function and also produce “neoantigens” that can be recognized by the immune system as immunogenic.

In this study, we propose to study and correlate the expression of iNOS and nitrotyrosine in pancreatic islets from human cases with early onset type 1 diabetes and evaluate its role in the immunopathogenesis of the disease.

Techniques taught: Multi-label immunohistochemistry, pancreatic pathology, immunology, advanced microscopy,digital image acquisition and quantification.
A knowledge of basic statistics and ImajeJ desirable.

Using human iPSC-derived kidney organoids to study renal cell carcinoma

Supervisor

Dr. Veronika Sander 

Discipline

Molecular Medicine and Pathology

Project code: MHS054

Kidney cancer is among the 10 most common cancers, with renal cell carcinoma (RCC) accounting for ~90% of all kidney cancers. Recent studies have revealed that loss of the transcription factor HNF1B confers an aggressive phenotype in RCC. Advanced (metastatic) RCC is associated with bad prognosis, and a major shortcoming in the field has been a lack of a relevant human model for developing new, efficient treatment.
Our lab has established a method for converting induced pluripotent stem cells (iPSCs) into kidney organoids (mini kidneys cultured in vitro). These organoids contain multiple kidney cell types and show a high degree of maturation. Kidney organoids allow us to recapitulate the development of kidney diseases in small scale in the lab. Using CRISPR/Cas9 gene editing we have created iPSC lines that carry a biallelic mutation of HNF1B. Kidney organoids derived from these HNF1B-deficient lines develop into RCC-like tissue after ~6 weeks in culture. The aim of this summer studentship is to characterise the cellular composition of the cancerous tissue in these organoids and unravel molecular events, e.g. additional loss of p53, that may contribute to tumorigenesis.
Our findings will enhance the overall understanding of the pathology of renal cell carcinoma. More specifically, kidney organoids as model for RCC could provide a platform for developing new therapies for this cancer.

Please send a CV and academic transcript if interested.

Skills taught:
Students projects will involve analysis of kidney organoids, and specific techniques learned will include:
• Histology (paraffin embedding, sectioning, H&E- and trichrome staining)
• Fluorescence immunohistochemistry and imaging (light and confocal microscopy)
• RNA isolation and qPCR
• Data analysis and presentation

Mini kidneys in a dish – using organoids to study kidney fibrosis

Supervisor

Dr. Veronika Sander 

Discipline

Molecular Medicine and Pathology

Project code: MHS104

Kidney disease is a pertinent issue in New Zealand, with approximately 1 in 10 people suffering from some form of it. Patients have a high risk of progressing to chronic kidney disease and end-stage renal failure requiring dialysis and kidney transplantation. Many chronic and progressive nephropathies share a final pathway marked distinctly by fibrosis. Fibrosis can result from a range of aberrant conditions including ischemia, oxidative stress, ER stress or physical injury inferred to an organ. Fibrosis is characterised most distinctly by the accumulation of the extracellular matrix in place of functional tissue. Whilst initially being a part of tissue repair in mild injury, extracellular matrix deposition may continue without being counteracted, resulting in the reduction of organ structure and function and, eventually, organ failure. Treatments targeted at upstream events have yet to show success, and novel therapeutic strategies are urgently required.
Our lab has established a method for converting induced pluripotent stem cells (iPSCs) into kidney organoids (mini kidneys cultured in vitro). These organoids contain multiple kidney-specific tissues including the glomerular blood filter, tubules and collecting ducts that resemble the renal structures of the human organ. Kidney organoids allow us to recapitulate the development of kidney diseases in small scale in the lab. Preliminary observations on long-term organoid cultures have shown progressive accumulation of fibrotic tissue and upregulation of fibrosis-specific marker genes. The aim of this project is to unravel the molecular processes that lead to the development of fibrosis and test potential inhibitors of this process in human iPSC-derived kidney organoids. Fibrosis markers including alpha smooth muscle actin, transforming growth factor 1B, and interleukin 1B measured by qPCR and immunohistochemistry will be used as readout. The organoid model could be a significant step towards understanding the pathophysiology of fibrotic kidney disease and the development of novel therapies.

Please send a CV and academic transcript if interested.

Skills taught:
Students projects will involve analysis of kidney organoids, and specific techniques learned will include:
• Histology (paraffin embedding, sectioning, H&E- and trichrome staining)
• Fluorescence immunohistochemistry and imaging (light and confocal microscopy)
• RNA isolation and qPCR
• Data analysis and presentation

Uncovering the role of renal resident macrophages using IPSCs-derived kidney organoids

Supervisor

George Chang
Alan Davidson

Discipline

Molecular Medicine and Pathology

Project code: MHS156

Macrophages have long been recognised as the front line defenders against microbe infection. Interestingly, in addition to immunity, macrophages also play a crucial role in maintaining tissue homeostasis and health via microenvironment surveillance, scavenging apoptotic cells and cell debris clearance. While these tissue-resident macrophages have been studied in detail in organs such as brain, liver and lung, very little is known about their actual role in the kidney.
Our lab has established a novel method for converting induced pluripotent stem cells (IPSCs) into kidney organoids (mini kidneys grown in a dish). These organoids show a high degree of similarity to human kidneys and have emerged as a valuable tool for understanding kidney biology. The aim of this project is to investigate the functional importance of renal resident macrophages in kidney development and homeostasis by co-culturing monocyte-derived macrophages with these kidney organoids.
Key techniques used in this project include:
• Histology (paraffin embedding, sectioning, H&E- and trichrome staining)
• Fluorescence immunohistochemistry and imaging (light and confocal microscopy)
• RNA isolation and qPCR
• Data analysis and presentation

Modelling paediatric kidney cancer using IPSCs-derived kidney organoids

Supervisor

George Chang
Alan Davidson

Discipline

Molecular Medicine and Pathology

Project code: MHS158

Wilms’ tumour is the most common form of paediatric kidney cancer that arises from abnormally proliferative embryonic renal progenitors that fail to properly differentiate. Whilst advances in treatment have enhanced overall survival, severe toxicities and tumour recurrence remained the major drawbacks of the current regime. Thus, the discovery of novel therapeutic agents is still needed but efforts are hampered by a lack of a physiologically relevant model to study cancer progression in the laboratory.

Our lab has pioneered an innovative method for growing human kidney organoids (mini kidney cultured in vitro) from inducible pluripotent stem cells (iPSCs). In combination with CRISPR/Cas9 gene editing technology, we now have the opportunity to model kidney diseases with high in vivo resemblance. Here, we propose generating kidney organoids from iPSCs that have been engineered to carry a mutation in the WT1 gene and an inducible IGF2 transgene (two collaborating tumourigenic events commonly found in Wilms’ tumour). The goal of this project is to generate the first 3D in vitro model of Wilms’ tumour.

Techniques learned will include:
• Histology (paraffin embedding, sectioning, H&E- and trichrome staining)
• Fluorescence immunohistochemistry and imaging (light and confocal microscopy)
• RNA isolation and qPCR
• Molecular cloning
• Data analysis and presentation

Effect of TRPV4 missense mutation on calcium flux

Supervisor

Jennifer Hollywood
Alan Davidson

Discipline

Molecular Medicine and Pathology

Project code: MHS095

TRPV4 is an ion channel protein encoded by the TRPV4 gene. It is a calcium (Ca2+) permeable, nonselective cation channel that is involved in multiple physiological functions such as regulation of systemic osmotic pressure, vascular, liver, renal and bladder function. The channel is activated by various physical and chemical stimuli, such as cell swelling and heat which induce Ca2+ entry. Studies of whole genome and exome sequence data have identified a novel missense mutation in the TRPV4 gene that is highly frequent in certain NZ populations. At present it is unknown whether the high incidence of this mutation has beneficial or harmful effects.

The aim of this summer project will be to explore the effects this mutation has on calcium flux. To achieve this the student will perform site-directed mutagenesis on a TRPV4 expressing plasmid to introduce the missense mutation (G>A) observed in the effected population. These plasmids will be overexpressed in human cell lines and calcium flux will be measured to decipher the impact of this missense mutation on channel function.

Molecular techniques student will learn:
• Site-directed mutagenesis
• Cloning
• Transfection
• Calcium flux measurements

Unravelling mechanisms of lymphatic vessel development

Supervisor

Jonathan Astin

Discipline

Molecular Medicine and Pathology

Project code: MHS012

Aims
Lymphatic vessels are components of the vascular system, and play a key role in immune cell trafficking. Inappropriate lymphatic vessel growth contributes to the pathogenesis of many chronic inflammatory disorders and in cancer metastasis. Remarkably, we know almost nothing about the guidance cues that dictate where lymphatic vessels grow in different tissues and this knowledge is fundamental to the design of new therapies to treat these diseases.

Using transgenic zebrafish embryos in which lymphatic vessels are fluorescently labeled we have created the first map of the embryonic lymphatic vasculature. We used this map to identify lymphatic vessels that develop along cartilage or sensory neurons. The aim of this project will be to identify novel molecules involved in lymphatic vessel guidance by examining mutants in cartilage and neuronal development in which lymphatic vessels do not develop correctly. This work will uncover novel mechanisms of lymphatic vessel guidance that may be applied to develop treatments for lymphatic-related diseases. Skills include: Molecular Biology, Zebrafish Husbandry, Confocal Imaging (on both live and fixed specimens), Model Organism Genetics.