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Solving the problem of inconsistent meat tenderness is a top priority of the meat industry. This requires a greater understanding of the processes that affect meat tenderness and the adoption of such information by the meat industry. It is essential that we understand the mechanism of meat tenderisation of which, the calpain protease system is believed to play a central role. This thesis focuses on three aspects; characterisation of calpain activity, the effect of porcine μ-calpain on myofibril degradation and the effect of μ-calpain on specific proteins desmin and troponin-T. To study the effect of calpain activity, fluorogenic assays were used to determine: μ-calpain concentration for optimal peptide cleavage; calcium requirements and the effect of chelating substances on the activity of μ-calpain. In addition, the affinity of μ-calpain for substrates CalS-I and CalS-III were assessed. The effect of μ-calpain on myofibril degradation was evaluated through the use of myofibrillar fragmentation index and density marker beads. Myofibrils were digested at three different temperatures for varying time periods. Conflicting results were displayed and it was concluded that these methods are not accurate, thus further research should be conducted to ensure inconsistencies are eliminated. Specific proteins desmin and troponin-T have previously been shown to exhibit degradation in the presence of calcium and μ-calpain. SDS-polyacrylamide electrophoresis, western blotting and densitometry measurements were utilized to investigate this effect. It was concluded that μ-calpain plays a significant role in the post mortem proteolysis of myofibrillar protein. This thesis provides information and strives to give a better understanding of the proteolytic changes that occur within muscle. Understanding how these mechanisms affect meat on a cellular level, can help to control the influence they inflict on meat quality.

Yes / Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication and transcription. TOP1 is also a major driver of endogenous genome instability, particularly when its catalytic intermediate-a covalent TOP1-DNA adduct known as a TOP1 cleavage complex (TOP1cc)-is stabilised. TOP1ccs are highly cytotoxic and a failure to resolve them underlies the pathology of neurological disorders but is also exploited in cancer therapy where TOP1ccs are the target of widely used frontline anti-cancer drugs. A critical enzyme for TOP1cc resolution is the tyrosyl-DNA phosphodiesterase (TDP1), which hydrolyses the bond that links a tyrosine in the active site of TOP1 to a 3' phosphate group on a single-stranded (ss)DNA break. However, TDP1 can only process small peptide fragments from ssDNA ends, raising the question of how the ~90 kDa TOP1 protein is processed upstream of TDP1. Here we find that TEX264 fulfils this role by forming a complex with the p97 ATPase and the SPRTN metalloprotease. We show that TEX264 recognises both unmodified and SUMO1-modifed TOP1 and initiates TOP1cc repair by recruiting p97 and SPRTN. TEX264 localises to the nuclear periphery, associates with DNA replication forks, and counteracts TOP1ccs during DNA replication. Altogether, our study elucidates the existence of a specialised repair complex required for upstream proteolysis of TOP1ccs and their subsequent resolution. / Supported by the Medical Research Council programme grant (MC_EX_MR/K022830/1) to K.R. J.F. is supported by a CRUK DPhil studentship. K.W. was supported by an MRC studentship and A.R. is supported by a European Molecular Biology Organization (EMBO) long-term fellowship (ALTF 1109-2017). E.D. is supported by the S:CORT consortium which is funded by a grant from the MRC and CRUK. S.F.E. is funded by a Wellcome Trust Investigator award (103844) and a Lister Institute Fellowship.

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La protéolyse péricellulaire représente un phénomène commun à différentes pathologies vasculaires, notamment l’athérome et les anévrismes de l’aorte ascendante humaine (TAA). Le plasminogène est un zymogène plasmatique délivré à la paroi aortique sous l’effet de la conductance hydraulique transmurale où il s’active au contact des cellules musculaires lisses (CML) en plasmine. La protéase nexine-1 (PN-1), une serpine tissulaire exprimée et sécrétée par les CML est capable de réguler l’activité protéolytique des sérines protéases, notamment celle de la plasmine au sein de la paroi aortique. Notre hypothèse est donc que la PN-1 a un rôle protecteur au cours des pathologies athéromateuses et anévrismales via l’endocytose des complexes plasmine-PN-1 par le LRP-1 (LDL receptor related protein-1).Nous avons mis en évidence, dans les tissus des stades précoces de l’athérome humain, une présence du plasminogène, une augmentation d’activité plasmine et une surexpression de laPN-1 et du LRP-1. Les plaquettes et les cellules spumeuses, plus particulièrement les CML représentent les sites majeurs d’accumulation de la PN-1 à ces stades de la pathologie. L’expression de la PN-1 est également modulée par les LDL (low density lipoprotein). Nous avons observé dans les TAA, une augmentation d’expression du plasminogène, de la PN-1 et du LRP-1. L’activité plasmine est aussi augmentée et corrélée avec la production de la PN-1 dans les milieux conditionnés provenant de TAA. Nos données en immunocytochimie ont montré que les complexes plasmine-PN-1 et la PN-1seule sont internalisés via le LRP-1 dans les CML d’aortes saines et de TAA. En revanche, la plasmine seule n’est pas internalisée. En conclusion, nos travaux montrent que la PN-1 a un rôle protecteur dans l’athérome précoce humain et les TAA via l’inhibition de la plasmine et la clairance tissulaire des complexes plasmine-PN-1 par le récepteur scavenger LRP-1 dans les CML. / Pericellular proteolysis is a common phenomenon to various vascular diseases, includinghuman atheroma and aneurysms of the ascending aorta (TAA). Plasminogen is a plasmazymogen delivered to the aortic wall by radial transmural hydraulic conductance, where it isactivated in contact with smooth muscle cells (SMC) into plasmin. Protease nexin-1 (PN-1), atissue serpin expressed and secreted by SMC is capable of regulating proteolytic activity ofserine proteases, especially plasmin activity within the aortic wall. Our hypothesis is that PN-1 has a protective role in the atherosclerotic and aneurysmal pathologies via the endocytosisof plasmin-PN-1 complexes by LRP-1 (LDL receptor related protein-1).We showed in tissue of early stages of human atheroma, a presence of plasminogen, anincrease of plasmin activity and an overexpression of PN-1 and LRP-1. Platelets and foamcells including SMC are the major sites of PN-1 accumulation at those stages of the disease.PN-1 expression is also modulated by LDL (low density lipoprotein).In TAA, plasminogen, PN-1 and LRP-1 were overexpressed. The plasmin activity is alsoincreased and correlated with PN-1 production in conditioned media from TAA.Our immunocytochemical data showed that plasmin-PN-1 complexes and PN-1 alone areinternalized via LRP-1 in SMC from healthy aortas and TAA. However, plasmin alone is notinternalized.In conclusion, our work show that PN-1 has a protective role in early human atheroma andTAA via plasmin inhibition and tissue clearance of plasmin-PN-1 complexes by the scavengerreceptor LRP-1 in SMC.

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