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2018 Cohort

Alexandre Chappard portrait

Alexandre Chappard

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Developing single-molecule and super-resolution techniques to characterise alpha-synuclein aggregation

Aberrant protein aggregation is a predominant feature of many neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Evidence suggests that small oligomers formed from alpha-synuclein are the key cytotoxic species in Parkinson’s disease. These aggregates are challenging to study due to their high heterogeneity and low abundance. As such, I am developing a range of single-molecule and super-resolution techniques to investigate these aggregates. One such super-resolution technique is a new PAINT-based approach, which uses small, alpha-synuclein-specific fluorescent peptides to image aggregates from a range of samples at the nanoscale. In addition, I am also using these techniques to investigate the impact of the G51D mutation (which promotes a particularly aggressive early-onset, autosomal dominant form of Parkinson’s disease) on the aggregation pathway of alpha-synuclein, to gain a better understanding of this mutation in disease.

Ailsa Golightly portrait

Ailsa Golightly

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Effect of radiotherapy on primary tissue models of cancer

My research explores the use of Surface Enhanced Raman Spectroscopy (SERS) to probe the effects of radiotherapy on primary tissue models.

The main focus points for the project are:

  • applying SERS to 2D and 3D cell culture models to measure the health and viability of the cultures
  • developing the tissue models using tumour-on-a-chip technology to mimic the environment of tumour cells inside the human body
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Imaging tumour macrophage polarisation for non-invasive evaluation of anticancer therapy. Tumours are populated with not only malignant cells but also many other resident cells such as macrophages. Normally, macrophages act as the first defense line of the immune system and are specialised to kill and remove disease cells or foreign substances that may be harmful to human body. .

However, the macrophages in the tumour is a “double edged sword” and can either inhibit or promote tumour growth depending on their polarisation to different phenotypes including pro-inflammatory macrophages (M1; suppress tumour growth) or anti-inflammatory macrophages (M2; promote tumour development). It has been demonstrated that cancer therapy can affect the population of tumour macrophages. A high M1/M2 ratio is linked with extended survival of cancer patients. Therefore, the population of macrophages and the M1/M2 ratio is very important in assessing therapeutic outcome and status of tumour. Our goal is to develop a non-invasive fluorescence nanoprobe (materials science and chemical engineering) to specifically target M1/M2 tumour macrophages (cancer biology). Through this, the population of tumour macrophages and the M1/M2 ratio can be determined via optical imaging (physical science). The information will be used to assess the outcome of anticancer therapy and the status of tumour to guide doctors in treatment.

To achieve the goal, our strategy is to: develop two types of organic nanoparticles of different fluorescent colours, with one type to coat with monoclonal CD86 antibody or MHC class II antibody to target macrophage M1, and the other to coat with monoclonal CD204 or CD206 antibody to target macrophage M2, followed by fluorescence microscopy analysis of tumour. The details will be as follow.

The organic nanoparticles are made from Bis(4-(N-(2-naphthyl) phenylamino) phenyl)-fumaronitrile (NPAPF) containing either NIR712 dye (NIR fluorescence) for M1 targeting or C545T (green fluorescence) for M2 targeting, based on our previously published work1,2. These nanoparticles have high brightness and photostability. Importantly, the biocompatibility is much better than inorganic fluorescent nanomaterials, which significantly increases the potential of being used in clinic.

Subsequently, NPAPF/NIR712 nanoparticles are coated with monoclonal CD86 antibody or MHC class II antibody for macrophage M1 targeting; and NPAPF/C545T nanoparticles are coated with monoclonal CD204 or CD206 antibody for M2 targeting. The size and surface of nanoparticles will be carefully tuned for high stability, efficient delivery to tumour, good penetration to deep tumour, and efficient uptake by macrophages. These nanoparticles can be administered through intravenous injection and evidences have shown that the nanoparticles can be delivered to tumour with high delivery efficiency. Once in tumour, the nanoparticles will specifically be uptaken by macrophages M1 or M2 and present different fluorescent colours. In mouse model, the nanoparticles can be directly imaged to analyse macrophage population and M1/M2 ratio. In human patients, the observation will need to be done through endoscopic fluorescence imaging system.

The novelty of our project is that we will be the first to perform in-situ imaging of macrophage population and analyse M1/M2 ratio to assess the outcome of anticancer therapy and provide guidance for subsequent cancer treatment. Organic nanoparticles will be developed for high potential of being used in clinic in the future. The interaction of these nanoparticles with tumour microenvironment will be investigated as well to understand the working mechanism of nanomedicine and provide guidance of nanomedicine design.

Daisy Dickinson portrait

Daisy Dickinson

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This PhD project will customise new microscope systems using 2nd generation versions of novel laser sources for specific modes of medical imaging. In particular, the laser has potential as an enabling source for new modes of light-sheet microscopy, super-resolution imaging, ratiometric imaging and fluorescence lifetime imaging (FLIM), with significant clinical advantages.

As a first clinical target, we believe that these lasers, used with these techniques, will
offer new opportunities for the analysis of biomedical signalling mechanisms. The unique enabling properties of the laser sources include:
• Tuneable wavelengths across the visible and near-infrared spectrum, enabling improved
fluorophore targeting, lower cross-talk and noise;
• Nanosecond-pulsed operation, enabling fluorescence lifetime imaging;
• Rapid temporal control of pre-programmed wavelength patterns, enabling multiplexing of multiple targets in a single sample and applications such as ratiometric imaging;
• Simultaneous single or multi-colour emissions (i.e. monochromatic or polychromatic arrays), for wide field-of-view excitation and fast data acquisition

Emma Alexander portrait

Emma Alexander

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Development of a fluorogenic platform for imaging second messenger ion flux in macrophages.

This proposal seeks to establish a new bio-orthogonal reaction platform for the formation of fluorophores in response to the flux of metal ions, such as Ca2+. GAB’s group has recently identified aromatic ynamines as a chemoselective substrate for copper-catalysed azide-alkyne cycloaddition (CuAAC) reactions. Ynamines react faster with azides than regular alkynes and require significantly lower catalytic loading of copper. Furthermore, this cycloaddition reaction can be catalyzed by Ca2+. Ca2+ for example, is a known second messenger in all cells. The development of imaging agents which couple the formation of a fluorescent product from a non-fluorescent precursor in response to metal ion flux are unprecedented and their development could form a powerful tool for non-invasive bio-imaging of fundamental cellular function.

In vivo imaging data showed that macrophage interacting with pre-neoplastic cells (PNCs) promote their growth during tumour initiation (YF’s group). Ca2+ signalling is involved in mediating this interaction. However, a lack of appropriate imaging probes are available for Ca2+ imaging, which prevents detailed analysis of when, how and what Ca2+ signals are triggered during PNC macrophage interactions, which might hold a key for the development of preventative agents for cancer.

Giulia Rinaldi portrait

Giulia Rinaldi

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Development of novel imaging probes to detect and investigate ETotic cell death in mammalian and non-mammalian models.

Granulocytic neutrophils play a key role in host defence and often contribute to tissue damage associated with chronic inflammatory diseases (Robb et al., 2016 Semin Immunopathol). NETosis (when occurring in neutrophils) or ‘ETosis’ (when occurring in other cells) is an evolutionary ancient cell death process (Robb et al., 2014 Nat Commun) thought to be important in defence against invading organisms but, when dysregulated, has been implicated in numerous inflammatory diseases. NETosis occurs when stimulated neutrophils cast a web of extracellular fibril matrix containing DNA, histones and a variety of anti-bacterial proteins that serve to ensnare and neutralize invading microorganisms. In terms of composition, the core histones (H2A, H2B, H3 and H4) constitute the majority of neutrophil extracellular trap (NET)-associated proteins of which histone H2A is the most abundant protein studded on externalised DNA. Indeed, histone decoration of chromatin on NETs is a prominent and distinctive feature of NETosis.

This project proposes to use the biology, chemistry and clinical expertise afforded by the supervisors to develop bespoke chemical optical imaging probes to detect and visualise hallmark ETotic markers using established mammalian (human) and non-mammalian models (e.g. zebrafish, protists and archaea). By analyses of chemical modifications of DNA and histones (and other key markers) released from ETotic cells we will build upon and use as structural backbones as a platform for novel probe development.

Having developed optical probes to detect ETosis, ultimately, we will test these probes in primary human neutrophils, human tissue samples including ex vivo human lung tissue, lavage from acutely sick patients in intensive care and children with inflammatory lung disease and innate immune cells of lower vertebrates (zebrafish) and evolutionary ancient organisms (amoeba and archaea). If probe detection is achievable, these probes will represent a novel detection method for ETotic cell death that will be of huge clinical benefit as they could be used for efficient detection of ETosis in a multiplicity of pro-inflammatory diseases states, where ETosis is prevalent. Furthermore, detection of ETosis in lower vertebrates and evolutionary ancient organisms will also be integral to further understand the detrimental effects ETosis exerts in higher mammals.

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Grant Cumming portrait

Grant Cumming

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Environmentally Sensitive Raman Probes for the Molecular Level Imaging of DNA Damage and Repair in Relation to Cancer.

This project proposes a new approach to understand the local environment of DNA, including damaged DNA, within cancer cells using an imaging approach. In particular, we are interested in understanding if we can image changes in the local DNA environment using a new class of environmentally sensitive probes and Raman spectroscopy. This project will focus on the development of these probes, which is based on a specific change in vibration of a tagged molecule, when DNA found within cancer cells changes environment. This change in environment could come about due to different types of DNA damage, DNA base mismatches (e.g. G:C to G:T), or structural rearrangements, following exposure to external stimuli such as radiation or oxidative or alkylating agents.

We propose to build on some exciting preliminary data to test the hypothesis of generating a specific environmentally sensitive probe in a number of different cell models and then in vivo imaging environments using Raman spectroscopy with the view to ultimately moving to stimulated Raman scattering microscopy to provide a faster imaging approach. This is an ambitious collaborative project between a group specialising in spectroscopy and a group with expertise in DNA mismatch repair and its relationship to cancer.

Layla Mathieson portrait

Layla Mathieson

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Optical Imaging of Fibroblast Activation Protein in Humans.

Fibroblast activation protein (FAP) is a cell surface serine protease found on ‘activated’ fibroblasts in many disease states including solid organ malignancies, pulmonary fibrosis and hepatic fibrosis. Importantly, FAP+ cancer-associated fibroblasts are implicated in immunotherapy failure in non-small cell lung cancer (NSCLC).
This interdisciplinary PhD will develop bespoke imaging compounds targeting FAP and will follow the bench-to-bedside journey of:
i) In-house chemical synthesis of FAP optical imaging compounds
• Synthesis of FAP cleavable peptide sequences and labelled boronic acid inhibitors and assessment in vitro to confirm FAP specific binding/cleavage, concentrations, kinetics and specificity.
ii) Lead compound optimisation
• Fluorophore/quencher choices, binding site/peptide modifications for specificity and affinity and scaffold/secondary structure modifications for stability and affinity.
iii) Biological validation in translationally relevant systems
• FAP+CAFs from cancer resections, on ex vivo NSCLC samples and in an ex vivo large animal lung model.
iv) Translational development
• Delivery to a patients undergoing diagnostic bronchoscopy for NSCLC.

This will consolidate the additional commercially-relevant training provided in OPTIMA, as well as providing close collaboration with advanced optical imaging technologies developed from within the PROTEUS project.
Imaging FAP dynamically in humans will allow the use of personalised adaptive treatment regimens in NSCLC to improve patient outcome but the imaging compounds will have applicability in other cancers and inflammatory conditions

Matthew Berry portrait

Matthew Berry

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Through Tissue Imaging of Bacterial Infections using SESORS Imaging.

This project proposes to use specifically designed functionalised metal nanoparticles as optical imaging probes to target bacteria and biofilms at depth through skin and tissue for example deep tissue or joint infections. This will be achieved using functionalised nanoparticles which will be designed to display a unique Raman response as well as having a biorecognition moiety, which targets bacteria specific biomarkers. The advantage of using Raman rather than fluorescence for optical imaging is the molecular specificity of the optical response and the ability to detect multiple SERS responses from multiple targets to be imaged for simultaneously if multiple bacteria exist in, for example, a biofilm. However, perhaps more importantly in this case, is the combination of surface enhanced spectroscopy and spatially offset Raman (SESORS), which allows detection of Raman signals at depth, with our recent work allowing detection of a breast tumour model at depths of 15 mm using a handheld instrument.
In this project we will develop new Raman-active chromophores as highly sensitive surface enhanced Raman probes. The specific reporters will be designed such that they will display Raman responses upon interaction with the target bacteria. We will explore chromophores that undergo a change in their chemical structure when reacting with the target as well as chromophores with shifts in their wavelengths upon target binding which will also change the SERS response. However, SESORS is a new approach and we will initially use model systems in the lab with live bacteria in tissue phantoms to optimise the nanoparticles and response to give a strong SERS response to allow them to be imaged at greater depths and also allow us to develop data analysis methodologies to create images in 2 and potentially 3 dimensions before moving to in vivo experiments. In addition, it may be possible to develop responsive nanoparticle systems that could potentially detect and treat the bacteria in a localised manner reducing the over use of antibiotics
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Photonic Mediated Therapeutics and Imaging.

The project brings together the skills of synthetic chemistry, cell biology, bacteriology and medicine. It aligns with the drive of the AMR agenda, while opening up new opportunities and activities. This PhD has 3 strands:

(a) The synthesis of chemical probes that bind bacteria, become fluorescent when they do so and when illuminated by NIR light cause bacterial destruction. This will include:
(i) The use of dyes that can generate singlet oxygen.
(ii) The use of a redox photo-catalyst to allow optical, catalytic, generation of a “killing” species.
These probes will be applied to tissue models of infection with a drive to apply them within a frugal innovation setting.

(b) An investigation of how/if light activates immune cells, alters their mobility and improves their efficacy of bacterial engulfment and/or killing. This will include an exploration of what wavelength is optimal? (Ref: Scientific Reports, 2016 6:39479/srep39479).

(c) The loading of isolated immune cells with photo-sensitizers (see (i)) as a means of improving their bacterial killing efficacy by the use of illumination. The photosensitizers will be either surface mounted (e.g. via the use of hydrophobic anchors) or internalised (using peptide delivery ligands) – to give an “optically enabled cellular assassin”.

These approaches will be evaluated with immune cells on lab strains of bacteria initially before moving onto clinical isolates (e.g. from tracheal tubes).

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Assessment of diabetes control by imaging skin autofluorescence.

Complications of diabetes are closely associated with diabetes control and substantially reduce the quality of life and life expectancy of sufferers. On a molecular level, these complications are due to protein glycation, ie their modifications by interactions with sugars. Developing a non-invasive clinical tool for assessment of the structure and amount of glycated protein in human tissue is relevant particularly in the following patient groups with diabetes: children, needle phobic and vulnerable patients where obtaining blood samples is problematic. Such an approach would allow frequent monitoring of diabetes via a simple non-invasive measurement through the skin.
Protein-sugar interactions modify local environments of fluorescent aminoacids (Trp,Tyr,Phe) leading to changes in their fluorescence responses. Further reactions give rise to advanced glycation end-products (AGEs), some of them being fluorescent (eg pentosidine and crossline). This gives an opportunity to explore changes in skin autofluorescence to determine different forms and extent of glycation in a patient.

In this project we aim to explore a variety of fluorescence imaging and spectroscopy techniques to investigate the relationship between diabetes control (measured by glucose and HbA1c), protein glycation in vitro and in vivo and develop a portable device for quick and non-invasive determination of the skin glycation condition for potential clinical use.
Initially, we will apply time-resolved fluorescence, including fluorescence lifetime-imaging microscopy (FLIM), to investigate the protein-sugar samples in vitro to establish the changes in the “fluorescence signature” of individual proteins during glycation in controlled conditions. This data will help us in the later stage to interpret fluorescence responses of the skin samples from subjects (GCU Skin Research Tissue Bank) and establish the key features of the fluorescence images of skin from healthy controls and subjects with diabetes. Finally, the measurement procedure will be optimised/simplified to provide quick and clinically useful information on diabetes control as reflected by the HbA1c which is a standardised measure of glycaemic control used in practise. By using time-resolved spectroscopy combined with wavelength selection we aim to discriminate against other endogenous fluorophores such as melanin.