Biosciences MRes extended research projects 2020 entry

Those applying to study MRes Biosciences at Aston University will be required to select which extended research project they would like to complete as part of their studies. This will need to be clearly highlighted in your application in the personal statement section of the application from. 

We offer a variety of projects each year in a wide range of specialist biosciences fields. Please take a look through the list below to find out more. If you have any questions about a specific project please contact the project supervisor directly via the contact details provided. 

 

Dissecting the autophagic dysfunction in diabetes and Alzheimer’s Disease

Supervisor: Zita Balklava

Email: z.balklava@aston.ac.uk

Project Overview: Alzheimer’s disease (AD) and Type 2 Diabetes Mellitus (T2DM) are two of the most prevalent diseases in the elderly population worldwide and share common pathological features. People with T2DM are at a higher risk of developing AD. Likewise, AD brains are less capable of glucose uptake resembling a condition of brain insulin resistance. 

Intracellular accumulation of misfolded protein aggregates is a prominent feature of most neurodegenerative diseases. Autophagy, a lysosomal degradation pathway for damaged organelles or long-lived proteins ensures the clearance of these aggregates to maintain neuronal homeostasis. Autophagy is also crucial in the pathophysiology of diabetes where it regulates the normal function of pancreatic beta cells. Insulin resistance might impact autophagy impairment and aggravate AD pathogenesis. The main aim of the project is to characterise autophagic function in C. elegans T2DM and AD models and establish the connection between AD and T2DM pathogenesis in terms of autophagic dysfunction.

Project Aims:

  • Quantify autophagosomes in AD and T2DM models of C. elegans by SDS-PAGE and Western blotting.
  • Characterise autophagosomes in AD and T2DM models of C. elegans by fluorescence microscopy in different stages of development.
  • Characterise autophagic function in AD and T2DM models of C. elegans following exposure to antidiabetic drug metformin.

Core techniques: C. elegans maintenance and genetic crosses; genotyping (PCR, agarose gel electrophoresis); light and fluorescence microscopy; SDS-PAGE and western blotting.

How is Aquaporin-4 phosphorylated?

Supervisor: Roslyn Bill 

Email: r.m.bill@aston.ac.uk

Project Overview: Aquaporin-4 (AQP4) is the main water channel protein in the mammalian central nervous system (CNS). Influx of water into CNS tissue from the blood following stroke or head trauma causes life-threatening swelling of the brain or spinal cord (known as cytotoxic oedema). AQP4 is known to be the major route for water to enter the CNS and it is therefore a promising drug target for preventing death and disability in sufferers of stroke and head trauma. We have recently discovered that AQP4 is dynamically trafficked between the plasma membrane and intracellular vesicles, and that blocking this effectively reduces cytotoxic oedema in animal models of CNS trauma. We know that this trafficking requires phosphorylation of the AQP4 protein by Protein Kinase A. A-Kinase Anchoring Protein 5 (AKAP5) is a scaffold protein that facilitates the interaction between Protein Kinase A and some of its protein substrates. We have recently discovered that AQP4 is one of these substrates that is bound by AKAP5. The overall aim of this project is to characterise the interaction between AQP4 and AKAP5 both in cultured human cells and in vitro using purified proteins, with a view to better understanding of AQP4 membrane trafficking to aid future drug discovery projects. 

Project Aims:

  • Make mutations to AQP4 in potential AKAP5 binding regions.
  • Express AQP4 mutants in human cells and use immunoprecipitation to determine whether they bind AKAP5.
  • Optimise expression and purification of AKAP5 protein for in vitro binding and phosphorylation studies.

 Core Techniques: Protein mutagenesis; mammalian cell culture; immunoprecipitation; protein expression and purification.

A role for cellular senescence in dysregulation of insulin signalling and nutrient homeostasis in peripheral metabolic cells

Supervisor: James Brown

Email: j.e.p.brown@aston.ac.uk

Project Overview: Our group has evidence that senescence-associated secretory phenotype (SASP), the term for the secretome of senescent cells, can alter glucose homeostasis in metabolic cells. Other research has suggested that selectively clearing an animal of senescent cells improves glucose utilisation.

Project Aims:

  • Investigate the impact of SASP on insulin signalling in adipocytes, hepatocytes and skeletal muscle cells.
  • Investigate the impact of SASP on nutrient homeostasis in all 3 cell types.
  • Identify the signalling pathways associated with loss of nutrient homeostasis.

Core Techniques: cell culture; biochemical assays; cell viability analysis; microscopy; Luminex; immunohistochemistry; western blotting.

Antibiotic discovery from the Eden Project’s tropical plants

Supervisor:  Jonathan Cox

Email: j.a.g.cox@aston.ac.uk

Project Overview: The Eden Project is a huge, multiple greenhouse complex in Cornwall with two climatically regulated biomes that are optimized to grow plants from diverse environments. The tropical plant house contains plant species imported from rainforests around the world and represents an untapped resource in drug discovery. We have received 12 of the most exotic plants, selected by the head botanist at Eden, Dr Rachel Warmington. The aim of this project is to screen chemical extracts from these plants for novel antibiotic properties against a wide range of pathogenic bacteria. The project will begin with extracting compounds from the plants by maceration and solvent extraction, followed by recovery of the extracted compounds which will be tested for antimicrobial properties. Extractions will then be fractionated and re-tested to identify the specific compounds that are antimicrobial and the structure of the compounds elucidated by chemical analysis. New natural product antimicrobial compounds identified in this project will contribute to antibiotic drug discovery and could offer a future treatment for antibiotic resistant infections.

Project Aims:

  • To chemically extract compounds from 12 tropical plant species.
  • To screen extractions for antibacterial activity against a panel of clinically relevant bacteria.
  • To fractionate individual compounds from the mix and establish the compounds exhibiting antibacterial activity.

Core techniques: This project will involve the handling, culture and manipulation of clinically relevant BSL2 microorganisms. Compounds will be chemically extracted using basic biochemical organic extraction methods. Bacteria will subsequently be tested for susceptibility to the compounds by disk diffusion and broth microdilution techniques using a microtiter plate. Plates will be continuously read using a spectrophotometric plate reader and the end points established by a resazurin spectrophotometric assay. 

Extracellular vesicles - towards defining the functional surface of EV from cells of the neurovascular unit

Supervisor: Andrew Devitt

Email: a.devitt@aston.ac.uk

Project Overview: Cell communication is central to the development of multicellular organisms and their effective physiological functioning. One novel mechanism is mediated by extracellular vesicles (EV). These EV are released from cells when viable, stressed or undergoing cell death and they are taken up by recipient cells where they mediate a broad range of effects including the induction of cell death, cell survival and inflammatory responses. Consequently, these EV are now established as contributing to a range of pathological conditions, including those associated with traumatic brain injury, dementia and diseases of the central nervous system (CNS). It is therefore essential that we understand the composition of these EV released from different cells under different conditions. This will enable us to define how these EV are taken up and how they exert their effects (both desirable and undesirable).

This project will characterise EV release from cells of the neurovascular unit and subsequently identify and characterise key factors associated with EVs that mediate their inflammation-controlling function.  Using flow cytometry we will define molecules present on the surface of EV as these are the surface molecules that will facilitate EV uptake by recipient cells and may propagate pathology, as seen in dementia. By defining these molecules, it will be possible to modulate EV uptake for therapeutic gain (e.g. via antibody or small molecule inhibition) and will also highlight potential biomarkers for disease. 

Project Aims:

  • To characterise EV release from cells of the neurovascular unit, in isolation or co-culture.
  • To identify key proteins, present on EV that mediate EV function in modulating the innate immune response. Lead proteins for study may be selected from our existing large dataset of mass-spectrometry results of the EV proteome.
  • To define the function of selected proteins in EV activity. Using a variety of cell biological and molecular approaches, this will assess proteins as key ligands for EV binding, uptake or active function.

Core Techniques: tissue culture for the harvesting of EV; single particle analyses to characterise EV (number and size); live cell imaging to identify the capacity of EV to recruit macrophages; flow cytometry to reveal the EV surface proteome and flow cytometry of macrophages to define their phenotype after treatment with EV. 

Extracellular vesicles – structure/function relationships in the control of inflammation

Supervisor: Andrew Devitt

Email: a.devitt@aston.ac.uk

Project Overview: Inflammation is a key defense mechanism though its benefits are only realised when the response is turned off and tissue homeostasis restored. This process relies on intercellular communication mediated by extracellular vesicles (EV). These EV can attract macrophages, drive a change in macrophage phenotype and provide additional signals that drive the tissue repair. Relatively little is known about how these EV act or the key molecular factors that underpin their function. Our extensive preliminary work reveals that EV carry a large range of factors that may underpin their activity.

This project will identify and characterise key factors associated with EVs that mediate their function. The results from this project will therefore enable a better understanding of the molecular players and mechanisms involved in EV function providing important insight for the control of inflammation and regenerative medicine applications.

Project Aims:

  • To identify key proteins, present on EV that mediate EV function in modulating the innate immune response. Lead proteins for study will be selected from our existing large dataset of mass-spectrometry results of the EV proteome.
  • To define the function of selected proteins in EV activity. Using a variety of cell biological and molecular approaches, this will assess proteins as key ligands for EV binding, uptake or active function.

Core Techniques: tissue culture for the harvesting of EV; single particle analyses to characterise EV (number and size); live cell imaging to identify the capacity of EV to recruit macrophages; flow cytometry to reveal the EV surface proteome and flow cytometry of macrophages to define their phenotype after treatment with EV. 

Scaling-up of keratinocyte expansion for the treatment of large burn wounds

Supervisor: Patricia Perez-Esteban 

Email: p.perez-esteban@aston.ac.uk

Project Overview: The multi-layered epidermis in healthy skin is preserved due to the proliferation and differentiation of stem and progenitor cells within the basal layer, i.e. basal keratinocytes. When skin is damaged, its self-repairing mechanism involves the migration of keratinocytes from adjacent epidermis into the wound to promote re-epithelialisation. For large wounds, this process is not always efficient enough for rapid closure. In order to improve the prognosis of patients suffering from large wounds, more rapid and efficient methods for regenerating epidermal coverage must be developed. In order to overcome the challenges of resurfacing large wounds, new options to produce a clinically relevant number of keratinocytes from a patient’s biopsy are needed. Hence, there is an opportunity here for optimisation processes and the introduction of well-established technologies (such as stirred tank bioreactors and hollow fibre bioreactors) in the bioprocessing industry with a key clinical translation to improve health.

Project Aims:

  • To determine the optimal seeding density of keratinocytes on hollow fibre bioreactors.
  • To determine the optimal flow rate of media on hollow fibre bioreactors.
  • To screen a selection of microcarriers for keratinocyte attachment.
  • To determine the optimal seeding density in microcarriers.
  • To compare keratinocyte proliferation and phenotype when grown on the two different platforms.

Core Techniques: adherent human cell culture; cell counting; metabolic analysis (glucose and lactate); phase contrast microscopy; immunocytochemistry; Image J analysis.

Effect of butanol on biological membranes

Supervisor: Alan Goddard

Email: a.goddard@aston.ac.uk

Project Overview: Increasing concerns over climate change have driven the move towards sustainable “green” biotechnological alternatives to traditional petrochemical processes. A key example of this is production of biobutanol from Clostridia from waste products. However, this process is inhibited by butanol itself as it damages the bacterial cell membrane and ultimately kills the cells.   Our group, and others, have demonstrated that this damage can be reduced by tuning the lipid composition, or corresponding physical properties, of the cell membrane. However, nearly all model systems for studying such interactions rely on the use of lipid-only bilayers.

Biological membranes are approximately 50% protein by mass and, as such, proteins represent a significant membrane component. The interaction of butanol with membranes appears to occur via intercalation between the individual lipids in the bilayer which is likely to be significantly altered by the presence of proteins. We wish to address this knowledge gap using defined in vitro systems. 

Project Aims:

  • Purification of 3 model bacterial membrane proteins of various sizes and architectures. This will use detergents and/or polymer-based approaches as relevant and proteins will be characterised by SDS-PAGE.
  • Reconstitution of these proteins into liposomes of various lipid compositions to generate biological membrane mimics.
  • Biophysical characterisation of these proteoliposomes in the presence of butanol e.g. size, surface charge, porosity, lateral pressure and fluidity.

Core techniques: membrane protein expression in bacteria; membrane protein purification and chromatography approaches; generation of model lipid membranes; biophysical assays including dynamic light scattering, membrane fluidity and membrane integrity assays.

Deciphering the role of metastasis-inducing proteins in placental development implantation

Supervisor: Stephane R. Gross 

Email: s.r.gross@aston.ac.uk

Project Overview: Our lab has, over the years, been interested in different protein markers whose aberrant expressions relate to increased cellular motility. These proteins are key and now considered to be significant markers for cellular metastasis and poor prognosis when related to cancer biology. Recently we have shown that these same factors also play vital roles in regulating motility of specific cells known as trophoblasts in a much more physiological context, that of placental development during the stage of implantation.

The project will aim to study, at the molecular levels how specific proteins regulate changes in the cell architecture, mainly the cytoskeletal framework to module key aspects of cellular motility and invasion

Project Aims:

  • Regulate expression of metastatic inducing proteins or MIPs (the lab currently focuses on S100 proteins, ezrin and IQGAP1).
  • Establish the expression changes in these MIPs during placental development.
  • Determine how high levels of these MIPs affect cellular motility and invasion.
  • Use an array of microscopy analysis, including fluorescence, to decipher the mode of action of these proteins.
  • Use whenever possible animal samples or ex vivo placental samples to establish the importance of these factors in vivo.

Core Techniques: This project aims to further elucidate how these proteins regulate both cellular motility and invasion in trophoblasts, using an array of both molecular and cellular biology techniques including tissue culture, gene expression manipulation and imaging.

Scalable production of neural progenitor cells (NPCs) for spinal cord injury

Supervisor: Petra Hanga

Email: m.hanga@aston.ac.uk

Project Overview: Spinal cord injury is a devastating event that often results in permanent loss of motor and sensory function. In such cases, regeneration is difficult or absent. Recently, the first human clinical trial using induced pluripotent stem cells (iPSCs) for spinal cord injury was approved in Japan. Moreover, several studies demonstrated functional recovery post-spinal cord injury could be promoted when iPSC-derived neural progenitor cells (NPCs) were transplanted. NPCs can self-renew and differentiate into neurons, astrocytes and oligodendrocytes, thus making them important cell sources for a wide range of applications including therapies for spinal cord injury. However to date, these cells have only been produced in 2D planar cultures which are highlylaborious and not able to satisfy the current needs for volume and reproducibility.  There is a need for developing technologies that can promote the consistent large scale production of NPCs to support their availability as therapeutic products.

Project Aims:

  • Expand human iPSCs on microcarriers or as aggregates (embryoid bodies) in small bioreactors.
  • Differentiate iPSCs towards NPCs in small bioreactors.
  • Characterise the NPCs produced.

Core Techniques: iPSC culture in monolayer and in small bioreactors on microcarriers or as aggregates;  iPSC differentiation to neural lineage; automated cell counting; metabolic analysis; phase contrast and fluorescent imaging; flow cytometry; immunofluorescence staining.

Tissue engineered approaches for scalable production of skeletal muscle

Supervisor: Petra Hanga

Email: m.hanga@aston.ac.uk

Project Overview: Skeletal muscle comprises a large proportion of our body and plays an important role in movement. This tissue has the ability to regenerate by activating satellite cells. However, this regeneration is limited to small wounds, whereas muscle loss caused by trauma requires surgical reconstruction. Tissue engineering could be highly beneficial in such situations. Muscle satellite cells have limited proliferation and differentiation potential limiting their clinical applicability. Mesenchymal stem cells (MSCs) may represent a more promising alternative cell source for muscle tissue engineering since they can easily be harvested, expanded and differentiated to myogenic lineage by using small molecules or biomaterials of specific stiffness. The hypothesis is that encapsulating MSCs will result in increased myogenic efficiency. We will test this hypothesis by comparing 3D with 2D approaches. Different media formulations and hydrogels will be tested and the optimal combination will be taken forward for developing a scalable production for muscle cells.

Project Aims:

  • Establish an optimal myogenic differentiation media formulation.
  • Select a suitable biomaterial for encapsulating MSCs.
  • Develop a production protocol for muscle cells in small bioreactors in agitated conditions.

Core Techniques: MSC culture and characterisation; bioreactor culture; automated cell counting; cell encapsulation in hydrogels; metabolic analysis; phase contrast and fluorescent imaging; immunofluorescence.

3D culture of stem cell derived neural networks

Supervisor: Eric Hill 

Email: hillej@aston.ac.uk

Project Overview: Whilst stem cells are providing scientists with the ability to generate human neuronal cultures, these systems lack the three-dimensional complexity of human brain tissue, and therefore have reduced potential to accurately model human disease. The aim of this project is to engineer 3D neuronal networks of human stem cell-derived neurons by generating defined tissue architectures that will allow modelling of network function and pathology in vitro.

Project Aims: 

  • Optimise production of biocompatible hydrogels.
  • Determine optimal properties for neuronal growth.
  • Assess connection of neuronal cells in 3D cultures.

Core Techniques: 3D printing; cell culture; biochemical assays; cell viability analysis; microscopy.

Crosstalk between mesenchymal stem cells and immune cells

Supervisor: Jill Johnson 

Email: j.johnson1@aston.ac.uk

Project Overview: Mesenchymal stem cells are stromal/structural cells found in many adult tissues, including the bone marrow, but they are also found throughout the body, associated with capillaries (known as pericytes). These cells are an attractive progenitor cell source for the renewal of damaged tissues due to their ability to self-renew, their high proliferative capacity and their differentiation potential. Under certain circumstances, usually associated with chronic inflammation, MSCs/pericytes are capable of differentiating into another cell type known as myofibroblasts; these cells release excessive amounts of extracellular matrix proteins and ultimately lead to tissue destruction through the process of fibrosis. This is a normal bodily reaction to an injury (for example, skin scarring after an injury), but if left unresolved, it can also lead to the loss of organ function. As fibrosis has no currently available treatment, this loss of function has serious detrimental effects.

Project Aims:

  • To determine the mechanisms by which MSCs/pericytes impact on the function of macrophages, an immune cell type closely associated with wound healing and fibrosis.
  • To determine the role of cell-to-cell contact and soluble factors in modulating macrophage function.
  • To specifically identify the mediators produced by healthy pericytes as well as pericytes that have been grown under pro-fibrotic or oxidative stress conditions, and the impact of these products on macrophage activation, polarization, and cytokine secretion.

Core Techniques: cell culture; ELISA; immunostaining; microscopic imaging and digital data analysis.

Growing a better MSC: Investigating the immuno-modulatory properties of cultured MSCS

Supervisor: Ewan Ross

Email: e.ross1@aston.ac.uk

Project Overview: Cell based therapies have become an increasingly effective clinical approach to promote tissue repair, wound healing or reduce inflammation. Mesenchymal stem cells (MSCs) are a key cell type in this approach as they can restore damaged tissues (fat, bone, muscle) and reduce inflammation through direct cell-cell contact or release of soluble mediators (proteins, extracellular vesicles (EVs)). Therefore, the ability to grow and maintain functional MSCs in the laboratory is a crucial research goal.

Producing MSCs for patients in the laboratory is difficult as they spontaneously differentiate in culture over time, losing their immuno-modulatory abilities. We have recently demonstrated growing MSCs on specialised surfaces or treating with certain compounds can maintain their therapeutic potential by modulating their metabolism. This results in turn maintains their ability to suppress lymphocyte proliferation. This project will further develop this work by investigating the mechanisms by which MSCs are educated by these surfaces or compounds to promote their immuno-modulatory effects.

Project Aims:

  • To investigate physiological changes to the MSC as it adheres to the immunized surface. Preliminary data on changes to the cytoskeleton and mitochondrial function will form the starting point of this study.
  • Co-culture of MSCs grown on surfaces or treated with compounds with lymphocytes to monitor effects on immune-modulation. T cell proliferation and the development of regulatory T cells will be assessed.
  • MSCs EVs will be purified and their effects on macrophage and T cell function will be assessed.
  • Physiological and phenotypic changes to MSCs after treatment with compounds will be investigated to monitor their naïve, therapeutic potential.

Core Techniques: Primary bone marrow MSCs will be grown on surfaces or treated with compounds using standard tissue culture techniques. Changes to MSC cytoskeleton and mitochondrial function will be assessed by immunofluorescent staining and microscopy. T cell proliferation and regulatory T cell staining will be analysed by flow cytometry. EVs will be harvested from cultures and analysed by single particle analysis for number and size. EVs effects on macrophage biology will assessed by phagocytosis assay and phenotyping by flow cytometry. Alterations to MSCs physiology and phenotype will be revealed using a combination of assays; flow cytometry, PCR, western blotting and in vitro differentiation assays.

Investigating the mechanism of clathrin cage disassembly

Supervisor: Alice Rothnie 

Email: a.rothnie@aston.ac.uk

Project Overview: Clathrin mediated endocytosis is a fundamental function of eukaryotic cells, important for nutrient uptake, protein trafficking and cell signalling. In a coordinated process involving many different proteins, a clathrin coated vesicle carrying protein cargo buds off from the membrane. Once this clathrin coated vesicle has reached its intracellular target the cage has to be removed so that the vesicle can fuse with its target membrane. This cage disassembly is carried out by the small chaperone protein Hsc70 and its partner auxilin, in an ATP-dependent process. The overall aim of this work is to understand the molecular mechanism by which clathrin cage disassembly occurs. Specifically, in this project you will investigate the effect of a mutation within auxilin. The mutation D876A lies within the J domain of auxilin and was predicted to abolish binding to Hsc70. However preliminary studies show disassembly can still occur, albeit at a reduced rate.

Project Aims:

  • Determine whether the D876A mutation of auxilin does completely abolish interaction with Hsc70, or just decrease the affinity very significantly, and whether it can still stimulate Hsc70 ATPase activity.
  • Examine if the D876A mutation of auxilin affects its interaction with clathrin.
  • Measure the effects of D876A auxilin on the kinetics of clathrin cage disassembly and the stoichiometry of the three components.

Core Techniques: protein expression, purification and SDS-PAGE; light scattering, binding and ATPase assays. 

Novel polymers to extract and purify membrane proteins

Supervisor: Alice Rothnie 

Email: a.rothnie@aston.ac.uk

Project Overview: Membrane proteins are vitally important, controlling what enters and leaves a cell, mediating cellular communication and cell identification. It’s estimated that half of all prescribed drugs bind to membrane proteins. However their membrane environment can make it challenging to study their structure & function. Detergents have been used to solubilise membrane proteins from the lipid bilayer, but they can alter protein structure and function, as well as stripping away important lipids. Recently styrene-maleic acid co-polymer (SMA) has been used instead of detergents to extract small discs of bilayer containing the membrane protein whilst maintaining its lipid environment. This polymer approach has revolutionised the study of membrane proteins, making them technically much simpler to work with, but it does still have some limitations. This project will investigate novel polymer variants to try to overcome these limitations.

Project Aims:

  • Screen a series of polymer variants for solubilisation of a range of membrane proteins.
  • Purify proteins using the novel polymers, to investigate relative yield and purity.
  • Measure the stability and function of purified proteins.
  • Investigate whether any of the current limitations are overcome.
  • Test which downstream methods/assays can be undertaken with the purified proteins.

Core Techniques: Bacterial protein expression, solubilisation, purification and SDS-PAGE; fluorescent and enzymatic assays.

Reconstituting proteins from SMALPs to proteoliposomes

Supervisor: Alice Rothnie 

Email: a.rothnie@aston.ac.uk

Project Overview: Membrane proteins are vitally important, controlling what enters and leaves a cell, mediating cellular communication and cell identification. However their membrane environment can make it challenging to study their structure & function. Detergents have been used to solubilise membrane proteins from the lipid bilayer, but they can alter protein structure and function, as well as stripping away important lipids. Recently styrene-maleic acid co-polymer (SMA) has been used instead of detergents to extract small discs of bilayer containing the membrane protein whilst maintaining its lipid environment. This polymer approach has revolutionised the study of membrane proteins. However, whilst the nanodisc-like shape of the SMALP is perfect for ligand binding assays it’s not useful for measuring transport across the membrane, which requires an enclosed space. We have developed preliminary methods for reconstituting proteins from SMALPs into proteoliposomes. The aim of this project is to develop and optimise this process.

Project Aims:

  • Test the reconstitution on a range of different target proteins.
  • Measure the function of reconstituted proteins.
  • Investigate the effect of different lipid components within the proteoliposome on efficiency of reconstitution and protein function.

Core Techniques: Bacterial protein expression, solubilisation, purification and SDS-PAGE; fluorescent and enzymatic assays.

A 3D printing approach for drug discovery

Supervisor: John Simms

Email: simmsj3@aston.ac.uk

Project Overview: Drug Discovery has reached a significant bottleneck over the last decade, which has seen many promising compounds fail in pre-clinical trials. While these failures have several underlying causes, drug screens using traditional 2D cell culture have often led to misleading results. 3D printing combined with advances in cell culture have revolutionised how cells are studied and provide an opportunity to replace studies performed in vivo. While methods to develop these devices (popularly referred to as ‘Organs on a Chip’) are still in their infancy, many systems mimicking heart, lung and bone have been successfully developed. These systems are able to perform a range of functions and provide an excellent platform for exploring drug target-ligand interactions as well and studying diseases. 

This project will provide training in biomaterials and cell biology to develop a 3D printed micro-physiological chamber (‘organ on a chip’; ‘pancreas on a chip’) containing encapsulated insulin secreting cells. This device will ultimately be used for drug discovery.

Project Aims:

  • Explore methods to genetically manipulate hydrogel encapsulated cells to either express new proteins or to suppress existing ones to aid drug discovery.
  • Explore surface modifications for 3D printed components to provide extra functionality to the chamber e.g. cell trapping, ‘in device’ cell signalling assays.
  • Explore ways that the 3D printed device(s) can be modified for high throughput drug discovery.
  • Explore alternative hydrogel formulations and other environmental factors to develop a native like environment for cell signalling, and proliferation.

Core Techniques: Computer Aided Design (CAD); FDM 3D printing; 3D cell culture; cell signalling assays; cell viability analysis; molecular biology and microscopy.

Using lipid nanoparticles for drug delivery in Drosophila melanogaster

Supervisor: Cathy Slack

Email: c.slack@aston.ac.uk

Project Overview: The fruit-fly, Drosophila melanogaster, has proved to be an invaluable model organism for studying the molecular mechanisms of disease. The Drosophila genome encodes functional homologues for around 75% of human disease-causing genes, it has substantial tissue complexity, with clearly formed organs such as the brain, heart, gut and kidney, has been extensively characterised genetically and is easily manipulated with numerous reagents for genetic studies readily available from publicly accessible strain repositories. Yet they are not used effectively in high-throughput drug screening because drug delivery is problematic. This project aims to investigate the use of lipid nanoparticles or liposomes for drug delivery in Drosophila. Liposomes are spherical, closed vesicles made up of phospholipid bilayers surrounding an aqueous core, allowing hydrophobic and hydrophilic compounds to be carried in each compartment, respectively. This encapsulation of compounds decreases overall drug toxicity and enhances uptake into cells, whilst also preventing degradation and dilution within the circulation.

Project Aims:

  • Encapsulate the well-characterised mTOR inhibitor, rapamycin, within liposomes and feed to adult flies.
  • Measure drug efficacy and dose-dependent physiological and biochemical responses.

Core Techniques: Drosophila culture; western blotting; quantitative RT-PCR; immunohistochemistry; fluorescent microscopy. 

Studying the effects of O-GlcNAc glycosylation on Amyloid beta pathology using a Drosophila model of Alzheimer’s Disease

Supervisor: Cathy Slack

Email: c.slack@aston.ac.uk

Project Overview: A prominent feature of Alzheimer’s Disease (AD) progression is a substantial reduction in glucose metabolism within the brain. This often precedes the onset of clinical symptoms or histopathological changes by decades, suggesting that metabolic dysfunction within the brain is a central event in the onset of AD pathology. However, the molecular mechanisms by which impaired glucose metabolism in the brain drives AD progression are unclear. This project aims to investigate the link between neuronal glucose uptake and AD pathology and effects on the nutrient-responsive glycosylation of proteins by the addition of O-GlcNAc. O-GlcNAc modifications have been associated with protective responses to cellular stress and importantly, levels decline with AD progression in mammalian models of disease.

Project Aims:

  • Investigate this link between O-GlcNAc and neuronal pathology in AD in vivo using a Drosophila model of Amyloid beta (Aβ) toxicity.
  • Modulate O-GlcNAc levels using transgenic expression techniques and examine the effects on Aβ-related pathologies.

Core Techniques: Drosophila culture and behavioural assays; western blotting; quantitative RT-PCR; ELISA; immunohistochemistry; fluorescent microscopy. 

Investigating the effect of lipoxidation on mammalian cell function

Supervisor: Corinne M. Spickett

Email: c.m.spickett@aston.ac.uk

Project Overview: Lipoxidation is the covalent modification of proteins by lipid oxidation products such as short-chain aldehydes, which can be formed under conditions of oxidative stress, for example in inflammatory conditions. It is thought that lipoxidation can change the activity and localization of cellular proteins, and thus the behaviour of the cells. This may be important in determining the balance of cell proliferation, apoptosis or altered metabolic responses in cancer. This project will build on our previous research with pyruvate kinase in this area.

The project will involve treatment of the breast cancer cell line MCF-7 with different lipid oxidation products to analyse changes in cell morphology, viability, metabolic status and stress responses. Enzymatic assays will be used to investigate changes in activity of pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase or the signalling phosphatase PTEN, all of which are known to be redox-sensitive. Alterations at a proteomic level will also be determined.

Project Aims:

  • To determine which lipid oxidation products alter the balance of cell proliferation versus cell death.
  • To determine if pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase or PTEN are inhibited or activated by lipoxidation.

Core techniques: mammalian cell culture; enzymatic assays; SDS-PAGE; western blotting; mass spectrometry.

Regulating MSC-derived exosome secretion in scale up culture

Supervisor: Ivan Wall 

Email: i.wall@aston.ac.uk

Project Overview: MSCs have been widely examined in regenerative medicine applications but their mode of action remains controversial. Studies have revealed that MSCs secrete exosomes in culture and that the exosomes have similar potency to parent MSCs. To scale up exosome production, there is a need to scale up cell culture so that the “producer” cells are in abundance. Several methodologies exist to increase exosome secretion, including addition of heparinase. This study will ask the question, does timing and duration of heparanase addition to MSC cultures affect MSC and exosome quantity and quality?

We will test the hypothesis that addition of heparanase at a late time in culture increases exosome production in comparison to addition at an earlier stage.

Project Aims:

  • To determine whether heparanase increases exosome secretion by MSCs in scale up culture and whether this comes at the expense of cell division.
  • Establish 100mL microcarrier cell cultures for MSCs in spinner flasks.
  • Establish a matrix of experimental measurements for MSC and exosome attributes.
  • Measure quantify MSCs and exosomes in the presence of absence of heparinase.

Core Techniques: 2D planar cell culture for mesenchymal stem cells; bioreactor cell culture for mesenchymal stem cells on microcarriers; bioreactor sampling and measurement of cell number, viability and surface markers; harvest and concentration of exosomes and subsequent quantification.