As part of the BBSRC's priority to enhance the Cell Supply Chain (Engineering and Biological Systems Committee) we wish to investigate methods of improving the interactions between biomaterials and mesenchymal stem cells. The use of mesenchymal stem cells in orthopaedic applications is established in the use of bone marrow aspirate and more recently in the use of isolated, purified and expanded homogenous cell populations. Within the body, the local niche within which mesenchymal stem cells reside is important in maintaining the cell phenotype. In response to injury the stem cell niche changes and the cells are stimulated to play a central role in bone repair. Future success of medical applications of mesenchymal stem cells will be dependent on the delivery of cells into the body in a manor that recreates a suitable niche to maintain cell viability and to boost regeneration. To underpin this work we wish to use BBSRC funding to explore how the surfaces of injectable cell delivery systems can be modified to stimulate appropriate cell adhesion and subsequent activities. Cell delivery systems are a new class of biomaterials that carry stem cells into the body and then create a 3D porous environment around the cell population. The biomaterial is normally highly porous and therefore presents a massive surface area to the cell population. The available surface can be engineered to present whole proteins and peptides that bind to specific integrin receptors. Other growth factor mediated effects on cells can also be stimlated with such a surface engineering approach. The student supervisor will be Professor Kevin Shakesheff, School of Pharmacy, The University of Nottingham. Under this project the student will collaborate with Ms Brigitte Scammell (Orthopaedic Surgeon and Academic) to isolated and characterise human mesenchymal stem cell populations. Following a careful literature review the student will surface engineer polymer surfaces with peptides that bind to the cells (this builds on work by Professor Shakesheff in collaboration with the University of Southampton, BONE 29 (6): 523-531 DEC 2001). The ability to augment osteoconductive behaviour (with additional ceramic components added to the biomaterial) will be used as in vitro quantification methodology. The student will work with Ms Scammell to develop animal models to quantify the effect of surface engineering on angiogenesis and bone repair.

Recent studies indicate RNA modification is an important biological process, relevant to RNA fate, and interactions with other molecules. RNA modification is thought to play a role in multiple cellular, developmental and disease processes. It is estimated that there are more than 170 different RNA modifications, and their significance has yet to be understood. This project involves experiments to characterise and understand the role of RNA modification in transcript processing using cell lines in which specific transcripts are retained within the nucleus prior to degradation. We have established cell lines from patients with a condition called myotonic dystrophy (DM) which is associated with progressive muscle weakness and wasting, in addition to cognitive decline and cataracts. Many aspects of the condition are associated with premature ageing. DM is dominantly inherited, caused by an expanded DNA sequence (CTG) which is transcribed but the mutant RNA is trapped in the nucleus where it forms distinct ribonuclear foci that can be visualised by in situ hybridisation. Recent digital PCR data indicate the mutant transcript is present in a 7x excess compared to the wild-type transcript. This transcript is tagged for degradation by a mechanism that is not understood, but which likely involves RNA modification. This project involves studies based on the unique cell lines from which the mutant RNA will be captured using complementary bead-pull down and analysed by a series of molecular, cellular and chemical methods. The sequencing part of the project will involve RNA-modified standards prepared in the School of Chemistry. In addition to the cell lines described above, the project will involve CRISPR Cas9 genome engineering to introduce an inducible promoter upstream of the DNA repeat expansion such that expression of the mutant RNA can be controlled by doxycycline induction. This will permit higher levels of the mutant transcript to be generated for capture and analysis. The repeat expansion RNA will be analysed using a series of techniques including super resolution microscopy, Nanopore sequencing, thin layer chromatography and mass spectrometry. The aim of the project is to study RNA modification at single nucleotide resolution to provide an insight to the molecular processes underpinning RNA metabolism.

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To compete with existing chemical manufacturing processes based on petrochemical derived raw materials, low cost feedstocks for biological fermentation processes are essential, since the feedstock typically equates to >60% of the overall production cost. In addition, the yield based on carbon needs to be high, which is very difficult to achieve with sugars or cellulosic feedstocks with a high oxygen content, since the oxygen is lost as CO2. The same holds true for the production of biofuels. Methane (CH4) and methanol(CH3OH) are one of the lowest cost carbon sources available in the abundance required to produce bio-based commodity chemicals on a scale that could replace existing chemical manufacturing processes. Efforts to use natural gas in transportation, either directly or by conversion to a liquid fuel, have been spurred by recent increases in available supply and a growing price spread between natural gas and petroleum, especially in the United States. A disruptive production process based on CH4 and/or CH3OH would accelerate the growth and market penetration of biobased chemicals and fuels considerably, not only replacing existing chemical processes, but also 1st and 2nd generation sugar/cellulosics processes. Aerobic methanotrophs represent the only available route for methane bioconversion, activating methane to methanol via methane monooxygenase(MMO) and subsequently converting methanol to formaldehyde en route to chemical and fuel production. AIM: In this project, we will explore the possibility of using methane as a feedstock for the production of high value chemicals; to improve the rates and energy efficiencies of methane uptake, as well as approaches to engineer high-productivity methane conversion organisms. STRATEGY: Our aim will be progress through the following activities:- (i) identifying the most appropriate methane/methanol-utilising chassis; (ii) implementing the requisite gene technologies for modifying the organism; (iii) using synthetic biology to engineer the strain to produce an exemplar platform chemical, and; (v) optimising the fermentation process to be used in a continuous stirred tank reactor (CSTR) as benchmark for the evaluation of production strains in various gas fermentation reactor designs.