The role of extracellular vesicles in ALL treatment resistance and disease relapse

Fluorescence microscopy offers us a look into the world of extracellular vesicles.
Top-left - a Jurkat-derived microvesicle labelled with WGA-555
Bottom-left - the nucleus of an isotropically expanded BaF3 cell
Right - exosomes (red) interacting with BaF3 cells

Microvesicles and exosomes are small bodies created by cells. These extracellular vesicles are involved in intercellular communication and have already been implicated in several diseases. While work to characterise these extracellular vesicles has been completed in other cancers, including chronic myeloid leukaemia, very little work has been conducted on their role in acute lymphoblastic leukaemia. T-cell acute lymphoblastic leukaemia is a disease characterised by its heterogeneity. Thus far, tailored, targeted treatments have not entered clinical use and high-dose chemotherapy with possible bone marrow transplant are the only available treatment options.

Extracellular vesicles interact with other cancer cells and the surrounding microenvironment in an extremely unique way. Microvesicles, formed by the outward budding of the plasma membrane, can readily transfer receptors between cells. Exosomes, purpose-built vesicles formed intracellularly and subsequently released into extracellular space, carry with them nucleic acids and proteins that can change the microenvironment in their releasing cell's favour.

Study of these extracellular vesicles could dramatically shift current treatment approaches to disease, not just T-ALL. If purpose-built vesicles packed with contents that could make the cell vulnerable to treatment can be developed that capitalise on these systems, it could potentially revolutionise treatment in all cancers, not just T-ALL.

Imaging of Plasmodium falciparum rhoptry and rhoptry interacting proteins during merozoite invasion

P. falciparum merozoite stage parasites were allowed to invade, fixed, proteins visualised, imaged using super-resolution microscopy, and 3D models created in Imaris

Malaria is a parasitic infection of the blood endemic to several countries globally. P. falciparum is the main cause of malarial disease and related death in African countries, accounting for ~405,000 deaths in 2018. Malarial disease and death have been significantly reduced over the course of decades thanks to routine use of insecticide treated bed nets and the first line anti-malarial drug artemisinin in combination with other potent anti-malarial drugs. Unfortunately, insecticide resistance has developed in mosquitoes and artemisinin resistance has cropped up in P. falciparum parasites. As such new therapeutic targets and drugs are desperately needed to counter this shift.

Accurate and reliable treatment of a disease is reliant upon sound understanding and knowledge of the biological processes that contribute to that disease. Malaria parasites undergo several morphological stages. The merozoite stage is purpose built to invade RBCs where they reside within a purpose-built vacuole and grow by eating haemoglobin rich within the RBC cell body. The process of invasion is fast, and the parasite employs unique, purpose-built machinery called the rhoptries to successfully complete invasion. Several proteins involved in invasion such as Apical Membrane Antigen 1 (AMA1) and Rhoptry Associated Protein 1 (RAP1) have been well characterised by previous work. P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting (PfCERLI) protein 1 has been characterised as sitting around the rhoptry membrane and as being essential to parasite survival and growth, likely having a hand in rhoptry discharge, an essential step in the merozoite’s invasion of a RBC.

Clonal lines developed by members of the Wilson lab were confirmed as still having a functional HA tag and functional knockdown of PfCERLI1 via the riboswitch system was still possible. Super-resolution fluorescence microscopy analysis of AMA1, RAP1, and PfCERLI1 in fixed invading merozoites was possible and yielded consistent results however these processes will require further optimisation if accuracy is to be improved. Expansion microscopy of fixed invading merozoites was possible and yielded results that were consistent though further development of protocols will be required to visualise PfCERLI1 and the rhoptries. Optical tweezer manipulation of RBCs was achieved, and protocols have been developed that will support future attempts at manipulation of invading merozoites. Future endeavours of PfCERLI1 visualisation and analysis will be supported by the data contained in this thesis. 

South Australian hospital admissions outcomes of cases with a positive infection of Neisseria meningitidis 

Invasive meningococcal disease (IMD) results in sepsis, or meningitis, or both and is characterised by high rates of mortality and sequelae. IMD is caused by the bacteria Neisseria meningitidis; serogroup B is the most prevalent strain in Australia. IMD has a bimodal age distribution; the first peak is in those aged <1 year; the second peak in those aged 17-18 years. Despite this, there is a lack of literature detailing the long-term health, economic and psychosocial burden the pathogen has. 

Case notes of 96 individuals aged 15-25 admitted to Australian hospitals 2-10 years prior to study commencement with confirmed IMD infection were audited for demographics, symptoms, and outcome data. Logistic regression was completed looking at characteristics associated with intensive care unit (ICU) admission and developing one or more sequelae. 

Non-blanching rash; stiff neck; and photophobia were the commonest meningitis specific symptoms. A triage category <=2 compared to categories >2 and the patient presenting to hospital via ambulance compared to self-presenting to the Emergency Department were associated with admission to ICU. Living outside a major city, and socio-economic status were not associated with the severity of IMD. More work is required to understand what role age may play in sequela risk.

Neuronal toxicity of Hypericum perforatum containing teas on model neuronal cells

Hypericum perforatum, commonly known as "St. John's Wort", is a modestly effective herbal remedy for depression and related symptoms. Packed full of various active compounds, H. perforatum is most commonly thought of by clinicians for its effects on metabolism where it induces the production of enzymes, speeding up the metabolic process. in doing so, this herb can wreak havoc on various prescription medications such as birth control, possibly opening a window where conception and implantation could occur. 

H. perforatum is currently unregulated in Australia and as such is available over the counter and could be taken with a myriad of prescription medications. Since herbal remedies are frequently seen as "harmless" and are used as adjunct medications cases have been reported of people with depression taking the herb concurrently with their SSRI medication. Furthermore, the herb is available in teas from various tea retailers with packaging containing no warnings of potential pharmaceutical interactions. It is currently unknown if these teas have an effect on neuronal activity when taken in combination with commonly prescribed SSRIs.

Model rat neuronal cells were treated with H. perforatum and H. perforatum containing teas by themselves and in combination with fluoxetine. Cells were then treated with the potent neurotoxin 6-hydroxydopamine and cell death assays conducted to ascertain the effect the compounds had on neuronal survivability. This project was undertaken as a part of a summer research scholarship and as such time constrains were severe. I laid the groundwork for the student who has taken over the project and data will likely be collected and analysed by the end of 2023.