21 research outputs found
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Engineering rFVIIa-loaded platelets; a novel approach to treating acute bleeding
Haemorrhage remains a leading cause of mortality around the world, resulting from both trauma and surgery. Current treatments for acute haemorrhage include blood products such as fresh frozen plasma, as well as platelet transfusion and recombinant clotting factors such as rFVIIa. However, the use of recombinant clotting factors is limited due to cost and adverse side effects resulting from increased thrombosis, such as increased rate of myocardial infarction and cerebrovascular accidents.
Platelets are small anucleate cells that exist in very large quantities in our blood and, along with cross-linked fibrin from the coagulation cascade, are involved in forming a haemostatic plug upon exposure to damaged endothelium in a wound. They are metabolically active and undergo a process of platelet activation when stimulated by pro-thrombotic agonists such as thrombin resulting from the coagulation cascade. This then triggers a process whereby the contents of their granules are released locally to facilitate coagulation.
This thesis explores the possibility of loading these platelet granules with recombinant clotting factors, in this case rFVIIa which has already been shown in clinical trials to result in a significant decrease in mortality from acute haemorrhage. The targeted delivery of this drug is explored, both through endocytosis and genetic engineering of megakaryocytes differentiated in vitro from induced pluripotent stem cells (iPSCs). These are evaluated with in vitro assays to model the process of clot formation, as well as an in vivo model of haemostasis to compare their efficacy to conventional treatments. Overall, this serves as a proof of concept of the engineering of platelet granules as a novel drug delivery system.Cambridge School of Clinical Medicine, the Rosetrees Trust and the Frank Edward Elmore Fun
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Forward programming of human pluripotent stem cells to a megakaryocyte-erythrocyte bi-potent progenitor population: an in vitro system for the production of platelets and red blood cells for transfusion medicine.
There exists a need to produce platelets in vitro for use in transfusion medicine, due to increased platelet demands and short shelf life. Our lab uses human induced pluripotent stem cells (iPSCs), as an attractive alternative supply, as iPSCs can be cultured indefinitely and differentiate into almost any cell type. Using a technique called forward programming, we over express three key haematological transcription factors (TFs), pushing iPSCs towards the megakaryocyte lineage, to produce mature megakaryocytes, the platelet precursor cell type.
A major limitation of the forward programming technique is a reliance of lentiviral transduction to overexpress the three TFs, which leads to a number of issues including heterogeneity and high experimental costs. To overcome this, I have developed an inducible iPSC line by inserting the forward programming TFs into a genomic safe harbour, using genome editing techniques. TF expression is strictly controlled, with the TFs expressed only after chemical induction. Inducing forward programming is an efficient method for producing mature megakaryocytes and these cells maintain higher purity in long-term cultures, when compared to cells produced by the lentiviral method.
Removing the requirement of lentiviral transduction is a major advancement, making forward programming more amenable to scaling-up, thus moving this technology closer towards our goal of producing in vitro platelets for use in transfusion medicine. I have also shown that forward programming generates a bi-potent progenitor population, from which erythroblasts can be generated, by altering only media conditions. As for megakaryocyte cultures, inducing forward programming improves the purity of erythroblasts produced, compared to the lentiviral method.
I have developed single cell progenitor assays combined with index sorting of different cell surface markers, to allow retrospective analysis of cells which successfully generate colonies. The aim of this work is to better characterise the progenitor cells produced by forward programming, to allow further study of this cell type. Single cell RNA-seq of megakaryocytes revealed heterogeneity in long-term cultures and also identified novel candidate surface markers that may help to further characterise the progenitor cell population.British Heart Foundation funded PhD
Correlation of influenza infection with glycan array
Poster Presentation: SPB1 / SPB2 - Virus Host Interaction/Pathogensis/Transmission: abstract no. B109PINTRODUCTION: The past 6 years has seen the introduction of glycan arrays containing large numbers of sialic acid (Sia) containing compounds and these arrays have been used to demonstrate the relative binding affinity of influenza viruses to different glycans. Though infor...postprin
Molecular and computational approach to the link between nutrition and cancer
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Física Aplicada. Fecha de lectura: 22-11-201
An analysis of target recipient groups for monovalent 2009 pandemic influenza vaccine and trivalent seasonal influenza vaccines in 2009-10 and 2010-11
Poster Presentation: SPA5 - How to Evaluate Vaccine Effectiveness and Efficacy?: abstract no. A513PINTRODUCTION: Vaccination is generally considered to be the best primary prevention measure against influenza virus infection. Many countries encourage specific target groups of people to undertake vaccination, often with financial subsidies or a list of priority. To understand differential patterns of national target groups for influenza vaccination before, during and after the 2009 influenza pandemic, we reviewed and identified changes in national target groups for trivalent seasonal influenza and the monovalent 2009 pandemic influenza vaccines dur...postprin
Efficacy of live attenuated seasonal and pandemic influenza vaccine in school-age children: a randomized controlled trial
Poster Presentation: SPA5 - How to Evaluate Vaccine Effectiveness and Efficacy?: abstract no. A508PBACKGROUND: A novel pandemic influenza A(H1N1) virus emerged in North America in early 2009 and rapidly spread worldwide. Monovalent pH1N1 vaccines were licensed later in 2009 based on preliminary studies demonstrating their immunogenicity and safety. In this study we report the efficacy of live attenuated monovalent pH1N1 vacc...postprin
The Impact of Flow on the Nuclear Translocation of NF-kB
In this dissertation, the impact of flow on the nuclear translocation of NF-kB in vascular endothelial cells was investigated. NF-kB is a key promoter of inflammatory responses and its mis-regulation is related to the development of vascular diseases. The aim was to establish a link between hemodynamic forces and NF-kB to gain insight in cardiovascular disease mechanisms such as aneurysms. Human umbilical vein endothelial cells (HUVEC) were transfected with two plasmids: H2B-mCherry and GFP-RelA. The nuclear translocation of NF-kB within the transfected primary cells was verified with TNF-a stimulation and compared to immunohistochemistry of TNF-a stimulated non-transfected cells. In the first part of the thesis, transfected HUVECs were exposed to different flow environments, including uniform low shear stress, uniform high shear stress and a shear stress gradient, and imaged live for 6 hours. Computer vision techniques were applied to track each individual cell and each nuclear NF-kB concentration was evaluated as a function of time. In each experiment, more than 1000 single cells were tracked and analysed. TNF-a stimulation caused a synchronised population response with a nuclear NF-kB peak at 30 minutes. The population mean of cells under static conditions remained constant, while spontaneous nuclear translocation of NF-kB in individual cells was observed. Uniform low shear stress stimulation increased translocating activity after 5 hours of flow. Alternatively, uniform high shear stress promoted increased nuclear translocation, directly after onset of flow and after 5 hours. Small differences of nuclear translocation of NF-kB at different shear stress magnitudes within a shear stress gradient were observed. The percentage of cells experiencing early nuclear translocation increased with increased shear stress. It is believed that high shear stress induces nuclear translocation early, while for low shear stresses, responses are delayed. In the second part of the thesis, a numerical model was developed to predict cell population responses of the NF-kB pathway. The model is deterministic but includes extrinsic noise to mimic stimulus dependent cell-to-cell variability. Population responses to different TNF-a concentrations were predicted in close agreement with live-cell measurements. The model was extended with a shear dependent activation module to predict small variabilities observed in nuclear translocation of NF-kB under different shear conditions. The close agreement between nuclear translocation of NF-kB in a shear stress gradient and the measurements allowed prediction of inflammatory responses in different flow environments such as a backward facing step channel. This work provides a first insight in the temporal dynamics of nuclear translocation of NF-kB in a large population of endothelial cells exposed to different flow environments. Although the effects were much weaker than with TNF-a stimulation, differences between static and flow conditions were observed, which indicate that hemodynamic forces affect intracellular signalling
The Retina in Health and Disease
Vision is the most important sense in higher mammals. The retina is the first step in visual processing and the window to the brain. It is not surprising that problems arising in the retina lead to moderate to severe visual impairments. We offer here a collection of reviews as well as original papers dealing with various aspects of retinal function as well as dysfunction. New approaches in retinal research are described, such as the expression and localization of the endocannabinoid system in the normal retina and the role of cannabinoid receptors that could offer new avenues of research in the development of potential treatments for retinal diseases. Moreover, new insights are offered in advancing knowledge towards the prevention and cure of visual pathologies, mainly AMD, RP, and diabetic retinopathy