Effect of myosin heavy chain isoform composition on tenderness of bovine muscles from carcasses of two skeletal maturities

De La Zerda, Michael James Texas A&M University 2001 해외박사(DDOD)

Bovine muscles (<italic>m. semitendinosus</italic> and <italic>m. longissimus lumborum</italic>) from carcasses of A- and E-skeletal maturity (SMAT) were evaluated for percentage composition of myosin heavy chain isoforms (MHC) while in the pre-rigor state, pre-rigor calpastatin and m-calpain activity, Warner-Bratzler shear force (WBS) at 7 and 14 d of postmortem aging, and gravimetric fragmentation index (GFI) at 0, 7, and 14 d of postmortem aging. Percentage composition of MHC-I isoform did not (<italic>P</italic> > 0.05) change between muscles of A- and E-SMAT carcasses. However, muscles of E-SMAT carcasses contained (<italic>P</italic> < 0.001) a lower percentage composition of MHC-IID compared to muscles of A-SMAT carcasses. Muscles of A-SMAT carcasses contained a lower (<italic>P</italic> < 0.0001) percentage of MHC-IIA isoform compared to muscles from carcasses of E-SMAT. The eye of round contained a lower (<italic>P</italic> < 0.0001) percentage of MHC-I isoform compared to the loin, while percentage MHC-IIA did not differ (<italic> P</italic> > 0.0001) between the two subprimals. Percentage MHC-IID (<italic> P</italic> < 0.0001) was higher in the eye of round compared to the loin. MHC composition can be used as an indicator of tenderization, but it is important to clarify that other factors such as connective tissue also contribute to the increased toughening exhibited by muscles of advanced skeletal maturity carcasses. Calpastatin activity was lower (<italic>P</italic> < 0.05) in muscles from carcasses of E-SMAT, but m-calpain activity was similar (<italic>P</italic> > 0.05) in muscles of A- and E-SMAT carcasses. There was no difference (<italic> P</italic> > 0.05) in calpastatin or m-calpain activity between subprimals. Lower calpastatin levels in carcasses of E-skeletal maturity may be due to the degenerative machinery associated with the aging process. Results indicated that carcasses of advanced skeletal maturity produced muscle that was higher (<italic>P</italic> < 0.0001) in WBS than youthful carcasses, but that postmortem aging period of 14 d helped decrease (<italic> P</italic> < 0.0001) the Warner-Bratzler shear force of subprimals across all skeletal maturity groups. The eye of round (<italic>m. semitendinosus </italic>) produced a lower (<italic>P</italic> < 0.05) WBS compared to the loin (<italic>m. longissimus lumborum</italic>). It is possible that this was the result of a longer sarcomere length.

How Mechanical Properties of the Pancreas Signal the Immune System in Type 1 Diabetes

De la Zerda, Adi ProQuest Dissertations & Theses Stanford Universit 2018 해외박사(DDOD)

The goal of this work is to investigate the role of mechanobiology in type 1 diabetes (T1D) using hyaluronic acid (HA)-based hydrogels that mimic the physiological mechanics and biophysical cues in pancreatic islets. This thesis shows each stage of the research, which focuses on characterizing the mechanical properties of the pancreatic islet matrix and the recapitulation of these properties through in-depth in vitro experiments to study their influence on T cell function. The thesis begins with a review in chapter 2 discussing the current challenges in the field of T cell mechanobiology and the importance of elucidating the field by using bioengineering strategies that can dissect the role of mechanical cues on T cell function and behavior while keeping the experimental set-up physiologically relevant. This review provides a short introduction to T cell biology, and covers the design criteria for effective experiments, followed by discussion of the bioengineering tools used to date for studying T cell mechanotransduction. Specifically, bioengineering strategies are categorized into 2- dimensional, 2.5-dimensional and 3-dimensional culture systems. This chapter also proposes and discusses some newer solutions that have been introduced in other fields but have not yet been applied to study T cell mechanotransduction. This chapter serves as background and motivation for bio-mimicking the endogenous T cell microenvironment using biomaterials in which the stiffness and ligand density can be independently tuned. The third chapter presents the role of HA content in governing tissue stiffness in diabetic pancreatic islets. It begins with a quantified characterization of the pancreatic islets’ size, volume, and HA content and composition during T1D in a double transgenic DO11.10 RIPmOVA (DORmO) mice model. Confirmation of HA accumulation in diabetic mice led to examining the effect of HA inhibition on halting T1D progression. The HA content was perturbed using the small-molecule inhibitor 4-methylumbelliferone (4-MU). Then the stiffness of the pancreatic islets was examined using atomic force microscopy (AFM). A novel “bed of nails” approach using quartz glass nanopillars was developed to facilitate this AFM measurement. We found that pre-diabetic islets had significantly reduced mechanical stiffness compared to healthy islets. Treatment of mice with 4-MU reduced HA accumulation, diminished swelling, and led to stiffening of the pancreatic islets to the basal tissue stiffness. Finally, to decouple all the components that may contribute to stiffness changes within pancreatic islets and to study solely the contribution of HA, we exploited an in vitro HA-based system in which the only parameter tuned was HA content. The fourth chapter reports work that continued the effort to mimic the physiological mechanics and biophysical cues that T cells encounter in pancreatic islets. We used in vitro 2D HA-based hydrogels that allow for the modulation of matrix stiffness within the physiological range of pancreatic islets (<1 kPa). Assays monitoring the proliferation and upregulation of activation markers were performed to characterize the T cell activation response. In addition, a polyacrylamide (PAA) gel platform with comparable stiffness to the pancreatic islets was fabricated to assess the role of stiffness on T cell activation independent from the possible confounding effects of HA-specific biochemical signals. To identify mechanosensory proteins relevant in the mechanotransductive pathway, we conducted Western blots with antibodies against 4G10, focal adhesion kinase (FAK) and protein tyrosine kinase 2 (Pyk2). The results of the work in chapters 3 and 4 included an inconsistency: In chapter 3, T cells were more activated in softer pancreatic islets, whereas in chapter 4 higher activation occurred in the stiffer 2D hydrogel substrates. The fifth chapter describes our attempt to resolve this discrepancy by repurposing the HA-based chemistry used in chapter 4 to encapsulate T cells using a 3D culture system. The system was made of chemically crosslinked HA of different stiffness to recapitulate the biophysical cues of pancreatic islets in a controlled fashion. Investigations using this system led to findings on the importance of having a gel chemistry that allows T cell mobility as well as a surprising result on the system’s cytotoxicity. Each of chapters 2 through 5 includes a statement about whether the work has been previously published in a peer-reviewed journal as well as a list of collaborators. The following section describes the specific contributions each collaborator made to the work. Each of these chapters also includes a methods and materials section that briefly describes the experimental procedures, reagents, and equipment used. Finally, chapter 6 presents recommendations for future work around this research topic and conclusions from the work presented. References are in chapter 7. .