<p>Historically, the meat industry has struggled to provide consumers with consistent beef tenderness. Various post-harvest technologies have been used in industry; however, there is still a need to develop a natural and safe post-harvest processing system that can be used to create consistently tender products for consumers. In addition to postmortem aging being a time-consuming process, literature has suggested that it is not a sufficient method to achieve tenderization in certain cull cow muscles. This has resulted in the large supply of cull cow beef to be underutilized due to its inferior quality, specifically tenderness. Applying a combination of mechanical tenderization with additional postmortem aging may be an effective strategy to overcome deficiencies in beef tenderness. Recent studies have found that tumbling without brine addition can be successful at improving instrumental tenderness and consumer liking of tenderness of fresh beef loin. The physical disruptions of muscles, which likely occur during tumbling, may enhance activity of proteolytic enzymes and thus induce more tenderization. The overall objective of this thesis was to investigate the effects of fresh beef tumbling methods and postmortem aging times on the tenderness and proteolysis of loin muscles from both A maturity cattle and cull cows.</p> <p>The first chapter of this thesis is a literature review that will address the factors affecting tenderness and the methods used by the industry to improve tenderness, specifically focusing on meat tumbling and cull cow beef. The second chapter is a study that investigated the effects of fresh beef tumbling at different postmortem times on meat quality attributes and proteolytic features of loins. The results from this study suggest that early postmortem tumbling coupled with aging can synergistically impact the improvements of beef loin tenderness and proteolysis, shortening the necessary aging period. The third and final chapter of this thesis is a study that aimed to determine the effect of fresh beef tumbling and postmortem aging on the quality and proteolysis of loins from cull cows. The results from this study indicate that aging would be effective at improving the quality and palatability of cull cow beef loins, although tumbling could improve consumer liking of tenderness at earlier postmortem times.</p>

<p>  </p> <p>Tenderness is a key sensory trait influencing beef palatability. Tumbling is a value-adding process that has been extensively applied and studied within the realm of processed meats. Various post-harvest strategies to ensure fresh beef reaches acceptable levels of tenderness have been employed, often with the aim of physically disrupting myofibrillar structure or enhancing the rate and extent of postmortem proteolysis. One such method would be the application of postmortem aging; however, the effectiveness of aging on tenderization is well-known to differ throughout individual muscles of the beef carcass. For inherently tough cuts, physical interventions such as mechanical tenderization are often used, although several detriments to quality attributes may be induced. Further, some modern consumers prefer meat products with no added non-meat ingredients. An alternative method of applying tumbling in the absence of a brine solution followed by additional postmortem aging could be a practical means to facilitate tenderization while potentially minimizing detriments to other eating quality attributes.</p> <p>To evaluate the efficacy of tumbling without brine a method of beef tenderization, the process was first assessed in the <em>longissimus lumborum </em>muscle (n=9). In this study, muscles were allocated among 0, 60, and 90 minutes of tumbling, after which aging for 0, 7, and 14 days was conducted. Immediately after the application of the tumbling process, steaks from muscles that had been tumbled were considerably more tender (24.7 N and 21.6 N for 60 and 90 minutes, respectively) than non-tumbled controls (34.8 N). Steaks from the tumbled groups maintained greater instrumental tenderness throughout the course of the aging period. These results were supported by increases in myofibril fragmentation index, as well as increased troponin-T degradation during aging. However, cooking loss was increased in tumbled steaks, which could have implications for sensory juiciness. Considering this study demonstrated that tumbling without brine inclusion followed by postmortem aging resulted in profound changes to sensory traits, further study regarding its impacts on sensory attributes and proteolysis among different beef muscles was warranted.</p> <p>The following study evaluated the combined tumbling and aging process on the quality, palatability, and proteolytic attributes of beef <em>longissimus lumborum </em>and <em>semitendinosus </em>muscles (n=16). Muscle sections were allocated among 0, 40, 80, and 120 minutes of tumbling, as well as 0 or 10 days of subsequent aging. Regardless of aging duration, tumbling for any duration increased instrumental tenderness of the <em>longissimus lumborum</em> but not <em>semitendinosus</em> muscle. Similar to the previous study, increased cooking loss was induced through tumbling. In both muscles, obvious fragmentation of the myofibrillar structure with tumbling was observed through increases in myofibril fragmentation index and transmission electron microscopy. Tumbling with aging favored the degradation of myofibrillar proteins including troponin-T and desmin; however, calpain-1 autolysis appeared mostly unchanged. Neither tumbling nor aging influenced the amount and properties of collagen, which may indicate why the process did not influence instrumental tenderness of the <em>semitendinosus </em>despite myofibrillar fragmentation and degradation. <em>Longissimus lumborum </em>muscles tumbled for any durations were rated by consumers (n=120) to be more tender with greater overall liking than control steaks. <em>Semitendinosus </em>steaks that were tumbled for 120 minutes and further aged had improved liking of tenderness with similar juiciness and flavor to control steaks at the same postmortem timepoint. These results indicated that tumbling without brine would result in myofibrillar fragmentation and favor the degradation of myofibrillar proteins during aging, while impacts on connective tissues would be minimal. Consequently, muscles without a high extent of background toughness would be effectively tenderized through tumbling, while the results would be more limited in inherently tough cuts.</p> <p>Considering these results, the process was then applied to muscles of intermediate tenderness from the sirloin, specifically the <em>gluteus medius, biceps femoris, </em>and <em>tensor fasciae latae </em>muscles (n=16). Muscles were tumbled for 0 or 120 minutes, then aged for 0 or 10 additional days. Tumbling increased the instrumental tenderness of the <em>gluteus medius </em>and <em>tensor fasciae latae </em>but not the <em>biceps femoris</em>, regardless of aging time. Cooking loss was increased with tumbling in all muscles. Similarly, myofibrillar fragmentation was also increased in all muscles, and there was some evidence to suggest that tumbling with subsequent aging would aid in the degradation of troponin-T in the <em>biceps femoris</em>. To further understand how tumbling might affect specific descriptive sensory attributes, a trained panel (n=8) was conducted on aged samples. Tumbled <em>gluteus medius </em>steaks had greater myofibrillar tenderness than non-tumbled controls; however, tenderness scores of other muscles were not affected. There was some evidence that tumbling with aging could induce the generation of off-flavors in the <em>gluteus medius </em>and <em>tensor fasciae latae</em>, as well as decrease juiciness of the <em>biceps femoris</em>.</p> <p>Taken together, these results support that tumbling without brine inclusion would be an effective strategy to improve beef tenderness and palatability, dependent on the traits of the individual cut. Improved tenderness would be primarily attributed to the fragmentation and degradation of myofibrillar structure. However, the results indicate that tenderization would be limited in cuts with a high extent of background toughness, which tumbling alone would be largely unable to disrupt. Future studies should focus on the effects of tumbling without brine inclusion with aging on oxidative stability and the potential introduction of hazards prior to industry application. Further elucidation of how the process could be optimized to maximize tenderization while minimizing potential negative impacts to flavor and juiciness would be beneficial to improving overall palatability.</p>

Innate immunity is the first line of defense against invading pathogens. It provides immediate protection by initiating both cellular and humoral immune reactions in response to a wide range of infections. It is also important to the development of long-lasting and pathogen-specific adaptive immunity. Thus, studying of the innate immunity, especially the pathogen recognition and signaling modulation, is crucial for understanding the intrinsic mechanisms underlying the host defense, as well as contributing the development of the fight against infectious diseases. Drosophila is an ideal model organism for study of innate immunity. Comparing to mammals, Drosophila immunity is relative conserved and less redundant. A variety of molecular and genetic tools available add further convenience to the research in this system. My work is focused on the signaling modulation by post-translational modification after activation. In these studies I demonstrated in the center of Imd pathway, the Imd protein undergoes proteolytic cleavage, K63-polyubiquitination, phosphorylation, K63-deubiquitination and K48-polyubiquitination/degradation in a stimulation-dependent manner. These modifications of Imd play a crucial role in regulating signaling in response to infection. The characterization of ubiquitin-editing event provides a new insight into the molecular mechanisms underlying the activation and termination of insect immune signaling pathway.

Les dépôts intracellulaires de la protéine Tau agrégée sont la caractéristique commune des tauopathies, une famille de maladies neurodégénératives dont fait partie la maladie d’Alzheimer. Alors que les isoformes de Tau contenant trois (3R) ou quatre (4R) domaines de liaison aux microtubules sont retrouvées à des niveaux similaires dans le cerveau des individus sains, elles diffèrent au sein des inclusions intracellulaires en fonction des tauopathies. Notre étude repose sur l’identification de déterminants structuraux communs et distincts de fibres de Tau 3R et 4R. Pour cela deux approches de protéomique structurale complémentaires ont été mises au point à partir de fibres de Tau 1N3R et 1N4R produites in vitro. La première stratégie, reposant sur l’utilisation de protéolyses ménagées, nous a permis d’identifier les fragments protéolytiques qui composent un « code-barre » moléculaire propre à chaque assemblage. La seconde stratégie a utilisé un marquage chimique covalent des lysines accessibles suivi de l’analyse qualitative et quantitative des acides aminés marqués par spectrométrie de masse. Nous avons ainsi pu montrer que la partie N-terminale de la protéine était accessible au sein des fibres 1N3R et 1N4R tandis que la région C-terminale de la protéine est protégée pour Tau 1N3R et accessible au solvant pour Tau 1N4R. Nos résultats ouvrent la voie à de nouveaux outils moléculaires pour discriminer les tauopathies. / Intracellular deposits of Tau protein aggregates are the common hallmark of tauopathies, a range of neurodegenerative diseases including Alzheimer's disease. Levels of tau with three (3R) or four (4R) microtubule binding repeats are found similar in the normal adult brain, whereas they differ in neuropathological intracellular Tau inclusions, according to the type of tauopathy. Our study consists of the identification of common and different structural molecular determinants of 3R and 4R Tau fibrils. To this end, two proteomic approaches were optimized using 1N3R and 1N4R recombinant fibrils. The first strategy, using limited proteolysis, allowed us to identify the proteolytic fragments composing the molecular “bar-code” for each type of fibril. The second strategy we optimized used chemical covalent surface labelling of accessible lysines, and qualitative and quantitative analysis of the biotinylated residues using mass spectrometry. We show that, while the N-terminal part of the protein remains accessible within 1N3R and 1N4R fibrils, the C-terminal region is protected within 1N3R yet solvent accessible for 1N4R assemblies. Our results pave the way to new molecular tools to discriminate tauopathies.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24801号 / 医博第4993号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 鈴木 実, 教授 溝脇 尚志, 教授 江木 盛時 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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In Parkinson's disease, aggregates of α-synuclein within Lewy bodies and Lewy neurites represent neuropathological hallmarks. However, the cellular and molecular mechanisms triggering oligomeric and fibrillary α-synuclein aggregation are not fully understood. Recent evidence indicates that oxidative stress induced by metal ions and post-translational modifications such as phosphorylation, ubiquitination, nitration, glycation, and SUMOylation affect α-synuclein conformation along with its aggregation propensity and neurotoxic profiles. In addition, proteolytic cleavage of α-synuclein by specific proteases results in the formation of a broad spectrum of fragments with consecutively altered and not fully understood physiological and/or pathological properties. In the present review, we summarize the current knowledge on proteolytical α-synuclein cleavage by neurosin, calpain-1, cathepsin D, and matrix metalloproteinase-3 in health and disease. We also shed light on the contribution of the same enzymes to proteolytical processing of pathogenic proteins in Alzheimer's disease and report potential cross-disease mechanisms of pathogenic protein aggregation.

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Les dynorphines sont des neuropeptides importants avec un rôle central dans la nociception et l’atténuation de la douleur. De nombreux mécanismes régulent les concentrations de dynorphine endogènes, y compris la protéolyse. Les Proprotéines convertases (PC) sont largement exprimées dans le système nerveux central et clivent spécifiquement le C-terminale de couple acides aminés basiques, ou un résidu basique unique. Le contrôle protéolytique des concentrations endogènes de Big Dynorphine (BDyn) et dynorphine A (Dyn A) a un effet important sur la perception de la douleur et le rôle de PC reste à être déterminée. L'objectif de cette étude était de décrypter le rôle de PC1 et PC2 dans le contrôle protéolytique de BDyn et Dyn A avec l'aide de fractions cellulaires de la moelle épinière de type sauvage (WT), PC1 -/+ et PC2 -/+ de souris et par la spectrométrie de masse. Nos résultats démontrent clairement que PC1 et PC2 sont impliquées dans la protéolyse de BDyn et Dyn A avec un rôle plus significatif pour PC1. Le traitement en C-terminal de BDyn génère des fragments peptidiques spécifiques incluant dynorphine 1-19, dynorphine 1-13, dynorphine 1-11 et dynorphine 1-7 et Dyn A génère les fragments dynorphine 1-13, dynorphine 1-11 et dynorphine 1-7. Ils sont tous des fragments de peptides associés à PC1 ou PC2. En plus, la protéolyse de BDyn conduit à la formation de Dyn A et Leu-Enk, deux peptides opioïdes importants. La vitesse de formation des deux est réduite de manière significative dans les fractions cellulaires de la moelle épinière de souris mutantes. En conséquence, l'inhibition même partielle de PC1 ou PC2 peut altérer le système opioïde endogène. / Dynorphins are important neuropeptides with a central role in nociception and pain alleviation. Many mechanisms regulate endogenous dynorphin concentrations, including proteolysis. Proprotein convertases (PCs) are widely expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous Big Dynorphin (BDyn) and Dynorphin A (Dyn A) levels has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild type (WT), PC1-/+ and PC2-/+ animals and mass spectrometry. Our results clearly demonstrate that both PC1 and PC2 are involved in the proteolysis regulation of BDyn and Dyn A with a more important role for PC1. C-terminal processing of BDyn generates specific peptide fragments Dynorphin 1-19, Dynorphin 1-13, Dynorphin 1-11 and Dynorphin 1-7 and C-terminal processing of Dyn A generates Dynorphin 1-13, Dynorphin 1-11 and Dynorphin 1-7, all these peptide fragments are associated with PC1 or PC2 processing. Moreover, proteolysis of BDyn leads to the formation of Dyn A and Leu-Enk, two important opioid peptides. The rate of formation of both is significantly reduced in cellular fractions of spinal cord mutant mice. As a consequence, even partial inhibition of PC1 or PC2 may impair the endogenous opioid system.