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Funders:
The Wellcome Trust

Results

Controlling phosphodiesterase activity in biofilm dispersal 27 Apr 2017

Biofilm formation and dispersal are controlled by the secondary messenger bis-(3’-5’) cyclic dimeric guanosine monophosphate (c-di-GMP); high levels are associated with biofilm formation, while a reduction induces dispersal. The enzymes catalysing formation of c-di-GMP are diguanylate cyclases, while phosphodiesterases catalyse the breakdown of c-di-GMP. Structural studies of the EAL type phosphodiesterases were observed in the host laboratory in the presence of substrate c-di-GMP, suggesting that these structures would require further steps to attain full catalytic activity. The host laboratory have recently demonstrated that dimerization and active site formation as well as formation of three metal binding sites are distinct activation steps required to tune the enzymatic activity of phosphodiesterases (Scientific Reports 2017). The proposed project deals with the motility regulator protein, MorA, and the mucoid alginate regulator, MucR, from Pseudomonas aeruginosa. Both proteins have dual activity as diguanylate cyclase and phosphodiesterase. Interestingly, the two linked catalytic domains influence each other's activity; different levels of activity are observed for the phosphodiesterases when comparing isolated domains with the linked conformation. The objective of the project is to purify these proteins and determine their catalytic activities, comparing the isolated domains, mixtures of the individual domains, and the cyclase-phosphodiesterase domains in linked conformation.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Southampton

A CRISPR/Cas9-Based Screen for Novel Actin Dynamic Factors 27 Apr 2017

The project will focus on understanding critical aspects involved in the biology of the apicomplexan parasite Toxoplasma gondii (T. gondii), an obligate intracellular parasite that can infect every warm-blooded animal including humans. T. gondii, the causative agent of toxoplasmosis, is of medical importance for human health as an infection can cause severe damage in the developing foetus and immunocompromised persons. In addition, T. gondii serves a model organism for Plasmodium falciparum, the aetiological agent of malaria. In order to invade the host cell apicomplexan parasites rely on their acto-myosin system. Furthermore, recent research highlights the importance of parasite actin during intracellular parasite development. It is thus important to identify and characterise factors involved in the regulation of actin dynamics. Here, cutting edge molecular tools including conditional CRIPSR/Cas9-systems will be applied to screen a subset of essential genes in T. gondii for novel factors involved in the regulation of actin dynamics. Investigating how the parasites regulate these filaments will give valuable insights into how apicomplexan parasites establish infections in their host.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Glasgow

Identification of genes important for susceptibility to taxol-induced apoptosis 27 Apr 2017

CRISPR-based genetic screens have emerged as cutting-edge tools to identify genes important for many processes. Microtubule-disrupting agents, such as paclitaxel (Taxol) and docetaxel (Taxotere), are widely used chemotherapeutic agents that kill highly replicating tumor cells. However, such agents also trigger NFkappaB-dependent responses that can negatively influence tumor clearance by promoting tumor survival and metastasis. Moreover, how transformed cells develop resistance to taxol and related compounds is poorly understood. Here, we wish to use CRISPR-based genome-wide screening methods to screen for genes that, upon inactivation, confer resistance to taxol and related compounds. Identification of genes required for taxol-induced cell death will generate new insights on how taxol initiates apoptosis in tumors as well as potential resistance mechanisms.

Amount: £0
Funder: The Wellcome Trust
Recipient: Trinity College Dublin

Evaluating the combination of Oncotype DX and Neutrophil/Leukocyte Ratios as prognostic indicators of chemotherapy response in breast cancer. 27 Apr 2017

This research will study the effect of the individual’s immune system on their reoccurrence of breast cancer after chemotherapy. This would be a correlation between the Neutrophil to Lymphocyte Ratio (NLR) and the Oncotype Dx score. Previous research (unpublished) has demonstrated the significance of the NRL in predicting breast cancer outcomes in HER 2 overexpressing and Luminal B breast cancer (accounting for approx. 20% of all breast cancers). We will extend our previous research to include all four cancer subtypes (Luminal A and Triple negative breast cancer). We aim to compare the immune system’s reaction to chemotherapy across all four cancer subtypes. A key goal is to determine if NLR predicts outcome or survival in Luminal A or TNBC. A further key goal is to determine if there is any predictive value of combining the NLR with Oncotype scores (in relevant cases).

Identifying Integrative and Conjugative Elements using DLIGHT 27 Apr 2017

Integrative and conjugative elements (ICEs) are mobile genetic elements present in both gram-positive and gram-negative bacteria. They mostly reside in the host chromosome and under certain conditions, will excise and transfer to a new host via the conjugation machinery. ICEs have been found to provide the host with a wide range of phenotypes, including antibiotic and heavy metal resistance and the ability to colonise a eukaryotic host, promote virulence and biofilm formation. The ability of ICE to spread to different species of bacteria through horizontal gene transfer is a major factor in bacterial evolution. Bioinformatics approaches have been increasingly used to identify possible ICEs through sequence similarity. In this project, we aim to find out the effectiveness of using an algorithm, DLIGHT (Distance Likelihood based Inference of Genes Horizontally Transferred) that was originally used to detect lateral gene transfer, to identify integrative and conjugative elements. We will achieve this by assessing DLIGHT's ability to recover already documented ICEs. We will also use DLIGHT to test certain sequences which we suspect to contain ICEs. The predictions of new ICEs will then be vetted through manual analysis and collaboration with experimentalists.

Amount: £0
Funder: The Wellcome Trust
Recipient: University College London

Unravelling Haemophilus influenzae and bacteriophage dynamics in the human upper respiratory tract 27 Apr 2017

Haemophilus influenzae is a pathogen that infect the human upper respiratory tract to cause diseases such as pneumonia and is associated with chronic diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) (Riesbeck, 2010). DNA sequence analysis of H. influenzae revealed the acquistion of novel genes mediated by phages which have not been identified; however, six H. influenzae phages have been identified of which HP1c1 is well characterised (Williams et al., 2002). The pathogen can change phenotype to escape HP1c1 but this makes it more vulnerable to the host immune system suggesting that phages impact the interaction of the of H. influenza with the host cell. During this project, we will isolate new H. influenzae phages from infected patients to better understand the pathogen dynamics in the respiratory tracts which could direct therapeutic towards treating these respiratory tract infections.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Leicester

Evaluation of meningococcal disease isolates for phase variation in pilin adhesion determinants 27 Apr 2017

Neisseria meningitidis is the leading cause of bacterial meningitis. Carriage rates range from 10% for healthy populations to >30% in at risk populations. Invasive serogroup W (MenW) disease is increasing and has led to inclusion of MenACWY vaccine in the national immunisation schedule. Phase variation allows for alterations in outer membrane protein surface expression through slippage across simple sequence repeats (SSRs), altering the coding frame and truncating the protein or reducing promoter activity. Phase variation within pilC genes affects adhesion and virulence. Preliminary data demonstrates that phase variation is responsible for switching between pilC1 and pilC2 expression during carriage raising the potential for an impact on carriage to disease transitions. Key goals of this project are to analyse and compare phase variation in carriage and invasive MenW isolates. Objective 1: reconstruction of pilC loci through bioinformatics and PCR. Objective 2: analysis of variability in SSRs of pilC genes by alignments and GeneScan analysis. Objective 3: expression states of pilC1 and pilC2 will be determined through bioinformatics. Due to highly similar nature of MenW ST-11 genomes, these data will provide an excellent platform to study pilC phase variation within Neisseria and the effect upon bacterial adherence.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Leicester

Ocular Drug Delivery by Using Phase Invertible Implants 27 Apr 2017

Age–related macular degeneration (AMD) currently affects more than 600,000 people in the UK and is the leading cause of vision loss. Intravitreal injections of anti-vascular endothelial growth factor (VEGF) is an effective treatment for AMD patients. However, frequent injections are required in the treatment, which is expensive and often associated with complications and poor patient tolerance. This study focuses on the development of novel ocular drug delivery using in situ forming implants to treat AMD. In this study, in situ formed implants composed of biodegradable polymers will be formulated using PLGA and biocompatible organic solvent. Various properties of the in situ forming systems will be investigated, such as thermal properties, rheological properties, degradation behaviors and in vitro drug release profiles. These in situ forming implants can sustain the release of incorporated drugs, reduce complications caused by frequent administration and ultimately improve patient comfort.

Amount: £0
Funder: The Wellcome Trust
Recipient: Queen's University Belfast

Investigating the mechanisms of Alzheimer’s disease-related epileptic activity 27 Apr 2017

Inflammatory-related epilepsy and aberrant neuronal activity are primary consequences of conditions including infection and brain trauma. The consequences of brain trauma have gained significant attention in recent years, given the association between neurodegenerative conditions like Alzheimer’s (AD) and Parkinson’s disease, and repeated concussive injuries such as those related to contact sport and military activities. The AD brain is characterised by the accumulation of the toxic protein beta-amyloid (Abeta). This acts as a chronic inflammatory stimulus, leading to the excessive activation of microglia and subsequent deterioration in neuronal integrity. Abeta induces many of its inflammatory effects through activation of toll-like receptors (TLRs), similarly to some bacterial infections and trauma-induced inflammation; the adverse effects of which are widely recognised. Microglial TLR expression is increased under such conditions, rendering them more sensitive to infectious agents and Abeta. We have previously highlighted the importance of TLR2 in mediating the negative impact of Abeta on microglial activation and neuronal function. We now propose to examine the cellular mechanisms through which TLR2 stimulation conveys sensitivity to the neuronal dysfunction and excitotoxicity associated with Abeta exposure. Elucidating these mechanisms holds the key to identifying strategies for reducing vulnerability to AD in high-risk individuals.

Amount: £0
Funder: The Wellcome Trust
Recipient: University College Dublin

Deep Gray Matter Changes in Alzheimer's Disease 27 Apr 2017

The septal nuclei and septum pellucidum have not received attention in relation to the study of Alzheimer’s disease(AD), despite evidence that they are involved in memory function. They are connected to the hippocampus & amygdala, which are key regions altered in the earliest stages of AD. Other structures (nuclei) within the basal forebrain have been investigated. The hypothesis that is to be tested is that the septal nuclei and septum pellucidum is smaller in AD patients compared to healthy controls (HC). The second hypothesis is that these structures are also smaller in Mild Cognitively Impaired (MCI) subjects compared to healthy controls. The initial step will be establishing the method for volumetric measurement of these structures in a small cohort of healthy and Alzheimer’s disease patients, in order to demonstrate that volume can be measured reliably. This protocol will then be used to measure the volume of the structures in AD patients, MCI subjects and HC. The three groups will be matched for age, education level, and sex. The study will advance our understanding of the neurodegenerative processes in the basal ganglia in the earliest stages of the disease, while translating what is known already from animal research.

Amount: £0
Funder: The Wellcome Trust
Recipient: Trinity College Dublin

Immunomodulatory effect of cigarette- and electronic cigarette- exposed respiratory pathogens on macrophages in vitro 27 Apr 2017

It has been established that cigarette smoke and E-cigarette vapour can activate inflammatory response and impair antimicrobial functions of human immune cells. However, the direct effects of cigarette smoke and E-cig vapour on respiratory pathogens is less well known. The aim of this project is to further explore the effect of cigarette smoke extract (CSE) and e-cigarette smoke extract (ECSE) on important human respiratory pathogens and their interaction with the host. The immunomodulatory effects of CSE/ECSE exposed respiratory pathogens have on macrophages in vitro will be determined. Western blot analysis will be used to detect activation of the canonical NF-kappaB pathway and caspase-1 activation whilst ELISA assays will be utilized to measure the expression of the cytokines IL-8, IL-12, TNF-alpha and IL-1beta. In addition, susceptibility of bacteria to macrophage phagocytosis and killing will be measured. The findings of this project will provide valuable information about the direct effect of cigarette smoke and e-cigarette on the lung microbiome and its interaction with host immune cells.

Amount: £0
Funder: The Wellcome Trust
Recipient: Queen's University Belfast

The response of the gut microbiota to a range of anti-diabetic medications and its impact on glucose control 27 Apr 2017

The key goal of this project is investigating the effect dietary intake and gut microbiota have on glucose regulation in established diabetic patients prescribed a range of anti-diabetic medications. Evidence from the literature suggests an altered microbiota is associated with changes in multiple heath parameters, including glucose regulation (Cani et al. 2014). Previous studies have illustrated that dietary intake impacts the gut microbiota profile and can enhance its diversity, an independent marker of improved functionality in vivo (Cotillard et al. 2013). This project involves microbial DNA extraction to study the effect of various anti-diabetic medications on the gut microbiota profile (from time of prescribing to 8 week follow up). Dietary intake will be assessed to determine the role of diet on the baseline microbiota in diabetic candidates. Dr. O’Connor’s team have extensive expertise in analysing gut microbiota profiles and determining the role of diet in shaping microbiota in specific populations (Power et al. 2015, O’Connor et al. 2014, Power et al. 2014, O’Connor et al. 2013), especially during aging and aging-related health loss (Claesson et al. 2012). Dr.O'Connors group have numerous ongoing dietary intervention trials to determine the role of specific food ingredients on gut microbiota in specific population groups.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Limerick

Human Skeletal Muscle Wound Repair: An Association between Genetic Variation and Stem Cell Migration? 27 Apr 2017

Insufficient recovery following exercise-induced muscle damage (EIMD) can lead to muscle injury, with natural genetic variation perhaps influencing the extent of both. Skeletal muscle regeneration is a complex process that is mediated by muscle stem cells (called satellite cells). However, little is known about whether specific genetic variations (polygenic profile) influence dynamics of satellite cells and their impact on skeletal muscle regeneration. For that reason, we aim to conduct an in vitro muscle cell damaging study on primary skeletal muscle stem cells (previously derived from participants with specific polygenic profiles) to provide potential mechanisms regarding the individual response to injury in vitro. We hypothesise that activated satellite cells from individuals with a specific genetic profile will display better rates of recovery, following mechanical injury, compared with cells from participants without this profile. Our objective is to utilise stem cell-based research to provide oversight of the in-depth genetic mechanisms underlying the adaptations of muscle cells to injury repair. Our ultimate aim is to use current technology to prevent injury and promote recovery initially in the sporting world, but ultimately in the wider population as exercise becomes more widely accepted as a health intervention.

Bovine Respiratory Syncitial Virus Vaccine 27 Apr 2017

Bovine respiratory syncitial virus (BRSV) is a leading cause of enzootic pneumonia in calves and significantly contributes to the Bovine Respiratory Disease Complex found in feedlot cattle. It is proposed to develop a new vaccine using the Semliki Forest Virus (SFV) virus replicon particle (VRP) vaccine platform. The SFV VRP is a promising vaccine platform, due in part to its efficacious immunogenicity in cattle and its ability to express protein to high concentrations in the cells it infects. Furthermore, the replicon vectors are single cycle, propagation-defective particles that are not able to spread beyond the initial infected cells. The BRSV fusion (F) protein is known to be immunogenic thus making it a good candidate protein to target for gene expression. It is proposed to clone the F protein sequence into a VRP expression plasmid in place of the SFV structural proteins. The proposed project will involve generating the VRP plasmids, generation of VRP stocks and confirmation that this VRP can express the F protein. Human respiratory syncitial virus (hRSV), a closely related virus of human importance, also expresses the F protein. Thus BRSV infection in bovines is a relevant model for hRSV, as there is no current efficacious vaccine available.

Amount: £0
Funder: The Wellcome Trust
Recipient: University College Dublin

Does pharmacological intervention attenuate hallmarks of ocular cancer or blindness in a zebrafish model of Von Hippel-Lindau Disease? 27 Apr 2017

Von Hippel-Lindau (VHL) disease is an autosomal dominant condition affecting 1 in 36000 people. VHL is characterised by the formation of multiple benign and malignant neoplasms. Retinal hemiangioblastomas (RH) are the most common manifestation of VHL disease, presenting in up to 85% of patients. Vision loss is a common clinical presentation in VHL patients as current therapies are invasive and not effective. Furthermore, removal of RH is restricted to the peripheral retina. Although zebrafish do not develop RH, they are a useful in vivo model in the vhl studies as they possess many hallmarks associated with the disease. Defects in the vhl tumour suppressor gene leads to a systemic hypoxic response and subsequent uncontrolled blood vessel development in vivo. Ectopic and vasculature leakage has previously been shown in the vhl zebrafish model. This research aims to investigate the efficacy of a commonly used anti-angiogenic compounds (quininib analogues), in reducing the angiogenic hallmarks displayed by vhl patients. Its ability to rescue or improve visual function will be measured and endpoints will employ behavioural and morphological analysis.

Amount: £0
Funder: The Wellcome Trust
Recipient: University College Dublin

Can factors secreted by MUC1-ST educated monocytes influence breast cancer stem cell growth? 27 Apr 2017

Aberrant glycosylation of glycoproteins expressed by cancers can lead to tumour:immune cell interactions that are cancer specific. One such interaction is the binding of a tumour-associated glycoform of the mucin, MUC1-ST, to the sialic binding lectin, Siglec-9, expressed by monocytes and macrophages. The engagement of Siglec-9 by MUC1-ST, leads to the secretion of factors by monocytes associated with tumour-progression. Interestingly, one such factor that has been identified is growth hormone and the growth hormone receptor has been found to be associated with normal mammary stem cells. To investigate if growth hormone or other factors secreted by MUC1-ST educated monocytes can influence breast cancer stem cells we will examine the extent to which breast cancer cells lines show cancer stem cell properties by: 1. Determining the expression of the breast stem cell markers (CD44 and CD24) using flow cytometry, in the presence or absence of media from MUC1-ST educated monocytes. 2. Looking at mammosphere growth in the same medium, where we will also take into account the production of GH by blocking the GHR using specific blocking antibodies.

Amount: £0
Funder: The Wellcome Trust
Recipient: King's College London

Drug-eluting implants made of "green" materials for long lasting therapies 27 Apr 2017

Controlled release implants offer many potential advantages over traditional, periodic systemic injections including reduction or elimination of unwanted side-effects, enhanced efficacy and increased patient convenience and compliance. Implantable drug delivery devices also offer several advantages over conventional oral or parenteral dosage forms. Firstly, implantable devices allow site specific drug administration where the drug is needed most. For this application, biodegradable materials are particularly attractive as they require no surgical retrieval. Naturally-derived biodegradable materials generally offer superior biocompatibility and milder processing compared with synthetic degradable materials. The main aim of this project is to develop drug eluting implants based on natural biopolymers providing long lasting drug release. The devices will be formulated using two natural ocurring polymers zein and lignin. These two biopolymers can be obtained from renewable sources and consequently are a more environmental-friendly option and will reduce significantly the cost of the implants.Model molecules will be used to evaluate drug release: nile red as a model of hydrophobic drugs and methylene blue as a model for hydrophilic drugs. This will be achieved by: Formulation and characterization of zein/lignin implants containing hydrophobic and hydrophilic molecules. Evaluating biocompatibility of the developed systems. Evaluating drug release from the developed systems.

Amount: £0
Funder: The Wellcome Trust
Recipient: Queen's University Belfast

The impact of systemic inflammation on the brain. 27 Apr 2017

Brain tissue from adult mice has already been collected, with a control group treated with saline and another treated with an attenuated strain of Salmonella typhimurium, triggering a systemic immune response including production of pro-inflammatory mediators that communicate with the brain triggering neuroinflammation and neuronal dysfunction. Selected groups of mice have been treated with two different inhibitors that block the activity of the cytokine IL-1beta, in order to investigate if this pro-inflammatory cytokine plays a direct role in neuroinflammation during systemic infection. In this project we will compare a small molecule with a biologic, both targeting cytokines, but via different mechanisms. The brain tissue will be examined using antibodies to stain activation markers of microglial cells (CD11b, FcRI, MHCII) and activation markers of cerebral endothelial cells (VCAM, ICAM, MHCII). Synaptic activation markers, from both the pre- and post-synaptic membranes, will be investigated by isolated mRNA from hippocampal enriched tissue. This investigation into phenotypic changes in microglial, cerebral endothelial cells and BBB function will be used to determine if the inhibition of IL-1beta is beneficial in reducing neuroinflammation as a result of systemic infection. This could later be applied to AD models, taking into consideration behavioural changes.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Southampton

Investigating bacterial transporters that convey antibiotic resistance 27 Apr 2017

The rise of antimicrobial resistance (AMR) threatens many of the major advances in modern healthcare as the treatments rely on the implicit ability of the antibiotic to allow the body to heal from a serious infection. Antibiotic efflux transport is central to the development of AMR. At the single cell level, it is becoming apparent that the use of efflux pumps is the first line of defence against an antibiotic. These pumps decrease the intracellular level of antibiotic while the cell activates the various other levels of protection. This frontline of defence involves a coordinated network of efflux transporters. In the future, inhibition of this efflux transporter network, as a target for novel antibiotic therapy, will require the isolation and then biochemical/biophysical characterisation of each pump against all known and new antibiotics. This depth of knowledge is required so that we can fully understand and tackle the mechanisms of developing antimicrobial resistance. The key goals of this project are to over-express, IMAC purify a hexahistidine-tagged versions of a number of the efflux transporter and then characterise ligand binding against a set of clinically relevant antibiotics. The method used to measure ligand binding is thermophoresis (Nonotemper, Monolith).

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Southampton

Automation of the genotyping process for the myotonic dystrophy type 1 CTG repeat locus using next generation sequencing. 27 Apr 2017

Myotonic dystrophy type 1 (DM1) is a severe inherited neurological disorder caused by an expansion of CTG repeats in the DMPK gene. More repeats cause more severe disease and an earlier age at onset. Genotyping of the DMPK CTG repeat is usually determined by fragment length analysis. The DMPK CTG repeat is not usually genotyped in standard next generation sequencing protocols due to a combination of problems with read length and mapping. We have solved most of these problems developing new methods for high-throughput sequencing of DMPK CTG repeat alleles. In this project we will develop an automated bioinformatic pipeline for genotyping repeat lengths at the DMPK locus by adapting a pipeline we have already developed for automated genotyping of the Huntington disease CAG repeat. Adapting the pipeline to automated genotyping of the DMPK locus will require optimisation of the trimming and alignment parameters and reprogramming in Python of the machine learning component used to automatically call the diploid genotypes from the read length distributions. This new method will facilitate research to investigate the causes of variability of myotonic dystrophy, lead to an improved diagnostic test, and to new methods for screening for this disorder in the general population.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Glasgow