Development of an in vitro Model to Explore the Impact of mtDNA Mutations on the Metabolism and Epigenome of Myogenic Cells

The work presented in this thesis describes the successful establishment of an in vitro model of mitochondrial disease using hiPSC technology and targeted differentiation towards disease-relevant skeletal muscle cell types, which was then utilised to explore the impact of mtDNA disease pathogenic variants on histone acetylation modifications. hiPSC lines harbouring a number of different disease-associated mitochondrial DNA (mtDNA) pathogenic variants were first established, through reprogramming of mitochondrial disease patient fibroblasts. By taking advantage of the random segregation of mutant mtDNA in patient fibroblast populations, hIPSC lines with high heteroplasmy for disease modelling were successfully established, alongside isogenic control lines with low/undetectable levels. Whilst the m.8344A>G pathogenic variant is detrimental to cell reprogramming and pluripotency maintenance, the m.3243A>G does not overtly impact the reprogramming process. A targeted differentiation protocol was then optimised in order to differentiate selected mtDNA disease hIPSC lines into disease-relevant myogenic cell types including terminally differentiated myotubes which show characteristic deficits in mitochondrial function. Whilst mid-range m.8344A>G mutation loads do not overtly impact the myogenic differentiation process, impairments in both myogenic differentiation and subsequent maturation of myotubes are observed in lines harbouring mid-range and higher m.3243A>G mutation loads. Assessments of metabolically sensitive histone acetylation modifications at early and later stages of the myogenic differentiation process revealed cell lines harbouring the m.3243A>G pathogenic variant display widespread deficits in a histone acetylation deposition and/or maintenance. Together, the data presented in this thesis show that hiPSC-derived myogenic cell types represent a useful in vitro model for exploring early pathomechanisms contributing to myopathic phenotypes in mitochondrial disease states. These data show for the first time that mitochondrial deficits caused by mtDNA pathogenic variants can negatively impact metabolic fluxes contributing to histone acetylation deposition and associated physiologically relevant myogenic cell-identity transitions. Modulators of histone acetylation modifications might therefore represent a novel target for therapeutic intervention in human mitochondrial diseases

In memoriam: Ralph McInerny (1929-2010)

Obituari

Separatism in Brittany

The introduction to the thesis attempts to place the separatist movement in Brittany into perspective as one of the various separatist movements with in France, It contains speculation on some possible reasons for the growth of separatist feeling, and defines terms that are frequently used in the thesis. Chapter One gives an account of Breton history, tracing Brittany's evolution as an independent state, its absorption by France, the disappearance of its remaining traces of independence, and the last spasms of action to regain this independence after having become merely part of a centralised state. Chapter Two examines the beginnings and development of a renewed effort to regain some measure of independence, and covers in some detail the period from the early nineteenth century to the end of the Second World War, known in Brittany as that of the first and second emsavs. To clarify a complicated period of development, a lexicon, a list of parties, groupings and devices of the Breton movement, and two flow charts summarising the movement's development from 1914 to I939 are given at the beginning of this chapter. Chapter Three deals with the period from I945 to the present day, known in Brittany as that of the third emsav, and examines in some detail the present state of the Breton movement. Chapter Four examines the work of various Breton writers who have played some part in expressing or shaping the Bretons' awareness of their separate identity, and shows to what extent their writings reflect the historical and political development of Brittany, Chapter Five contains the writer's conclusions and one detailed examination of Breton attitudes to the Breton movement, which helps to put it into its overall Breton perspective. The most important of the appendices to the thesis is the latest available detailed breakdown of the movement

The application of process analytical technologies (PAT) to the dairy industry for real time product characterization - process viscometry

peer-reviewedThe ideal PAT tool is an inline instrument that can monitor and measure process parameters simultaneously in real time while operating in a highly automated environment. Instruments must be of sanitary design, operate robustly within the full process cycle (production and cleaning). Inline determination of the rheological properties of moving fluids (i.e. dairy concentrates) is one of the process parameters where PAT tools can be add real value in terms of optimising process control. Measurement of process viscosity is crucial in the monitoring and control of a variety of concentration processes in the dairy industry. Continuous monitoring of the rheological behaviour of the fluid can allow for optimisation of the process e.g. pumping (avoid pump blockage and failure), evaporation (limit fouling and maximise water removal) and spray drying (avoidance of nozzle fouling). This review concentrates on the state of the art developments being made in the area of process viscometry

A comparison of pilot-scale supersonic direct steam injection to conventional steam infusion and tubular heating systems for the heat treatment of protein-enriched skim milk-based beverages

peer-reviewedDirect supersonic steam injection, direct steam infusion, and indirect tubular heating were each applied to protein-enriched skim milk-based beverages with 4, 6 and 8% (w/w) total protein, and the effect of final heat temperature on the physical properties of these beverages was investigated. Supersonic steam injection resulted in significantly lower levels of denaturation of β-lactoglobulin (34.5%), compared to both infusion (76.3%) and tubular (97.1%) heating technologies. Viscosity, particle size and accelerated physical stability of formulations did not differ significantly between the heating technologies, while noticeable colour differences due to heat treatment (mainly attributed to increasing b* value) were observed, particularly for tubular heating. Overall, the extent of protein denaturation in high-protein dairy products was significantly influenced by the particular heating technology applied. The application of supersonic steam injection technology, with rapid heating and high shear characteristics, may enable differenciated product characteristics for ready-to-drink ambient-delivery high-protein dairy beverages. Industrial relevance: The design and application of novel direct supersonic steam injection technology was comprehensively studied and found to provide significant benefits over direct steam infusion and indirect tubular heating technologies for skim milk-based protein beverages. This type of injection heating system resulted in heat-treated formulations with lower levels of denatured whey proteins, compared to tubular and infusion heating, offering an alternative opportunity to the industry in terms of producing shelf-stable dairy protein beverages

Managing Household Waste in Ireland: Behavioural Parameters and Policy Options. ESRI WP295. May 2009

Ireland has signed up to ambitious targets for diverting municipal solid waste from landfill. These targets are likely to be very difficult to meet without substantial changes to the way household waste is collected and managed. Data on household waste management behaviour in Ireland is scarce, and policymaking could benefit from improved data and market analysis. In this paper we use data from the EPA and CSO to estimate econometric models of household waste collection in Ireland, providing national estimates of income elasticities of demand, price elasticities where unit charges are in place, effects of imposing weight-based charging and effects of other important changes to service characteristics. These results are then used in a simulation model to illustrate the likely effects of some current policy options

PINK1: From Parkinson's disease to mitophagy and back again

The genetics of Parkinson's disease has been key to unravelling the PINK1-dependent mitophagy process. Here, we discuss the implications of a 2010 PLOS Biology paper that shed light on the functional importance of PINK1 in the mitophagy cascade

Osmotic stress tolerance mechanisms in Lactococcus lactis

The response of Lactococcus lactis subsp. cremoris NCDO 712 to low water activity (aw) was investigated, both in relation to growth following moderate reductions in the aw and in terms of survival following substantial reduction of the aw with NaCI. Lc.lactis NCDO 712 was capable of growth in the presence of ≤ 4% w/v NaCI and concentrations in excess of 4% w/v were lethal to the cells. The presence of magnesium ions significantly increased the resistance of NCDO 712 to challenge with NaCI and also to challenge with high temperature or low pH. Survival of Lc.lactis NCDO 712 exposed to high NaCI concentrations was growth phase dependent and cells were most sensitive in the early exponential phase of growth. Pre-exposure to 3% w/v NaCI induced limited protection against subsequent challenge with higher NaCI concentrations. The induction was inhibited by chloramphenicol and even when induced, the response did not protect against NaCI concentrations> 10% w/v. When growing at low aw, potassium was accumulated by Lc. lactis NCDO 712 growing at low aw, if the aw was reduced by glucose or fructose, but not by NaCI. Reducing the potassium concentration of chemically defined medium from 20 to 0.5 mM) produced a substantial reduction in the growth rate, if the aw was reduced with NaCI, but not with glucose or fructose. The reduction of the growth rate correlated strongly with a reduction in the cytoplasmic potassium concentration and in cell volume. Addition of the compatible solute glycine betaine, partially reversed the inhibition of growth rate and partially restored the cell volume. The potassium transport system was characterised in cells grown in medium at both high and low aw. It appeared that a single system was present, which was induced approximately two-fold by growth at low aw. Potassium transport was assayed in vitro using cells depleted of potassium; the assay was competitively inhibited by Na+ and by the other monovalent cations NH4+, Li+, and Cs+. There was a strong correlation between the ability of strains of Lc. lactis subsp. lactis and subsp. cremoris to grow at low aw and their ability to accumulate the compatible solute glycine betaine. The Lc. lactis subsp. cremoris strains incapable of growth at NaCI concentrations> 2% w/v did not accumulate glycine betaine when growing at low aw, whereas strains capable of growth at NaCI concentrations up to 4% w/v did. A mutant, extremely sensitive to low aw was isolated from the parent strain Lc. lactis subsp. cremoris MG 1363, a plasmid free derivative of NCDO 712. The parent strain tolerated up to 4% w/v NaCI and actively accumulated glycine betaine when challenged at low aw. The mutant had lost the ability to accumulate glycine betaine and was incapable of growth at NaCI concentrations >2% w/v or the equivalent concentration of glucose. As no other compatible solute seemed capable of substitution for glycine betaine, the data suggest that the traditional; phenotypic speciation of strains on the basis of tolerance to 4% w/v NaCI can be explained as possession or lack of a glycine betaine transport system